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TUMOUR SITE CONCORDANCE

AND MECHANISMS OF
CARCINOGENESIS
EDITED BY ROBERT A. BAAN,
BERNARD W. STEWART, AND KURT STRAIF

IARC SCIENTIFIC
PUBLICATION NO. 165
TUMOUR SITE CONCORDANCE
AND MECHANISMS OF
CARCINOGENESIS
EDITED BY ROBERT A. BAAN,
BERNARD W. STEWART, AND KURT STRAIF

IARC SCIENTIFIC
PUBLICATION NO. 165
Published by the International Agency for Research on Cancer,
150 cours Albert Thomas, 69372 Lyon Cedex 08, France

©International Agency for Research on Cancer, 2019

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About the cover: The background image (credit: Qweek/E+/Getty Images) reflects the concept of “overlap” discussed in this
volume, where agents cause tumours in the same target organs in humans and in experimental animals (see Chapter 21 and
Annex 1). The bar graph (credit: Daniel Krewski) shows a “mechanistic profile” of the 86 carcinogens included in the analysis
described in Chapter 22. The 10 bars represent the key characteristics (see Chapter 10); the height of a bar indicates the
number of agents that display that particular characteristic. Genotoxicity (the second bar from the left) is the most
prominent characteristic.

This book is available in electronic format from


http://publications.iarc.fr.

IARC Library Cataloguing in Publication Data

Tumour site concordance and mechanisms of carcinogenesis / edited by Robert A. Baan, Bernard W. Stewart, Kurt Straif

(IARC Scientific Publications; 165)

1. Neoplasms – etiology 2. Neoplasms – epidemiology 3. Neoplasms, Experimental 4. Carcinogenicity Tests 5. Risk


Assessment 6. Public Health 7. Carcinogenesis
I. Baan, R. A. II. Title III. Series

ISBN 978-92-832-2215-6 (NLM Classification: W1)


ISSN 0300-5085
Table of contents

Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Consensus statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

Part 1
Concordance between cancer in humans and in experimental animals
Chapter 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Electrophilic agents

Chapter 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Aromatic amines and aristolochic acids

Chapter 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Arsenic and metals

Chapter 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Arsenic and metals

Chapter 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Arsenic and metals

Chapter 6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Anticancer agents: qualitative and quantitative aspects

Chapter 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Polycyclic aromatic hydrocarbons and associated occupational exposures

Chapter 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Benzene and haematological cancers

Chapter 9. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Human tumour viruses
Part 2
Mechanisms of carcinogenesis
Chapter 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Key characteristics of carcinogens

Chapter 11 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks

Chapter 12. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107


The role of genotoxicity in carcinogenesis

Chapter 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117


Alterations in cell proliferation, cell death, or nutrient supply

Chapter 14. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129


Receptor-mediated mechanisms

Chapter 15. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153


Oxidative stress and radical-induced signalling

Chapter 16. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159


Immunosuppression

Chapter 17 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Inflammation

Chapter 18. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175


Ionizing radiation

Chapter 19. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185


Host susceptibility

Chapter 20. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201


Age and susceptibility

Part 3
Statistical analyses of concordance and key characteristics
Chapter 21. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Analysis of tumour site concordance

Chapter 22. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257


Analysis of key characteristics of human carcinogens

Annex 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Development of a data set on tumours and tumour sites in humans and in experimental animals for Group 1 agents
identified up to and including Volume 109 of the IARC Monographs

Disclosures of interests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291


Contributors

Editors Workshop Participants Maarten C. Bosland


Department of Pathology
Robert A. Baan Lawrence Banks (April meeting College of Medicine
Senior Visiting Scientist, Section of only) University of Illinois at Chicago
IARC Monographs Tumour Virology Laboratory Chicago, IL, USA
International Agency for Research International Centre for Genetic
on Cancer Engineering and Biotechnology John R. Bucher
Lyon, France Trieste, Italy National Institute of Environmental
Health Sciences
Bernard W. Stewart Frederick A. Beland (April meeting Research Triangle Park, NC, USA
Cancer Control Program only)
South Eastern Sydney Public Health Division of Biochemical Toxicology Jane C. Caldwell
Unit National Center for Toxicological National Center for Environmental
Randwick, NSW, Australia Research Assessment
Jefferson, AR, USA U.S. Environmental Protection
Kurt Straif Agency
Head, Section of IARC Monographs James A. Bond Research Triangle Park, NC, USA
International Agency for Research Santa Fe, NM, USA
on Cancer
Lyon, France

Contributors v
Vincent J. Cogliano Mark A. Hill Christopher J. Portier (April
Integrated Risk Information System Radiation Biophysics Group meeting only)
(IRIS) Gray Institute for Radiation National Center for Environmental
National Center for Environmental Oncology and Biology Health
Assessment University of Oxford Agency for Toxic Substances and
U.S. Environmental Protection Oxford, United Kingdom Disease Registry
Agency Centers for Disease Control and
Washington, DC, USA Charles William Jameson Prevention
CWJ Consulting, LLC Atlanta, GA, USA
David M. DeMarini Cape Coral, FL, USA
Integrated Systems Toxicology Jerry M. Rice
Division Agnes B. Kane Georgetown University Medical
U.S. Environmental Protection Department of Pathology and Center
Agency Laboratory Medicine Department of Oncology
Research Triangle Park, NC, USA Brown University Lombardi Comprehensive Cancer
Providence, RI, USA Center
Bice Fubini Washington, DC, USA
Department of Chemistry Robert J. Kavlock
Interdepartmental Center “G. National Center for Computational Ivan I. Rusyn
Scansetti” for Studies on Asbestos Toxicology Environmental Sciences and
and other Toxic Particulates U.S. Environmental Protection Engineering
Faculty of Pharmacy Agency Gillings School of Global Public
University of Turin Research Triangle Park, NC, USA Health
Turin, Italy University of North Carolina
Daniel Krewski Chapel Hill, NC, USA
Bernard D. Goldstein R. Samuel McLaughlin Centre for
Department of Environmental and Population Health Risk Assessment Martyn T. Smith (November
Occupational Health School of Epidemiology, Public meeting only)
University of Pittsburgh Health and Preventive Medicine Division of Environmental Health
Pittsburgh, PA, USA University of Ottawa Sciences
Risk Sciences International School of Public Health
Stephen S. Hecht Ottawa, Ontario, Canada University of California Berkeley
Masonic Cancer Center Berkeley, CA, USA
University of Minnesota Paul F. Lambert
Minneapolis, MN, USA McArdle Laboratory for Cancer Leslie Stayner
Research Division of Epidemiology and
Uwe Heinrich (unable to attend) University of Wisconsin School of Biostatistics
Fraunhofer Institute of Toxicology Medicine and Public Health School of Public Health
and Experimental Medicine Madison, WI, USA University of Illinois at Chicago
Hanover, Germany Chicago, IL, USA
Ronald L. Melnick
Kari Hemminki Ron Melnick Consulting, LLC Bernard W. Stewart (Observer at
German Cancer Research Center Chapel Hill, NC, USA April meeting)
(DKFZ) Cancer Control Program
Heidelberg, Germany South Eastern Sydney Public Health
Unit
Randwick, NSW, Australia

vi
Robert L. Ullrich Representatives Observer for the Centre Léon
UTMB Cancer Center Bérard
University of Texas Medical Branch Representative of the French Béatrice Fervers
Galveston, TX, USA Agency for Food, Environment and Department of Cancer and the
Occupational Health Safety Environment
Harri Vainio Cécile Michel (November meeting Centre Léon Bérard
Finnish Institute of Occupational only) University of Lyon
Health French Agency for Food, Lyon, France
Helsinki, Finland Environment and Occupational
Observer for the R. Samuel
Health Safety (ANSES)
McLaughlin Centre for Population
Paolo Vineis (April meeting only) Maisons-Alfort, France
Health Risk Assessment
Department of Environmental Pascale Lajoie (November meeting
Epidemiology Representative of the World Cancer
only)
Imperial College London Research Fund International
R. Samuel McLaughlin Centre for
St Mary’s Campus Martin Wiseman (April meeting
Population Health Risk Assessment
London, United Kingdom only)
University of Ottawa
World Cancer Research Fund
Ottawa, Ontario, Canada
Michael P. Waalkes International
Department of Epidemiology
Inorganic Toxicology Group London, United Kingdom
Queens University
National Toxicology Program Kingston, Ontario, Canada
Laboratory
Division of the National Toxicology
Observers
Program IARC Secretariat
Observer for the R. Samuel
National Institute of Environmental
McLaughlin Centre for Population Robert Baan, Senior Visiting
Health Sciences
Health Risk Assessment Scientist (Co-Responsible Officer)
Research Triangle Park, NC, USA
Mustafa Al-Zoughool Lamia Benbrahim-Tallaa
R. Samuel McLaughlin Centre for Véronique Bouvard
Lauren Zeise
Population Health Risk Assessment Graham Byrnes
California Environmental Protection
University of Ottawa Fatiha El Ghissassi
Agency
Ottawa, Ontario, Canada Yann Grosse (Co-Responsible
Reproductive and Cancer Hazard
Department of Community and Officer)
Assessment
Environmental Health Neela Guha
Oakland, CA, USA
King Saud bin Abdulaziz University Zdenko Herceg (April meeting only)
for Health Sciences Marie-Pierre Lambert (April meeting
Riyadh, Saudi Arabia only)
Béatrice Lauby-Secretan
Observer for the R. Samuel Dana Loomis
McLaughlin Centre for Population Heidi Mattock
Health Risk Assessment Magali Olivier (April meeting only)
Michael Bird Ghislaine Scélo (April meeting only)
R. Samuel McLaughlin Centre for Chiara Scoccianti (April meeting
Population Health Risk Assessment only)
University of Ottawa Kurt Straif (Section Head)
Risk Sciences International Jiri Zavadil (November meeting only)
Ottawa, Ontario, Canada

Contributors vii
Additional Contributors Julian Little Administrative Assistance
School of Epidemiology, Public
The following people are co-authors Health and Preventive Medicine Sandrine Egraz
of chapters but were not present at University of Ottawa Elisabeth Elbers
the Workshop. Ottawa, Ontario, Canada Brigitte Kajo
Helene Lorenzen-Augros
Mélissa Billard M. Matilde Marques Annick Leroux
R. Samuel McLaughlin Centre for Department of Chemical and Karine Racinoux
Population Health Risk Assessment Biological Engineering Dorothy Russell
University of Ottawa Graduate Technical Institute
Ottawa, Ontario, Canada Technical University of Lisbon
Department of Epidemiology Production Team
Lisbon, Portugal
Queens University
Kingston, Ontario, Canada Karen Müller
Brittany Milton
English Editor
Risk Sciences International
Nicholas Birkett
Ottawa, Ontario, Canada
R. Samuel McLaughlin Centre for Sylvia Lesage
Population Health Risk Assessment Publishing Assistant
Jan M. Zielinski (deceased)
School of Epidemiology, Public
R. Samuel McLaughlin Centre for Elisabeth Elbers
Health and Preventive Medicine
Population Health Risk Assessment Publications Technician
University of Ottawa
School of Epidemiology, Public
Ottawa, Ontario, Canada
Health and Preventive Medicine Fiona Gould
University of Ottawa Publications Technician
Brian Collins
Healthy Environments and
Risk Sciences International
Consumer Safety Branch, Health Solène Quennehen
Ottawa, Ontario, Canada
Canada Publications Technician
Ottawa, Ontario, Canada
Kate Z. Guyton
National Center for Environmental
Assessment
Office of Research and
Development
U.S. Environmental Protection
Agency
Washington, DC, USA

viii
Acknowledgements

The two-part IARC Workshop on Tumour Site Concordance and Mechanisms of Carcinogenesis (held in April and
November 2012) was supported by grants from the European Commission (grant number VS/2012/0117) and from the
United States National Institute of Environmental Health Sciences (NIEHS).

Acknowledgements ix
Introduction Vincent J. Cogliano

The IARC Monographs to recommend agents for evaluation animals, and representative mech-
or re-evaluation. Otherwise, agents anistic and other relevant data, as
The IARC Monographs on the may be reviewed in response to an well as general information on the
Eval­
uation of Carcinogenic Risks urgent public health need. agent and human exposure to it.
to Humans, published by the International, interdisciplinary Work­ The Working Group meets at IARC,
International Agency for Research ing Groups of expert scientists de- in Lyon, France, for eight days to
on Cancer (IARC) of the World velop each volume of the IARC
discuss the critical reviews and to
Health Organization, are a series Monographs. IARC selects partici-
develop consensus evaluations that
of scientific reviews that identify the pants in the Working Groups based
classify each agent into one of the
causes of cancer in humans. Since on their knowledge and experience,
following categories:
its inception in the early 1970s, the and absence of conflicting interests.
• carcinogenic to humans (Group 1);
IARC Monographs Programme has Working Group members general-
• probably carcinogenic to humans
evaluated more than 1000 chemical, ly have published research on the (Group 2A);
physical, and biological agents and carcinogenicity of the agents under • possibly carcinogenic to humans
classified almost 500 of these as review. IARC also gives considera- (Group 2B);
carcinogenic, probably carcinogenic, tion to demographic diversity among • not classifiable as to its carcinogen-
or possibly carcinogenic to humans. Working Group members and to icity to humans (Group 3);
Agents are identified as subjects a fair balance of scientific findings • probably not carcinogenic to hu-
for IARC Monographs evaluations and views. The IARC Monographs mans (Group 4).
based on evidence of human expo- are a worldwide endeavour that, Each volume of the IARC Mono-
sure and some evidence or suspi- graphs opens with the Preamble,
since 1971, has involved more than
which describes the objective and
cion of carcinogenicity. Agents may 1200 scientists from more than 50
scope of the IARC Monographs
be re-evaluated when substantial countries.
Programme, the scientific principles
new information becomes avail- For each agent, the Working
and procedures used in developing
able. Periodically, IARC convenes Group writes a critical review of the a Monograph, the types of evidence
Advisory Groups of experts from na- pertinent studies of cancer in ex- considered, and the scientific crite-
tional and international health agen- posed humans, cancer after admin- ria that guide the evaluations (IARC,
cies and from research institutions istration of the agent to experimental 2006).

Introduction. Tumour site concordance and mechanisms of carcinogenesis xi


Volume 100: a review of and fibres; ionizing and non-ioniz- and mechanistic events, followed
human carcinogens ing radiation; viruses and other bio- by (ii) supplementary analyses of
logical agents; personal habits; and tumour site concordance between
For Volume 100 of the IARC pharmaceuticals. The precise num- humans and experimental animals,
Monographs, a review was undertak- ber of agents classified in Group 1 and of mechanistic events deemed
en of relevant information on all the cannot be given, because generic relevant to the carcinogenicity of
agents classified in Group 1 (carci- categories such as “nickel com- these agents. The reviews and an-
nogenic to humans). There was val- pounds” and “human papillomavirus- alyses were discussed during a
ue in such a comprehensive review, es” include multiple agents that were two-part Workshop on Tumour Site
because about half of the agents evaluated together. Concordance and Mechanisms of
classified in Group 1 had last been IARC explored ways to strengthen Carcinogenesis, which was con-
evaluated more than 20 years ear- the scientific outcome of Volume 100 vened by IARC on 16–18 April 2012
lier. For advice on the development (IARC, 2012a, b, c, d, e, f). For sever- and 28–30 November 2012 in Lyon.
of Volume 100, IARC convened al prominent human carcinogens, of This Scientific Publication is the re-
an Advisory Group (IARC, 2007) which asbestos and benzene are ex- port of that Workshop.
chaired by Dr Lorenzo Tomatis, who amples, active scientific debate was
had founded the IARC Monographs focused on the implications of mech- Scientific Publication on
Programme and later become the tumour site concordance
anistic studies that had not been con-
and mechanisms of
second IARC Director. ceived when these agents had last carcinogenesis
Volume 100 follows the prac- been reviewed, more than 20 years
tice within the IARC Monographs earlier. For other agents, including al- This Scientific Publication analyses
Programme of occasionally updat- coholic beverages and vinyl chloride, the information on cancer sites and
ing the evidence for a large num- there were questions about whether mechanistic events that was docu-
ber of agents from earlier Volumes. additional cancer sites in humans mented for the more than 100 agents
Supplement 1 updated the available had been established by more recent classified by IARC in Group 1 (car-
data for 54 agents from Volumes research. There were also cross-cut- cinogenic to humans). The corre-
1–20 for which studies of cancer in ting questions about the relevance to sponding Monographs are organized
humans were available, Supplement humans of certain cancer sites or by agent, with a separate Monograph
4 updated the information for 155 mechanistic pathways in animals. It for each agent or group of closely
agents from Volumes 1–29, and was recognized that there would be related agents. The chapters of this
Supplement 7 reviewed 189 agents scientific value in a systematic identi- Scientific Publication are organized
from Volumes 1–42 (IARC, 1979, fication of the cancer sites observed in other ways, in order to develop
1982, 1987b). Supplement 7 was in humans and those observed in insights about larger groupings of
preceded by Supplement 6, which experimental animals, and in a com- agents, cancer sites, and mechanis-
updated and summarized the find- pilation of mechanistic events for tic events.
ings from tests for genetic and relat- agents known to cause cancer in hu- Two data sets bring togeth-
ed effects of the same agents (IARC, mans. The outcome of Volume 100 er information across all Group 1
1987a). More recently, Volume 71 would thus encompass a bridge from agents evaluated up to and including
updated the evidence for 121 agents, the past focus on cancer studies in Volume 109. The first data set con-
most of them classified in Groups 2A humans and experimental animals tains the information about cancer
and 2B, by using a mini-Monograph to a future that promises increas- sites in humans and in experimental
format to present the findings for ing availability of mechanistic data. animals (see Annex 1, by Grosse et
most of these agents (IARC, 1999). Therefore, IARC initially planned the al.). It is organized to facilitate the
Group 1 agents are diverse and project in two phases: (i) a review investigation of tumour site concor-
include chemicals and chemical mix- of human carcinogens that would dance across species (see the con-
tures; occupations; metals, dusts, accrue information on cancer sites cordance analysis in Chapter 21,

xii
by Krewski et al.). It has long been To accommodate the different de- to determine whether there is coher-
recognized that concordance be- grees of precision with which cancer ence, which can be understood as
tween human and animal tumour sites have been identified (e.g. “liver concordance confirmed or discor-
sites is not evident for carcinogens cancer” for one agent and “hepato- dance explained.
of all types considered as a single cellular carcinoma” for another), the The chapters in this Scientific
category. Concordance can be ana- database uses designations that are Publication address what we have
lysed for a grouping of agents (e.g. more general in nature (“liver cancer” learned about some major mech-
aromatic amines) or for a cancer site in this example). Likewise, a detailed anisms for agents known to cause
(e.g. haematological cancers). Such list of 24 mechanistic events that was cancer in humans. The Consensus
analyses could explore the predictive initially proposed was subsequent- Statement was unanimously en-
value for human cancer of tumours in ly condensed to a set of 10 “key dorsed by the Workshop partici-
experimental animals, based on the characteristics” (see below). This pants. The chapters in Part 1 discuss
information collected to date. These level of aggregation is necessary to various groupings of carcinogenic
types of analyses may also identify avoid fragmenting the data into large agents, such as electrophilic agents,
human cancers for which there cur- numbers of categories with few data metals, constituents of tobacco
rently are no good animal models; points; this makes it possible to con- smoke, and human tumour viruses.
for these cancers, it might be advan- duct analyses across reasonable These chapters illustrate the types
tageous to focus on understanding numbers of agents. Further research of analysis that can be undertaken
mechanistic pathways to design new will change our understanding of for groups of carcinogenic agents,
experimental models that could iden- mechanistic events and will estab- including those that act at a com-
tify agents linked to these cancers. lish additional associations of agents mon site or through a common
The second data set contains the with mechanistic events in the future. mechanism.
information about established and Five additional human carcino- Chapter 10 (by Smith) discusses
our observation that all human carcin-
likely mechanistic events (see Al- gens, identified after the completion
ogens evaluated in Volume 100 (and
Zoughool et al., 2019 and Birkett et of Volume 100, are included in the
subsequent Monographs as men-
al., 2019). It is organized to facilitate data set: (i) diesel engine exhaust (re-
tioned above) display one or more of
the investigation of patterns across viewed in Volume 105; IARC, 2013),
what are called key characteristics of
mechanistic events and agents (ii) trichloroethylene (evaluated in
carcinogens: is electrophilic or can
(see the mechanistic analysis in Volume 106; IARC, 2014), (iii) poly-
be metabolically activated to elec-
Chapter 22, by Krewski et al.). Data chlorinated biphenyls (PCBs) and di-
trophiles; is genotoxic; alters DNA
can be aggregated for groupings of oxin-like PCBs (reviewed in Volume
repair or causes genomic instabil-
agents that involve common mech- 107; IARC, 2016b), and (iv) outdoor
ity; induces epigenetic alterations;
anistic events or common cancer air pollution and (v) particulate matter
induces oxidative stress; induces
sites. Such analyses could identify in outdoor air pollution (both evaluat-
chronic inflammation; is immunosup-
biomarkers that could be incorporat- ed in Volume 109; IARC, 2016a).
pressive; modulates receptor-medi-
ed into future epidemiological stud- The two data sets are linked. ated effects; causes immortalization;
ies. They also could identify popula- Concordance in the first data set and/or alters cell proliferation, cell
tions and developmental stages that may find support from the mech- death, or nutrient supply. Chapter 11
may be especially susceptible to the anistic information in the second. (by Stewart) places the key char-
occurrence of certain mechanistic Lack of concordance in the first data acteristics in the context of other
events. Ultimately, they could lead to set may or may not be explained by viewpoints, such as the hallmarks
the confident identification of human the mechanistic information in the of carcinogenesis. Subsequent
carcinogens based on mechanistic second. Accordingly, the analyses chapters in Part 2 discuss the role
information in the absence of ade- in this Scientific Publication do not of several of the key characteristics
quate cancer studies in humans or stop at a superficial analysis of con- individually, followed by chapters that
experimental animals. cordance or discordance. They seek discuss susceptibility.

Introduction. Tumour site concordance and mechanisms of carcinogenesis xiii


The analyses of concordance and a description of the databases of of the lessons to be learned from
mechanisms are presented in the concordance and mechanisms that the information in Volume 100 of the
chapters in Part 3. To facilitate simi- were developed from the informa- IARC Monographs. We encourage
lar analyses by cancer researchers tion compiled for Volume 100 and for all scientists to continue to analyse
worldwide, Annex 1 (by Grosse several subsequent Volumes. these data and to develop further
et al.), Al-Zoughool et al. (2019), We regard this Scientific Publi­ insights into the causes of cancer in
and Birkett et al. (2019) provide cation as the beginning, not the end humans.

References
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M, Birkett N, Krewski D, et al. (2019). chemicals, hydrazine and hydrogen peroxide. combustions. IARC Monogr Eval Carcinog
Development of a database of toxicological IARC Monogr Eval Carcinog Risks Hum. Risks Hum. 100E:1–575. PMID:23193840.
end-points and key characteristics of agents 71:1–315. PMID:10507919. Available from: Available from: http://publications.iarc.fr/122.
known to cause cancer in humans. J Toxicol http://publications.iarc.fr/89.
Environ Health B Crit Rev. (forthcoming) IARC (2012e). Pharmaceuticals. IARC
IARC (2006). Preamble to the IARC Monogr Eval Carcinog Risks Hum. 100A:1–
Birkett N, Al-Zoughool M, Bird M, Zielinski JM, Monographs, amended January 2006. 437. PMID:23189749. Available from: http://
Krewski D (2019). Carcinogenic mechanisms Available from: https://monographs.iarc.fr/ publications.iarc.fr/118.
for 109 human carcinogens. J Toxicol Environ previous-preamble/.
Health B Crit Rev. (forthcoming) IARC (2012f). Radiation. IARC Monogr
IARC (2007). Report of the Advisory Group Eval Carcinog Risks Hum. 100D:1–437.
IARC (1979). Chemicals and industrial to Plan Volume 100: a review of human PMID:23189752. Available from: http://
processes associated with cancer in humans. carcinogens. IARC Internal Report No. publications.iarc.fr/121.
IARC Monographs, volumes 1 to 20. IARC 07/001. Available from: https://monographs.
Monogr Eval Carcinog Risk Chem Hum Suppl. iarc.fr/ENG/Publications/internrep/07-001. IARC (2013). Diesel and gasoline engine
1:1–71. PMID:296141. Available from: http:// pdf. exhausts and some nitroarenes. IARC
publications.iarc.fr/133. Monogr Eval Carcinog Risks Hum. 105:1–
IARC (2012a). Arsenic, metals, fibres, and 704. PMID:26442290. Available from: http://
IARC (1982). Chemicals, industrial processes dusts. IARC Monogr Eval Carcinog Risks publications.iarc.fr/129.
and industries associated with cancer in Hum. 100C:1–499. PMID:23189751. Available
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IARC Monogr Eval Carcinog Risk Chem Hum ethylene and some other chlorinated agents.
Suppl. 4:1–292. PMID:6963265. Available IARC (2012b). Biological agents. IARC IARC Monogr Eval Carcinog Risks Hum.
from: http://publications.iarc.fr/136. Monogr Eval Carcinog Risks Hum. 100B:1– 106:1–514. PMID:26214861. Available from:
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IARC (1987a). Genetic and related effects: an publications.iarc.fr/119.
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volumes 1 to 42. IARC Monogr Eval Carcinog IARC (2012c). Chemical agents and related Monogr Eval Carcinog Risks Hum. 109:1–
Risks Hum Suppl. 6:1–729. PMID:3504843 occupations. IARC Monogr Eval Carcinog 448. Available from: http://publications.iarc.
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publications.iarc.fr/139.

xiv
Consensus statement

This statement was unanimous- required sufficient evidence in hu- (in 2007), benzo[a]pyrene (in 2010),
ly endorsed by the participants in mans to classify an agent as carcino- aristolochic acid (in 2012), and etopo-
the Work­shop on Tumour Site Con­ genic to humans (Group 1). Scientific side (in 2012). Mechanistic evidence
cordance and Mech­anisms of Car­ understanding of the mechanisms was also important in classifying
cinogenesis, which was convened
of carcinogenesis, accompanied by the carcinogenicity of several other
by IARC on 16–18 April and 28–30
the development of assays for stud- agents between 2004 and 2010, and
November 2012 in Lyon.
ying mechanistic events, has led to in revising the classification of car-
Introduction new ways of identifying human car- cinogenicity for several additional
cinogens. Some examples are the agents in Volume 100.
The IARC Monographs Pro­gramme
following agents that were classified For Volume 100 of the IARC
is an international consensus ap-
as carcinogenic to humans: ethylene Monographs, a review was undertak-
proach to the identification of chem-
icals and other agents that may oxide (in 1994), based on strong ev- en during 2008–2009 of relevant in-
pre­sent carcinogenic hazards to idence of genotoxicity and limited formation on all the agents classified
humans. The Monographs assess epidemiological evidence in exposed in Group 1 (carcinogenic to humans)
the strength of the published sci- humans; 2,3,7,8-tetrachlorodiben- in Volumes 1–99. There was value
entific evidence for such identifica- zo-para-dioxin (in 1997), based on in a comprehensive review, because
tions, which are based primarily on about half of the agents classified
strong evidence of binding to the
epidemiological studies of cancer in Group 1 had last been reviewed
aryl hydrocarbon receptor and sub-
in humans and bioassays for car- more than 20  years earlier. Volume
sequent events; neutron radiation (in
cinogenicity in laboratory animals. 100 was organized in six parts, each
2000), based on the underlying radi-
Information that may be relevant to prepared by a separate Working
ation physics; benzidine-based dyes
the mechanisms by which the puta- Group, covering: pharmaceuticals
tive carcinogen acts is also consid- (in 2010), because these substances
(Volume 100A); biological agents
ered in making an overall evaluation are metabolized to a carcinogen in (Volume 100B); arsenic, metals,
of the strength of the total evidence humans; and several compounds for fibres, and dusts (Volume 100C);
for carcinogenicity to humans. which single-agent exposure does radiation (Volume 100D); person-
The use of mechanistic data to not exist because they are compo- al habits and indoor combustions
identify human carcinogens is accel- nents of (complex) mixtures, for ex- (Volume 100E); and chemical agents
erating. Initially, the IARC Monographs ample tobacco-specific nitrosamines and related occupations (Volume

Consensus statement. Tumour site concordance and mechanisms of carcinogenesis xv


100F). Volume 100: A Review of Tumour site concordance bioassay); however, metabolic
Human Carcinogens was published or mechanistic considerations
in 2012 as a six-book set. 1. The results developed in Volume might explain tumour induction
IARC explored ways to strength- 100 of the IARC Monographs at different sites in separate ani-
en the scientific value of Volume confirm that the induction of mal models.
100, and embarked on a two-phase cancer in experimental animals 4. Descriptive statistics of tumour
project: (i) a review of the Group 1 is relevant to the identification sites identified to date may not
human carcinogens with respect to of a carcinogenic hazard to hu- be representative of future eval-
cancer sites and mechanistic events, mans: all human carcinogens uations or of the incompletely
followed by (ii) supplementary an- identified to date that have been characterized “universe of hu-
alyses of tumour site concordance adequately tested in animals man carcinogens”. The car-
between humans and experimental have also been shown to cause cinogens evaluated in Volume
animals, and of mechanistic events cancer in animals. 100 include several classes of
deemed relevant to the carcinogen- 2. For many human carcinogens, agents that have been relative-
icity of these agents. Accordingly, there is tumour site concordance ly straightforward to investigate;
this Scientific Publication on Tumour between humans and experi- some examples are: alkylating
Site Concordance and Mechanisms mental animals; for many others, agents that were used in ear-
of Carcinogenesis was proposed. there is not. At present, the state ly cancer chemotherapy; viral
To prepare for the supplemen- of the science does not support agents that infect hundreds of
tary analyses in this Scientific tumour site concordance as a millions of people; ionizing ra-
Publication, IARC had asked the general principle. For example, diation, which affects multiple
six Working Groups for Volume 100 there are four agents for which anatomical sites; widespread
to collect additional information, there is sufficient evidence for exposures, such as to tobacco
not routinely developed before, on
breast cancer in humans and smoke and its related agents
(i) cancer sites in humans for which
seven agents for which there and alcoholic beverages; and
there was sufficient evidence or
is sufficient evidence for breast chemical agents with long histo-
limited evidence in epidemiological
cancer in experimental animals, ries of occupational exposure at
studies, (ii) cancer sites for which
but only one of these agents high levels. Agents evaluated in
there was sufficient evidence in
causes breast cancer in both the future may have more subtle
experimental animals, and (iii) es-
humans and animals. effects and different character-
tablished and likely mechanisms
3. The analyses presented in this istics. Evidence from sources
involved in the cancers observed in
Scientific Publication are ex- other than human epidemiolo-
humans or experimental animals.
pected to underestimate concor- gy will need to be relied upon to
To further develop this Scientific
dance. One reason is the limited identify carcinogenic hazards to
Publication, the IARC Monographs
power and other limitations of humans.
Programme convened a group of
international scientific experts in a many observational epidemi- 5. Past evaluations have noted
two-part Workshop on Tumour Site ological studies that include cancer in experimental animals
Concordance and Mechanisms of populations and cancer sites at approximately 40 tumour sites
Carcinogenesis, held in Lyon in April that have not been adequate- in 15 organ and tissue systems.
and November 2012. The Workshop ly investigated. Another rea- Use of standard terminology
participants used the lists of mecha- son is that – for the purpose of for these sites can facilitate the
nistic events to develop a set of key this concordance analysis – an development of databases and
characteristics to define the mecha- agent was considered to cause their analysis and linkage to
nistic profile of the agents classified cancer at a site in animals only other sources of information.
in Group 1. if positive results were replicated The Workshop participants
The main points of consensus, at the same specific site in an- recommend that future IARC
the conclusions, and the recommen- other animal experiment (while Monographs Working Groups
dations of the Workshop participants recognizing the limitations of a consider the anatomically
are described below. single positive result in a cancer based taxonomy of tumour sites

xvi
that appears in this Scientific of systematic reviews of such The past practice of according
Publication in the analysis of evidence. The Workshop par- greatest concern to those agents
concordance between sites ticipants recommend that the demonstrated to be genotoxic,
where tumours arise in animals IARC Monographs Programme relative to agents whose carcino-
and in humans. use the key characteristics in its genicity appeared to be mediated
6. The Workshop participants also evaluations of carcinogenicity. by one or more other key char-
recommend that in future IARC 9. It is notable that in vivo or in vitro acteristics, appears to be overly
Monographs, the Evaluation mechanistic data are often avail- simplistic.
section for evidence of carci- able in humans. In most cases, 12. The objective of the IARC Mono­
nogenicity in experimental an- when animal data are available graphs Programme is to identi-
imals be expanded to include for a key characteristic, human fy carcinogenic hazards, not to
additional information for agents data for that characteristic are exhaustively list all mechanistic
generally available, too. This events and pathways that might
evaluated as exhibiting sufficient
supports the notion that carcin- contribute to carcinogenesis.
evidence. For such agents, an
ogens show their characteristics Future coverage of mechanis-
additional sentence after the
across species. tic data should increase as the
relevant evaluation should refer
10. There should be no expec- retrieval of such data becomes
to the recognized site or sites
tation that all, or even most, more systematic and the key
of tumorigenesis, by using the
key characteristics operate characteristics are used as a
specification system described
for any human carcinogen. No framework for organization and
in the chapter on concordance
key characteristic is neces- analysis of mechanistic data.
analysis (Chapter 21, by Krewski
sary for carcinogenesis, and 13. Descriptive statistics of mecha-
et al.).
negative results for one or nisms identified to date may not
more key characteristics are be representative of future eval-
Mechanisms involved in
not an argument against the uations. Although genotoxicity
human carcinogenesis
potential carcinogenicity of is the key characteristic most
7. With increasing scientific un- an agent. Observation of one exhibited by the human carcin-
derstanding and availability of or more key characteristics in ogens identified to date, this
information on mechanisms of exposed humans can increase may reflect the relatively great-
carcinogenesis, it is expected the biological plausibility of er attention paid in the past to
that the IARC Monographs will less-than-sufficient evidence the investigation of genotoxic
make even greater use of mech­ in humans. Observation of agents. Future evaluations of car-
anistic data in identifying human one or more key characteris- cinogenic agents may involve a
carcinogens. tics in experimental animals larger set of mechanistic events
8. Until now, there has been no can increase confidence and pathways that are not yet
generally accepted method for in the human relevance of well developed or understood.
organizing mechanistic data less-than-sufficient evidence Accordingly, future shifts in the
pertinent to the identification in experimental animals. In distribution of the key character-
of carcinogenic hazards to interpreting the biological rel- istics are to be expected. This
humans. The key character- evance of information pertain- does not detract from the value
istics presented here offer a ing to the key characteristics, of using these characteristics
promising foundation for the it is important to consider as- now in evaluations of carcino-
structured evaluation of mech- pects of metabolism and kinet-
genic hazards.
anistic information, and this ics in extrapolating between in
should increase the utility of vitro and in vivo systems.
mechanistic evidence in future 11. A human carcinogen may dis-
identifications of carcinogenic play multiple key characteristics
hazards and the transparency that may interact with each other.

Consensus statement. Tumour site concordance and mechanisms of carcinogenesis xvii


Abbreviations

ADH alcohol dehydrogenase


ADME absorption, distribution, metabolism, and elimination
AFB1 aflatoxin B1
AhR aryl hydrocarbon receptor
AIDS acquired immune deficiency syndrome
ALDH2 aldehyde dehydrogenase 2
ALL acute lymphoblastic leukaemia
AML acute myeloid leukaemia
AP-1 activator protein 1
AR androgen receptor
ARNT aryl hydrocarbon receptor nuclear translocator
ATLL adult T-cell leukaemia/lymphoma
ATP adenosine triphosphate
B[a]P benzo[a]pyrene
BCME bis(chloromethyl)ether
BL Burkitt lymphoma
BrdU 5-bromo-2′-deoxyuridine
bw body weight
CA chromosomal aberrations
CCL2 chemokine (C-C motif) ligand 2
CIN cervical intraepithelial neoplasia
CLL chronic lymphocytic leukaemia
CML chronic myeloid leukaemia
CMME chloromethyl methyl ether
COSMIC Catalogue of Somatic Mutations in Cancer
COX-2 cyclooxygenase 2
CT computed tomography

Abbreviations xix
CYP450 cytochrome P450
DCVC 1,2-dichlorovinyl-cysteine
DCVG (1,2-dichlorovinyl)glutathione
DDT dichlorodiphenyltrichloroethane
DES diethylstilbestrol
DMA(V) dimethylarsinic acid
DMBA 7,12-dimethylbenz[a]anthracene
DOHaD developmental origins of health and disease
DSBs double-strand breaks
eBL endemic Burkitt lymphoma
EBNA EBV nuclear antigen 1
EBV Epstein–Barr virus
EGF epidermal growth factor
EH epoxide hydrolase
ENCODE Encyclopedia of DNA Elements
ENU N-nitrosoethylurea
EPA United States Environmental Protection Agency
ER estrogen receptor
ERK extracellular signal-regulated kinase
ERα estrogen receptor alpha
F-344 Fischer 344
FANC/BRCA Fanconi anaemia complementation groups/breast cancer A
FISH fluorescence in situ hybridization
GLP good laboratory practice
GSH glutathione
GST glutathione S-transferase
GSTM1 glutathione-S-transferase M1
GTP guanosine-5′-triphosphate
GWAS genome-wide association study
H 2 O2 hydrogen peroxide
Hb haemoglobin
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HBx HBV X protein
HCC hepatocellular carcinoma
HCV hepatitis C virus
HER human epidermal growth factor receptor
HIF-1α hypoxia-inducible transcription factor 1 alpha
HIV human immunodeficiency virus
HIV-1 human immunodeficiency virus type 1
HMGB1 high-mobility group box 1 protein
HNSCC head and neck squamous cell carcinoma
HPB 4-hydroxy-1-(3-pyridyl)-1-butanone
HPV human papillomavirus
Hsp90 heat shock protein 90
HSV-1 herpes simplex virus type 1
HTLV-1 human T-cell lymphotropic virus type 1
IARC International Agency for Research on Cancer
ICD International Classification of Diseases
IGF-1 insulin-like growth factor 1

xx
IgM immunoglobulin M
IL-1α interleukin 1 alpha
InChI International Chemical Identifier
IRIS Integrated Risk Information System
IUPAC International Union of Pure and Applied Chemistry
JNK c-Jun N-terminal kinase
K14 keratin 14
KEGG Kyoto Encyclopedia of Genes and Genomes
KSHV Kaposi sarcoma-associated herpesvirus
LANA latency-associated nuclear antigen
LET linear energy transfer
LMP latent membrane protein
lncRNA long non-coding RNA
MALT mucosa-associated lymphoid tissue
MAPK mitogen-activated protein kinase
methyl-CCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea
MHV-68 Murid herpesvirus 68
miRNA microRNA
MMA monomethylarsenic
MMA(III) monomethylarsinous acid
MMPs matrix metalloproteinases
MN micronuclei
MNNG N-methyl-N′-nitro-N-nitrosoguanidine
MOCA 4,4′-methylenebis(2-chloroaniline)
NAB N′-nitrosoanabasine
NAD+ oxidized nicotinamide adenine dinucleotide
NADH reduced nicotinamide adenine dinucleotide
NADPH nicotinamide adenine dinucleotide phosphate
NAT N′-nitrosoanatabine
NDEA N-nitrosodiethylamine
NDMA N-nitrosodimethylamine
NF-κB nuclear factor kappa-light-chain-enhancer of activated B cells
NHL non-Hodgkin lymphoma
NK natural killer
NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
NNN N′-nitrosonornicotine

NO nitric oxide
NPV negative predictive value
NQO1 NAD(P)H:quinone oxidoreductase 1
Nrf 2 nuclear factor erythroid 2-related factor 2
O2•− superoxide
OAT organic anion transporter

OH hydroxyl radical
PAHs polycyclic aromatic hydrocarbons
PCB 77 3,3′,4,4′-tetrachlorobiphenyl
PCB 118 2,3′,4,4′,5-pentachlorobiphenyl
PCB 126 3,3′,4,4′,5-pentachlorobiphenyl
PCB 128 2,2′,3,3′,4,4′-hexachlorobiphenyl
PCB 153 2,2′,4,4′,5,5′-hexachlorobiphenyl

Abbreviations xxi
PCBs polychlorinated biphenyls
PCNA proliferating cell nuclear antigen
PeCDF 2,3,4,7,8-pentachlorodibenzofuran
PGHS-2 prostaglandin-endoperoxide synthase 2
PGRMC1 progesterone receptor membrane component 1
PI3K phosphoinositide 3-kinase
PPAR peroxisome proliferator-activated receptor
PPV positive predictive value
PR progesterone receptor
RET/PTC rearranged during transfection/papillary thyroid carcinoma
SCC squamous cell carcinoma
SCE sister chromatid exchange
SCID severe combined immunodeficiency
SIV simian immunodeficiency virus
SREBP1c sterol regulatory element-binding protein 1c
STAT3 signal transducer and activator of transcription 3
SULT1B1 sulfotransferase family cytosolic 1B member 1
SV40 simian virus 40
TCDD 2,3,7,8-tetrachlorodibenzo-para-dioxin
TCE trichloroethylene
TCF8 transcription factor 8
TD50 dose rate (in mg/kg body weight/day) that is estimated to reduce the proportion of
tumour-free animals by 50%
TGF-β transforming growth factor beta
TNF-α tumour necrosis factor alpha
ToxCast Toxicity Forecaster
TPA 12-O-tetradecanoylphorbol-13-acetate
UDP uridine-5′-diphosphate
UV ultraviolet
vFLIP viral Fas-associated death domain-like IL-1-converting enzyme inhibitory protein
vGPCR viral G protein-coupled receptor
WHO World Health Organization

xxii
CHAPTER 1
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 1.

Electrophilic agents
James A. Bond and Ronald L. Melnick

Introduction or mice and, except for ethylene chloride), the lung (sulfur mustard),
oxide, sufficient evidence of carci- the lymphohaematopoietic system
In this chapter, electrophilic agents nogenicity from studies of exposed (benzene, 1,3-butadiene, and eth-
include direct-acting electrophilic humans. For ethylene oxide, there ylene oxide), nasal tissue (formal-
chemicals and chemicals that are was limited evidence of carcinogen- dehyde), and the kidney (TCE). For
metabolized to reactive electro- icity in humans, but the classification bis(chloromethyl)ether (BCME), the
philes. All of the chemicals discussed of this chemical was raised to carci- lung and the nasal cavity were iden-
here are IARC Group 1 agents and nogenic to humans (Group 1) based tified as target organs in humans
as such can be characterized as on strong mechanistic evidence and rats, respectively. In addition,
carcinogenic to humans. Relevant of mutagenicity and clastogenici- angiosarcomas of the liver, which
carcinogens discussed in this chap- ty, including the induction of sister are rare tumours, were identified in
ter do not include pharmaceutical chromatid exchange (SCE), chro- humans, rats, and mice exposed to
drugs classified in Group 1, which mosomal aberrations (CA), and mi- vinyl chloride.
otherwise typically include alkylating cronuclei (MN) in workers exposed In several instances, tumour sites
cytotoxic agents. to ethylene oxide. identified in animals were not de-
Tumour sites identified in previous Among this group of chemicals, tected in epidemiological studies of
IARC evaluations of the carcinogen- there is remarkable concordance in exposed workers. These apparent
icity of several non-pharmaceutical tumour sites with sufficient evidence discrepancies may be due to dif-
organic compounds in humans and or limited evidence of carcinogenicity ferences in susceptibility between
laboratory animals are shown in in humans and sufficient evidence of humans and certain animal mod-
Table 1.1. For each of these agents, carcinogenicity in rats and/or mice, els, differences in exposure con-
there was sufficient evidence of car- for example for the liver (aflatoxins, ditions between studies in animals
cinogenicity from studies in rats and/ trichloroethylene [TCE], and vinyl and in humans, or limitations in

Part 1 • Chapter 1. Electrophilic agents 1


Table 1.1. Tumour sites in humans, rats, and mice for electrophilic agents

2
Agent Humans Rats Mice

Sufficient evidence Limited evidence Sufficient evidence Sufficient evidence

Aflatoxins Liver: hepatocellular Liver: hepatocellular carcinoma


carcinoma
Benzene Acute myeloid Acute lymphoblastic Oral cavity: carcinoma Lymphoid tissue: lymphoma
leukaemia leukaemia Forestomach: squamous cell Haematopoietic tissue: granulocytic leukaemia
Chronic lymphocytic carcinoma Mammary gland: adenocarcinoma
leukaemia Skin: squamous cell carcinoma Lung: bronchiolo-alveolar carcinoma
Multiple myeloma Zymbal gland: carcinoma Zymbal gland: carcinoma
Non-Hodgkin lymphoma Preputial gland: squamous cell carcinoma
Bis(chloromethyl)ether Lung Nasal cavity: olfactory Soft tissue: sarcoma
(BCME) neuroblastoma Skin: fibrosarcoma
1,3-Butadiene Lymphohaematopoietic Lymphoid tissue: lymphoma
Soft tissue: haemangiosarcoma
Liver: hepatocellular carcinoma
Mammary gland: adenocarcinoma
Lung: bronchiolo-alveolar carcinoma
Forestomach: squamous cell carcinoma
Harderian gland: carcinoma
Preputial gland: squamous cell carcinoma
Ethylene oxide Lymphohaematopoietic Brain: glioma Lung
(non-Hodgkin lymphoma, Lymphoid tissue: lymphoma
multiple myeloma, chronic Peritoneum: mesothelioma
lymphocytic leukaemia)
Breast
Formaldehyde Nasopharynx Paranasal sinuses Nasal cavity: squamous cell
Leukaemia carcinoma
Sulfur mustard Lung Larynx Lung
Trichloroethylene (TCE) Kidney Non-Hodgkin lymphoma Kidney Liver
Liver Lung
Vinyl chloride Liver: angiosarcoma Liver and extrahepatic: Liver and extrahepatic: angiosarcoma
Liver: hepatocellular angiosarcoma Lung: bronchiolo-alveolar carcinoma
carcinoma Liver: hepatocellular carcinoma Mammary gland: adenocarcinoma
Mammary gland: adenocarcinoma
Zymbal gland: carcinoma
epidemiological studies that reduce Table 1.2. Common among these 10 interactions of organic compounds,
their power to detect excess cancer agents is the electrophilic nature of or their metabolites, with DNA and

CHAPTER 1
PART 1
risk at particular sites. For example, the parent chemical or a metabolite other critical macromolecules or re-
the finding that mammary gland tu- thereof. All of these agents either ceptors are also important, because
mours were induced in female mice exist as direct-acting electrophilic these interactions can initiate the car-
exposed to benzene, 1,3-butadiene, species or can be metabolized to cinogenic process or affect its pro-
or vinyl chloride, whereas breast can- reactive electrophilic species. It is gression. Determinants for metabol-
cer risk was not elevated in exposed generally accepted that the abili- ism or molecular interactions are
workers may be due to the fact that ty of these electrophiles, whether dependent on the exposure history
women were not included in many alkylating agents, epoxides, or qui- (including the intensity, duration,
occupational cohort or case–control nones, to react with nucleophiles, frequency, and route or routes of
studies of these agents, sometimes such as DNA, is key to the carci- exposure, and the life stage of the
because there were very few or no nogenicity of this group of agents exposed individual) and may differ
women in relevant workforces, or (Table 1.1 and Table 1.2). according to the specific genotype of
because they were exposed to much Because of the diversity in the the individual (i.e. genetic polymor-
lower concentrations. In contrast, for biochemical and physical properties phisms in metabolic enzymes).
hospital-based sterilization workers of organic electrophilic compounds,
exposed to ethylene oxide, among it is useful to first examine, in a gen- Absorption
whom there is a high proportion of eral way, factors that are important
for their absorption, distribution, Organic compounds can be ab-
women, increases in breast cancer
metabolism, and elimination. These sorbed after inhalation, dermal ex-
incidence were observed. Exposure
factors include, but are not limited posure, or ingestion. The sites and
to ethylene oxide increased the inci-
to, (i) physiological, (ii) chemical, extent of absorption depend on the
dence of mammary gland tumours
and (iii) metabolic determinants. physicochemical characteristics of
in female mice but not in male mice.
Examples of physiological factors the compound. Highly reactive or-
Thus, limitations in human cancer
are alveolar ventilation rate, cardi- ganic chemicals typically interact
databases and sex-specific tumours
ac output, blood flow to organs, or- with tissues at the portal of entry,
in animal studies need to be consid-
gan volumes, and body mass and and toxicity and carcinogenicity are
ered in evaluations of site concor-
composition (e.g. percentage body often most evident at these sites.
dance for tumour induction between
fat). Examples of chemical factors Formaldehyde, for example, is a wa-
humans and animals.
include the stability and reactivity ter-soluble, reactive volatile organic
The carcinogenicity of organic
of the parent compound and its me- compound. After inhalation, form-
compounds and their organ specifici-
tabolites, partition coefficients that aldehyde interacts with the highly
ty in animal models and in humans
control the distribution of organic aqueous nasal mucosa, the first
are influenced by numerous factors.
compounds between blood and air respiratory tract tissue that is en-
This chapter focuses on factors
or blood and tissues, rates of uptake countered upon inspiration. In rats
that affect tissue dosimetry, factors
from the gastrointestinal tract, and chronically exposed to formaldehyde
that contribute to the multistep car-
absorption through the dermis. For vapours, the anterior portion of the
cinogenic process for each agent,
some organic chemicals, metabol- nasal cavity was the site of tumour
and factors that might account for
ic determinants such as the cellular induction.
interspecies and inter-individual
capacity for metabolism and the af- Sulfur mustard (also known as
differences in susceptibility.
finity of metabolic enzymes for the mustard gas) is another example of
Chemical disposition compound are critical, both in acti- a reactive volatile organic compound
vating the parent compound to the that can be absorbed after inhalation
The role of metabolism in the forma- putative carcinogenic electrophilic or through dermal exposure. It is a
tion of the putative carcinogenic inter- metabolite and in transforming the lipophilic substance that easily pene-
mediates of the non-pharmaceutical parent compound or the electrophilic trates into the skin and mucosal sur-
organic compounds that are carcino- intermediate into a metabolite that faces (Drasch et al., 1987; Somani
genic to humans is summarized in can be readily eliminated. Molecular and Babu, 1989), resulting in a high

Part 1 • Chapter 1. Electrophilic agents 3


Table 1.2. Mechanisms for formation of electrophilic species

Agent Mechanism Electrophilic species

Bis(chloromethyl)ether (BCME) Direct-acting Chloroalkyl ether


Chloromethyl methyl ether (CMME) Direct-acting Chloroalkyl ether
Ethylene oxide Direct-acting Epoxide
Formaldehyde Direct-acting Aldehyde
Sulfur mustard Direct-acting Sulfonium ion
Aflatoxins Metabolic activation Epoxide
Benzene Metabolic activation Quinone, epoxide, aldehyde
1,3-Butadiene Metabolic activation Epoxides
Trichloroethylene (TCE) Metabolic activation Epoxide, thioketene
Vinyl chloride Metabolic activation Epoxide

degree of bioavailability. Sulfur mus- among the most potent animal and large blood–tissue interface of the
tard is a bifunctional alkylating agent human carcinogens known. The fact alveoli in that region. The blood–air
(IARC, 1987). Like for formaldehyde, that BCME and CMME are powerful partition coefficient is a key factor in
there is evidence that DNA alkylation alkylating agents provides moderate determining the maximum levels of
by sulfur mustard leads to forma- to strong evidence that they oper- the organic compound that can be
tion of cross-links, inhibition of DNA ate by a genotoxic mechanism. This attained in the blood at any given
synthesis and repair, and induction mechanism is likely to be similar to concentration of the compound in air.
of point mutations and chromo- that of other strong alkylating agents, For example, the blood–air partition
some-type and chromatid-type ab- involving modification of DNA and coefficients for the carcinogens vinyl
errations (ATSDR, 2003). Some of resultant mutations. BCME can chloride, 1,3-butadiene, benzene,
these changes are observed in nasal also be absorbed through the skin, and TCE are 1.2, 1.5, 7.8, and 10
tissue, consistent with the nose be- and studies in which mice were ex- respectively. At equivalent air con-
ing a target organ for sulfur mustard. posed to BCME by dermal applica- centrations, higher levels of benzene
BCME and chloromethyl methyl tion demonstrated that this agent is and TCE will be present in the blood
ether (CMME) are highly reactive or- a potent complete skin carcinogen, at steady state, compared with vinyl
ganic compounds that belong to the producing both papillomas and squa- chloride and 1,3-butadiene.
group of chloroalkyl ethers, which mous cell carcinomas (Van Duuren The organic compounds dis-
are flammable, volatile, colourless et al., 1969). cussed here can also be absorbed
liquids with highly irritating odours. Ethylene oxide is a water-soluble after oral exposure. Ingestion and
In water and aqueous biological volatile organic compound that is rel- subsequent oral absorption repre-
fluids, these substances are rapid- atively stable in aqueous solutions at sent the greatest potential route of
ly hydrolysed to form hydrochloric neutral pH (half-life, approximately exposure to non-volatile organic
acid, methanol, and formaldehyde 72 hours). Because it is completely compounds. Organic chemicals may
(Nichols and Merritt, 1973; National miscible with water, inhaled ethylene enter the body via food or in liquids.
Toxicology Program, 2004). The car- oxide will dissolve in the aqueous lin- Aflatoxin is a compound for which
cinogenic effects of BCME are re- ing of the respiratory tract and diffuse ingestion is considered the most
stricted to the epithelial tissue where into the blood. Thus, uptake of this important route of exposure. For ex-
exposure occurs, namely the lung organic compound will be substantial ample, human uptake of aflatoxins
for humans and respiratory tract tis- throughout the respiratory tract. in quantities of nanograms to micro-
sues for laboratory animals exposed Volatile organic compounds that grams per day occurs mainly through
to BCME by inhalation. This por- are not reactive or water-soluble are consumption of contaminated maize
tal-of-entry effect is consistent with generally absorbed into the blood, and peanuts, which are dietary sta-
the short half-life of BCME in aque- primarily in the pulmonary region of ples in some tropical countries.
ous media (ATSDR, 1989). BCME is the respiratory tract because of the Uptake of aflatoxin M1 in quantities

4
of nanograms per day occurs main- reactive exo-epoxide; binding of the to be metabolized via two CYP450
ly via consumption of aflatoxin-con- exo-epoxide to DNA, resulting in enzymes, rather than just one,

CHAPTER 1
PART 1
taminated milk, including breast milk the formation of DNA adducts; and and that the putative, high-affinity
(IARC, 2002). Once absorbed from miscoding during DNA replication, enzyme is active primarily at ben-
the gastrointestinal tract, aflatoxins which leads to development of mu- zene concentrations below 1 ppm
are transported via the hepatic por- tations, with eventual progression to (Rappaport et al., 2009). Whereas
tal blood to the liver, where they are tumours. Aflatoxin B1, the most com- CYP2E1 is the primary enzyme re-
metabolized. As discussed below, mon and potent of the aflatoxins, is sponsible for mammalian metabol-
metabolism is key to understanding metabolized predominantly in the ism of benzene at higher levels of
the carcinogenicity of aflatoxins. liver. In humans, CYP1A2, CYP2B6, exposure (Valentine et al., 1996;
CYP3A4, CYP3A5, and CYP3A7, Nedelcheva et al., 1999), CYP2F1
Metabolic activation or as well as the phase II enzyme glu- and CYP2A13 have been proposed
detoxification and elimination tathione-S-transferase M1 (GSTM1; as enzymes that metabolize ben-
see below) are hepatic enzymes zene at environmental levels of expo-
Metabolism plays a key role in both that mediate aflatoxin metabolism. sure, i.e. below 1 ppm (Powley and
the activation and the detoxification In humans, the relative contribution Carlson, 2000; Sheets et al., 2004;
of many organic compounds. The of these enzymes in vivo depends Rappaport et al., 2009).
first step, generally called phase I not only on their affinity but also A large proportion of benzene
metabolism, is oxidation to a meta- on their expression level in the liv- oxide spontaneously rearranges to
bolic intermediate. This intermediate er, where CYP3A4 is predominant, phenol, which is either eliminated via
becomes the substrate for the sec- mediating the formation of the afla- phase II conjugation or further metab-
ond step, phase II, in which it is en- toxin B1 exo-epoxide and aflatoxin olized to hydroquinone and 1,4-ben-
zymatically hydrolysed or conjugated Q1. In humans, as in other species, zoquinone. The remaining benzene
with one of a variety of biological sub- the DNA binding and carcinogenicity oxide is either hydrolysed to produce
strates, such as sulfate, glucuronic of aflatoxin B1 result from its con- benzene-1,2-dihydrodiol (catechol),
acid, or glutathione (GSH). Phase II version to the exo-8,9-epoxide by which is further oxidized to 1,2-ben-
reactions increase the water solubil- CYP3A4 (Essigmann et al., 1982). zoquinone, or conjugated with GSH
ity of the chemical, which facilitates This epoxide is highly reactive and to produce S-phenylmercapturic
its excretion in urine, or increase the is the main mediator of cellular injury acid. Metabolism of the oxepin tau-
molecular weight, so that the agent (McLean and Dutton, 1995). In con- tomer of benzene oxide is thought to
is more readily eliminated in bile. trast, CYP1A2 can generate some open the aromatic ring; this yields the
Phase I metabolism of organic com- exo-epoxide but also a high propor- reactive muconaldehydes and mu-
pounds can also result in formation tion of endo-epoxide and aflatoxin conic acid. Benzene oxide, the ben-
of reactive intermediates that can M1. Aflatoxins M1 and Q1, produced zoquinones, the muconaldehydes,
spontaneously interact with critical from aflatoxin B1 by CYP1A2 and and the benzene dihydrodiol epox-
macromolecules. For many organic CYP3A4, respectively, are present ides (formed from CYP450-mediated
chemicals, this is the first key step in in the urine of individuals exposed oxidation of benzene dihydrodiol)
the carcinogenic process. to aflatoxins (Ross et al., 1992; Qian are electrophiles that react readily
Cytochrome P450 (CYP450) is et al., 1994). with peptides, proteins, and DNA
the collective name of the family of Benzene is also a substrate for (Bechtold et al., 1992; McDonald
enzymes responsible for the initial CYP450 enzymes. In common with et al., 1993; Bodell et al., 1996;
phase I metabolism of many organ- other compounds discussed in this Gaskell et al., 2005; Henderson et al.,
ic compounds. Metabolic activation section, benzene must be metabo- 2005; Waidyanatha and Rappaport,
by various CYP450 isozymes is lized to become carcinogenic (Ross, 2005), thereby interfering with cellu-
a key first step in the carcinogen- 2000; Snyder, 2004). The initial lar function (Smith, 1996). The role of
ic process for aflatoxin, benzene, metabolic step involves CYP450- these different metabolites in the car-
1,3-butadiene, TCE, and vinyl chlo- dependent oxidation to benzene ox- cinogenicity of benzene remains un-
ride. For example, in the mechanism ide, which exists in equilibrium with clear, but formation of benzoquinone
of carcinogenicity of aflatoxins, the its tautomer oxepin. It has been re- from hydroquinone via myeloperoxi-
key steps involve: metabolism to a ported that benzene is most likely dase in the bone marrow has been

Part 1 • Chapter 1. Electrophilic agents 5


suggested to be a key step (Smith, competition between 1,3-butadiene by alcohol dehydrogenase (ADH)
1996). Indeed, there is consider- and butenediol or epoxybutene for and ALDH, and glucuronidation.
able evidence for an important role CYP450-mediated metabolism may The initial step in TCE oxidation in
of the metabolic pathway that leads limit the extent to which the second both humans and laboratory ani-
to formation of benzoquinone, be- oxidation reaction may occur.
mals is catalysed by one of several
cause the benzoquinone-detoxifying Vinyl chloride is another volatile
CYP450 enzymes and results in
enzyme NAD(P)H:quinone oxidore- organic compound that is primarily
ductase 1 (NQO1) protects mice and rapidly metabolized in the liv- the formation of an enzyme-bound
against benzene-induced myelodys- er (Reynolds et al., 1975; Ivanetich intermediate (an oxygenated TCE-
plasia (Long et al., 2002; Iskander et al., 1977; Barbin and Bartsch, CYP450 transition state, TCE-O-
and Jaiswal, 2005) and protects 1989; Lilly et al., 1998; Bolt, 2005) by CYP), which is chemically unstable.
humans against the haematotoxicity CYP2E1 (WHO, 1999). The prima- This intermediate can be converted
of benzene (Rothman et al., 1997). ry metabolites of vinyl chloride are to (i) the electrophile TCE epoxide,
However, this does not rule out ad- the highly reactive chloroethylene
(ii)  N-hydroxy-acetyl-aminoethanol,
verse effects from other metabolites. oxide, which is formed in a dose-de-
or (iii) chloral, which is in equilibrium
Similarly to the metabolism of pendent process and has a half-life
with chloral hydrate. The majority of
benzene, the first step in 1,3-buta- of 1.6 minutes in aqueous solution
diene metabolism involves CYP450- at neutral pH (Barbin et al., 1975; the conversion is directed towards
mediated oxidation to epoxybutene Dogliotti, 2006), and its rearrange- chloral/chloral hydrate. TCE epoxide
(Himmelstein et al., 1997). At low ment product chloroacetaldehyde spontaneously generates dichloro-
concentrations of 1,3-butadiene, (Bonse et al., 1975). Both can bind acetyl chloride, another chemically
metabolism via the CYP2E1 isozyme to proteins, RNA, and DNA and form unstable and reactive species, or
predominates (IARC, 1999, 2008). etheno adducts in RNA and DNA. oxalic acid, which is readily excreted
Epoxybutene may be metabolized Chloroethylene oxide is more reac-
in urine. Dichloroacetyl chloride un-
by conjugation with GSH mediated tive with nucleotides than is chlo-
dergoes spontaneous dechlorination
by glutathione S-transferase (GST) roacetaldehyde (Guengerich and
to produce dichloroacetate. Chloral/
or by hydrolysis catalysed by epox- Watanabe, 1979).
ide hydrolase (EH) (Csanády et al., Conjugation of chloroethylene chloral hydrate undergoes either re-
1992; Himmelstein et al., 1997). It oxide and chloroacetaldehyde with duction by ALDH or CYP450 to gen-
may also be oxidized to multiple GSH eventually leads to the major erate trichloroethanol, or oxidation
diastereomers of diepoxybutane urinary metabolites N-acetyl-S-(2- by ALDH to form trichloroacetate.
(Seaton et al., 1995; Krause and hydroxyethyl)cysteine and thiodigly- Trichloroacetate is typically the major
Elfarra, 1997), and dihydroxybutene colic acid (Plugge and Safe, 1977). urinary metabolite of TCE that is re-
formed by hydrolysis of epoxybutene Chloroethylene oxide can also be de-
covered, although trichloroethanol is
may be oxidized to epoxybutanediol. toxified to glycolaldehyde by microso-
also an important metabolite in urine.
Diepoxybutane and epoxybutane- mal EH, and chloroacetaldehyde can
Trichloroethanol can be oxidized by
diol are also detoxified by GST or be converted to chloroacetic acid by
EH (Boogaard et al., 1996). Partial aldehyde dehydrogenase 2 (ALDH2) CYP450 enzymes to yield trichloro-
hydrolysis of diepoxybutane also (Guengerich and Watanabe, 1979; acetate, or can undergo glucuronida-
produces epoxybutanediol. Bone ATSDR, 2006; IARC, 2008). tion by uridine 5′-diphosphate (UDP)-
marrow myeloperoxidase can also Two metabolic pathways of TCE glucuronosyltransferases to produce
epoxidize 1,3-butadiene. Each of have been characterized in both trichloroethanol glucuronide. In all
the epoxide intermediates may con- humans and laboratory animals: species investigated, trichloroace-
tribute to the mutagenicity and car- oxidation and GSH conjugation
tate and trichloroethanol/trichloro-
cinogenicity of 1,3-butadiene. The (IARC, 2014). The major pathway
ethanol glucuronide are formed in
formation of epoxybutanediol or di- is CYP450-mediated oxidation, re-
epoxybutane requires a second oxi- sulting in the formation of a variety much larger quantities than other
dation of butenediol or epoxybutene, of short- and long-lived metabolites. oxidative metabolites. There are
respectively. At increasing exposure Subsequent processing of oxida- quantitative differences in the extent
concentrations of 1,3-butadiene, tive metabolites involves reduction of oxidative TCE metabolism among

6
species at higher exposures, but at dehyde dehydrogenase, and cat- is converted (i) by enzymatic and
lower exposures oxidation is limited alase, as well as CYP2E1. One- non-enzymatic hydrolysis to ethyl-

CHAPTER 1
PART 1
by the hepatic blood flow. carbon metabolism not mediated by ene glycol, which is partly excreted
Conjugation with GSH is another CYP450 is central to many biologi- as such and partly metabolized fur-
important metabolic pathway for TCE, cal processes. In aqueous solution, ther via glycolaldehyde, glycolic acid,
resulting in the formation of short- formaldehyde is rapidly converted and glyoxylic acid to oxalic acid, for-
lived and reactive metabolites. The to its dihydroxy form, methanediol mic acid, and carbon dioxide; and
initial conjugation reaction occurs (CH2(OH)2, also known as formalde- (ii) by conjugation with GSH.
primarily in the liver to form (1,2-di- hyde hydrate or methylene glycol), Sulfur mustard, BCME, and
chlorovinyl)glutathione (DCVG). which is in dynamic equilibrium with CMME can also react spontaneous-
Subsequent processing of DCVG formaldehyde.
ly with biological molecules without
occurs primarily in the kidney, which the need for metabolic activation.
is a tumour target site in rats and hu- Direct-acting compounds
For example, the reactivity of sulfur
mans. In the kidney, DCVG can be
Some organic compounds discussed mustard with a wide variety of cel-
hydrolysed by γ-glutamyltransferase
here are sufficiently reactive that they lular macromolecules is well docu-
and cysteinylglycine dipeptidase to
do not require metabolic activation mented (IARC, 1975, 1987; ATSDR,
1,2-dichlorovinyl-cysteine (DCVC),
as the first step in the carcinogenic 2003). The presence of two chlorine
which may be N-acetylated to form
process. Formaldehyde reacts read- atoms makes it a strong bifunctional
a mercapturate or converted by
ily and reversibly with amino groups alkylating agent with a high chemi-
β-lyase to generate a reactive thi-
to form Schiff bases, and with sulf- cal reactivity (Dacre and Goldman,
olate that rearranges to form either
hydryl groups resulting in the forma- 1996). The chlorine atom is typically
chlorothioketene or chlorothioacetyl
chloride (Dekant et al., 1988; Völkel tion of S-hydroxymethylglutathione, released under formation of a car-
and Dekant, 1998). Chlorothioketene which is oxidized by ADH3 to bonium ion, which then undergoes
and chlorothioacetyl chloride are S-formylglutathione. S-formylglu- intramolecular cyclization to create a
highly reactive and chemically un- tathione is further metabolized by highly reactive compound. Formation
stable, and are thought to be the S-formylglutathione hydrolase to of the carbonium ion is facilitated
molecular forms responsible for ad- generate formate and GSH. Formate in aqueous solutions (Somani and
duct formation with nucleic acids in can also be formed non-enzymati- Babu, 1989); this explains the sensi-
the kidney (Müller et al., 1998a, b). cally (Hedberg et al., 2002). Because tivity of mucosal tissues, such as the
For both humans and rodents, the formaldehyde reacts non-enzymat- eye, to its effect (Solberg et al., 1997).
information on urinary excretion of ically with critical macromolecules Elevated concentrations of thiodigly-
stable end-products is much more (DNA and others), many of these en- col, the major hydrolysis product of
extensive for the oxidative pathway zymatic processes can be viewed as mustard gas, were detected in hu-
than for the GSH conjugation path- detoxification steps, especially those man urine after exposure to mustard
way. However, this is not an accurate that lead to incorporation of the com- gas vapour and aerosol (Jakubowski
indication of the overall flux through pound into the one-carbon pool. et al., 2000). BCME and CMME are
each pathway, because it does not Ethylene oxide is another di- rapidly hydrolysed in water and in
account for the formation of reactive rect-acting alkylating agent that re- aqueous biological fluids to form hy-
and chemically unstable metabolites acts with nucleophiles without the drochloric acid, methanol, and form-
via the GSH conjugation pathway. need for metabolic transformation. aldehyde (Nichols and Merritt, 1973;
As noted above, CYP450 en- The direct reaction of ethylene ox- National Toxicology Program, 2004).
zymes are not the only enzymes ide with DNA is thought to initiate
involved in the metabolism of organ- the cascade of genetic and related Molecular interactions (DNA
ic compounds. Formaldehyde, an events that lead to cancer (Swenberg adducts, genetic alterations,
important intermediate in one-car- et al., 1990). The pathways of ethyl- etc.)
bon metabolism, is a substrate for ene oxide metabolism can thus be
several enzymes, including cyto- considered detoxification pathways A common feature of the above-men-
solic ADH, mitochondrial ALDH, that increase the elimination of the tioned agents is that they either are
cytosolic GSH-dependent formal- parent chemical. Ethylene oxide direct-acting electrophiles or are

Part 1 • Chapter 1. Electrophilic agents 7


metabolized to electrophiles. The cause mutations and cytogenetic and enzymes involved in mismatch
carcinogenicity of these chemicals is alterations. Entries with weak ev- repair and excision repair (Bernucci
considered to be initiated by reaction idence may reflect the availability et al., 1997).
of the electrophile with nucleophilic of few or no published studies for
Sulfur mustard
sites in DNA, leading to the induction certain characteristics of particular
of mutations, DNA strand breaks, agents in animal or human tissues, The elimination of a chloride ion from
and/or CA. However, additional pro- rather than negative responses sulfur mustard creates a highly re-
cesses may also be involved, for ex- (Table 1.3). active cyclic sulfonium ion that can
ample free-radical-mediated oxida- alkylate cellular macromolecules
tive stress, inhibition of DNA repair, BCME and CMME including DNA, RNA, and proteins.
inhibition of topoisomerase II, and Because of the presence of two chlo-
The chloroalkyl ethers BCME and
immunosuppression. In addition to rine atoms, sulfur mustard can act as
CMME are often referred to as pow- a bifunctional alkylating agent, pro-
time-dependent variation in tissue
concentrations of DNA-reactive me- erful alkylating agents. However, ducing DNA interstrand or intrastrand
tabolites of the chemicals described because these compounds are cross-links, for example by binding to
above, the likelihood that these short-lived in aqueous solution and guanines on opposite strands or to
compounds or their metabolites will undergo rapid hydrolysis, genotoxic- neighbouring guanines on the same
bind to DNA and induce site-spe- ity studies of BCME and CMME are strand (Roberts et al., 1971; Walker,
cific genetic alterations that lead to sparse and have produced mixed re- 1971; Shahin et al., 2001; Saladi
tumour development is a function sults (IARC, 1987). Both compounds et al., 2006). Such cross-links can in-
of the physicochemical properties were mutagenic in bacteria (Mukai hibit DNA synthesis and cell division.
of the reactive agent (e.g. binding and Hawryluk, 1973; Anderson Sulfur mustard-specific 2-hydroxy­
affinity for DNA or protein), sever- ethylthioethyl–DNA adducts have
and Styles, 1978) and caused an
been detected in in vitro systems
al cellular features (including tissue increase in the frequency of CA in
and in multiple tissues of exposed
concentrations of alternative binding peripheral lymphocytes of exposed
animals (Somani and Babu, 1989;
biomolecules such as GSH, rates of workers (Srám et al., 1983). BCME
Fidder et al., 1994; van der Schans
cell division and cell death, and the binds to guanine and adenine res- et al., 1994; Niu et al., 1996). Similar
activities and effectiveness of repair idues of calf thymus DNA in vitro to the binding pattern for other alkyl-
enzymes for DNA adducts), other (Goldschmidt et al., 1975). Both com- ating agents, sulfur mustard-derived
physiological characteristics (e.g. pounds induced unscheduled DNA DNA adducts have been identified
age, sex, health status, and immu-
synthesis in cultured human cells at N7 of guanine, N3 of adenine,
nocompetence), and lifestyle factors
(Agrelo and Severn, 1981; Perocco and O6 of guanine (Fidder et al.,
(e.g. other exposures). Thus, multiple
et al., 1983) and cell transforma- 1994). O6 -alkylguanine DNA alkyl-
factors and mechanistic processes
tion in Syrian hamster embryo cells transferase is ineffective in repairing
affect the tissue and species speci-
(Casto, 1983) and cultured human O6 -ethylthioethylguanine adducts
ficity for tumour development asso- (Ludlum et al., 1986). Thus, sulfur
fibroblasts (Kurian et al., 1990). The
ciated with exposures to each of the mustard can inhibit cell division by
carcinogenicity of BCME is widely
carcinogenic chemicals discussed in cross-linking of DNA strands and
thought to involve mutagenesis re-
this chapter. can produce mutations by inducing
Table 1.3 presents 10 key char- sulting from alkylation of DNA bases
errors in DNA replication or repair.
acteristics of carcinogens (see (Bernucci et al., 1997). BCME and
Sulfur mustard induced mutations
Chapter 10, by Smith) that have been CMME may act synergistically with
and CA in exposed animals and in
identified in in vivo and/or in vitro formaldehyde, one of their hydrolysis a variety of in vitro systems (IARC,
studies on the electrophilic agents products. The likelihood of BCME– 1987). Further, TP53 mutations (pre-
reviewed in this chapter. What is DNA adducts leading to mutations dominantly G  →  A transitions) were
most evident from Table 1.3 is that depends on the cellular content and detected in DNA extracted from
all these compounds produce DNA activity of DNA repair enzymes such lung tumours of individuals exposed
adducts in humans and animals, and as methylguanine methyltransferase, to sulfur mustard (Hosseini-khalili

8
Table 1.3. Levels of evidencea in humans and animals of key characteristics of carcinogens

Characteristic Aflatoxins Benzene 1,3-Butadiene Ethylene oxide

Humans Animals Humans Animals Humans Animals Humans Animals

Is electrophilic or can be metabolically activated to electrophiles 2 2 2 2 2 2 2 2


Is genotoxic 2 2 2 2 2 2 2 2
Alters DNA repair or causes genomic instability 1 1 1 1
Induces epigenetic alterations 0 0 1 0 0 0 1 0
Induces oxidative stress 0 0 1 1 0 0 0 0
Induces chronic inflammation 0 0
Is immunosuppressive 0 0 2 2 0 0 0 0
Modulates receptor-mediated effects 1 1
Causes immortalization 0 0 0 0 0 0 0 0
Alters cell proliferation, cell death, or nutrient supply 0 1 1 1 1 0 0 0

Characteristic Formaldehyde Sulfur mustard Trichloroethylene Vinyl chloride

Humans Animals Humans Animals Humans Animals Humans Animals

Is electrophilic or can be metabolically activated to electrophiles 2 2 2 2 2 2 2 2


Is genotoxic 2 2 2 2 2 2 2 2
Alters DNA repair or causes genomic instability 2 1 1 1 0 0
Induces epigenetic alterations 1 1 0 0 0 1 0 0
Induces oxidative stress 0 0 1 1 0 0 0 0
Induces chronic inflammation 0 0
Is immunosuppressive 0 0 0 0 1 1 0 0
Modulates receptor-mediated effects 0 1
Causes immortalization 0 0 0 0 0 0 0 0
Alters cell proliferation, cell death, or nutrient supply 2 2 1 1 0 1 1 1
a 2 = strong evidence, 1 = moderate evidence, 0 = weak evidence.

Part 1 • Chapter 1. Electrophilic agents


9
PART 1
CHAPTER 1
et al., 2009). The base excision re- Hong et al., 2007). Evidence was be the major determinants of the na-
pair and nucleotide excision repair also provided for the involvement of sal carcinogenicity of formaldehyde
pathways were activated in human mutations in p53. in humans and laboratory animals.
lymphoblastoid cell lines exposed to The carcinogenicity of ethylene A mechanism for formalde-
the sulfur mustard analogue 2-chlo- hyde-induced myeloid leukae-
oxide is thought to be due to the
roethyl-ethylsulphide (Jowsey et al., mogenesis might involve pancytope-
induction of gene mutations and/
2009). nia caused by genotoxicity leading
or chromosomal changes resulting
to damage of primitive progenitor
Ethylene oxide from the formation of ethylene ox-
cells in the bone marrow; mutation
ide-derived DNA adducts. Although of myeloid progenitor cells by form-
Ethylene oxide is a direct-acting al- evidence for the carcinogenicity of aldehyde and subsequent growth of
kylating agent that reacts with nu- ethylene oxide was sufficient in ex- a mutant phenotype may then lead
cleophiles, resulting in the formation to myeloid leukaemia. Evidence of a
perimental animals and limited in
of a variety of adducts in DNA, RNA, mild pancytopenic effect of formal-
humans, the observed increases in
and protein. Numerous studies have dehyde or changes in ratios of lym-
the frequencies of CA, SCE, and MN
demonstrated that ethylene oxide phocyte subsets has been reported
in lymphocytes of exposed workers
induces gene mutations and chro- in exposed workers (Kuo et al., 1997;
served as the basis for raising the
mosomal changes in in vitro systems Ye et al., 2005; Tang et al., 2009;
classification of this alkylating agent
and in prokaryotic and eukaryotic Zhang et al., 2010). In addition, col-
to carcinogenic to humans (Group 1)
organisms. 2-Hydroxyethyl–DNA ony formation by cultured progenitor
(IARC, 1994, 2008). cells that give rise to myeloid cells
adducts formed upon exposure to
ethylene oxide have been observed Formaldehyde is inhibited by low concentrations of
in vivo at N7 of guanine, N3 of ad- formaldehyde (Zhang et al., 2010).
enine, and O6 of guanine (Walker Formaldehyde can react directly with The observation of increased mon-
et al., 1992) and in vitro at N1 and cellular macromolecules including osomy (loss) of chromosome 7 and
N6 of adenine and at the N3 posi- proteins and nucleic acids. The for- trisomy (gain) of chromosome 8 in
tion of cytosine, uracil, and thymine cultured myeloid progenitor cells
maldehyde-specific DNA adduct N 6 -
(Tates et al., 1999). Genotoxicity re- obtained from the blood of workers
hydroxymethyl-deoxyadenosine has
exposed to formaldehyde may be rel-
sulting from ethylene oxide-induced been identified in lymphocytes of
evant to the potential involvement of
DNA adducts may involve mispairing smokers (Wang et al., 2009). The ge-
formaldehyde in leukaemogenesis,
of altered bases, formation of aba- notoxicity of formaldehyde is well es- because these types of cytogenetic
sic sites upon depurination of the tablished: it induces mutations (point changes are frequently seen in my-
adducts at N7 of guanine followed
mutations, deletions, and insertions), eloid leukaemia and myelodysplastic
by insertion of a different base dur-
CA, SCE, MN, DNA strand breaks, syndromes (Zhang et al., 2010).
ing DNA synthesis, or DNA strand
and DNA–protein cross-links in sev-
breaks and subsequent chromo- 1,3-Butadiene
eral in vitro and in vivo systems, in-
some breakage (Tates et al., 1999;
cluding CA, SCE, and MN in nasal 1,3-Butadiene can be metabolized to
Houle et al., 2006). In mice, ethylene
mucosal cells and/or lymphocytes of three different DNA-reactive epoxide
oxide induced large deletion muta-
exposed humans (IARC, 2006). In intermediates, which are direct-act-
tions, base-pair substitutions, and
ing mutagens (IARC, 2008). The ma-
frameshift mutations (Walker and animals exposed to formaldehyde,
jor DNA adducts formed from these
Skopek, 1993; Walker et al., 1997a, genotoxic effects were more con-
epoxide intermediates in rats and
b). In tumours obtained from mice sistently found in nasal tissues than
mice exposed to 1,3-butadiene are
exposed to ethylene oxide, increas- in blood lymphocytes. In addition,
at the N7 position of guanine. These
es in K-Ras mutations with frequent formaldehyde produces irritation of N7-guanine adducts can undergo
G  →  T transversions at codon 12 the nose and pharynx in humans and spontaneous or glycosylase-medi-
and G  →  C transversions at codon laboratory animals. Genotoxicity and ated depurination, which leaves an
13 were reported (Houle et al., 2006; increased cell proliferation appear to apurinic site in the DNA. Epoxide

10
metabolites of 1,3-butadiene can effects of 1,3-butadiene and its me- oxide and/or by the rearrangement
also react at sites involved in base tabolites are induction of CA, SCE, product chloroacetaldehyde (Bonse

CHAPTER 1
PART 1
pairing and form adducts at the N3 and MN. et al., 1975). Both intermediates can
position of cytosine, at N1 and N6 of Genetic alterations in 1,3-butadi- bind to proteins, RNA, and DNA
adenine, and at N1 and N2 of guanine ene-induced tumours in mice are of (Guengerich and Watanabe, 1979).
(Selzer and Elfarra, 1996a, b, 1997; the same type as those frequently Vinyl chloride is mutagenic in bac-
Zhao et al., 1998; Zhang and Elfarra, involved in the development of a va- teria and mammalian cells. It is also
2004). An increase in the number of riety of human cancers. The K-Ras, clastogenic in vivo and in vitro, caus-
N1-trihydroxybutyladenine adducts H-Ras, p53, p16/p15, and β-cate- ing increases in the frequencies of
was detected in lymphocytes of work- nin mutations detected in tumours CA, SCE, and MN (IARC, 2008).
ers exposed to 1,3-butadiene (Zhao from exposed mice are probably The major DNA adduct formed from
et al., 2000). Alkylation of N1-adenine the result of the DNA reactivity and chloroethylene oxide is at the N7 po-
by epoxybutene followed by hydro- the genotoxic effects of 1,3-butadi- sition of guanine. In addition, etheno
lytic deamination forms the highly ene-derived epoxides. Other DNA- DNA adducts (1,N 6 -ethenoadenine,
alkylating metabolites of 1,3-butadi- 3,N4-ethenocytosine, N2,3-etheno-
mutagenic deoxyinosine (Rodriguez
ene (hydroxymethylvinylketone and guanine, and 1,N2-ethenoguanine)
et al., 2001), which codes for incor-
crotonaldehyde) may also contribute have been identified after in vitro in-
poration of cytosine during DNA rep-
to the mutagenicity and carcino- cubations with chloroethylene oxide,
lication, leading to the generation of
genicity of this compound. A con- and levels of these adducts are in-
A → G mutations. Diepoxybutane is
sistent pattern of K-Ras mutations creased in multiple organs of rats ex-
a bifunctional alkylating agent that
(G  →  C transversions at codon 13) posed to vinyl chloride by inhalation
can form monoadducts in DNA simi-
was observed at multiple organ sites (Ciroussel et al., 1990; Guengerich,
lar to those formed by epoxybutane-
of 1,3-butadiene-induced cancers 1992; Swenberg et al., 2000). The
diol, or DNA–DNA cross-links by
(Hong et al., 2000; Sills et al., 2001; etheno adducts, which may be in-
binding at the N7 position of guanine
Ton et al., 2007). Alterations in the volved in base-pair substitutions, are
of one DNA strand and at another
p53 gene in brain tumours in mice much more persistent than the N7-
site elsewhere in the DNA, such as
were mostly G → A transition muta- guanine adduct (Fedtke et al., 1990)
the N7 of another guanine or the N1 tions (Kim et al., 2005) that probably and have demonstrated miscoding
of an adenine (Goggin et al., 2009). arose from miscoding at apurinic potential in vitro and in vivo, causing
Depurination of these interstrand sites resulting from depurination of A  →  G transitions, A  →  T transver-
or intrastrand lesions can induce N7-guanine adducts. Inactivation of sions, C →  A transversions, C  → T
point mutations and large deletions. the tumour suppressor genes p16 transitions, and G  →  A transitions
However, if diepoxybutane alkylates and p15 may also be important in the (Singer et al., 1987; Cheng et al.,
an adenine at N6 in DNA, an exocy- development of 1,3-butadiene-in- 1991; Mroczkowska and Kuśmierek,
clic adenine adduct is formed pref- duced lymphomas (Zhuang et al., 1991; Singer et al., 1991; Basu et al.,
erentially to DNA–DNA cross-linked 2000). Mammary gland adenocarci- 1993). The same types of mutation
products (Antsypovich et al., 2007). nomas induced by 1,3-butadiene in have been observed in the TP53
1,3-Butadiene is genotoxic at mice frequently had mutations in the and RAS genes in vinyl chloride-in-
multiple tissue sites in mice and rats, p53, H-Ras, and β-catenin genes duced tumours. TP53 mutations
and its epoxide metabolites are mu- (Zhuang et al., 2002). Overall, these associated with exposure to vinyl
tagenic in a variety of in vitro sys- observations point to a genotoxic chloride (frequently A → T transver-
tems. Deletion mutations and base mechanism underlying the devel- sions) were found in angiosarcomas
substitution mutations induced by opment of 1,3-butadiene-induced in both humans and rats, and muta-
these alkylating agents are consis- cancers. tions in K-RAS were also associated
tent with their DNA adduct profiles with vinyl chloride-induced angio-
Vinyl chloride
and include G → A transition muta- sarcomas in humans (IARC, 2008).
tions, G  →  C transversions, A  →  T The carcinogenicity of vinyl chloride Polymorphisms in XRCC1, a gene
transversions, and A → G transitions is probably caused by its highly re- that encodes an enzyme that repairs
(Lee et al., 2002). Other genotoxic active metabolite chloroethylene etheno DNA adducts, may account

Part 1 • Chapter 1. Electrophilic agents 11


for inter-individual differences human hepatocellular carcinomas is gene mutations, it has shown poten-
among exposed workers in suscep- associated with exposure to aflatoxin tial to affect DNA and chromosomal
tibility to genetic damage induced by B1 (Gomaa et al., 2008). G → T trans- structure. The formation of DNA ad-
vinyl chloride (Li et al., 2003). version mutations are predominant ducts (Mazzullo et al., 1992; Cai and
in cell culture systems and animal Guengerich, 2001) and the mutage-
Aflatoxins
models and are consistent with the nicity of TCE in vitro are dependent
Aflatoxin B1 is genotoxic in prokary- formation of the major aflatoxin B1- on the presence of metabolic acti-
otic and eukaryotic systems in vitro, derived N7-guanine adduct. This is vation systems (IARC, 2014). There
including cultured human cells, and because adenine is most commonly is strong evidence that the GSH-
in vivo in humans and in a variety of inserted opposite the apurinic site. conjugated metabolites of TCE, par-
animal species. Its metabolism to a However, other types of mutation ticularly DCVC, are genotoxic, and
reactive exo-8,9-epoxide results in have also been observed with afla- some of the oxidative metabolites
DNA binding and formation of DNA toxin B1, including G  →  C transver- (TCE epoxide, dichloroacetate, and
adducts that lead to gene mutations, sions and G → A transitions in DNA chloral/chloral hydrate) may also be
CA, SCE, MN, and mitotic recombi- repair-deficient xeroderma pigmen- genotoxic. Thus, biotransformation
nation in a variety of in vivo and in tosum cells (Levy et al., 1992); this of TCE can produce genotoxic me-
vitro systems (IARC, 2002). Adduct suggests that DNA repair deficien- tabolites, particularly in the kidney,
formation in DNA at the N7 position cy may influence the frequency and where in situ metabolism occurs
of guanine represents more than distribution of mutations within a (IARC, 2014).
98% of the total adducts formed by gene. Aflatoxin B1 may contribute to Both genotoxic and non-genotox-
the exo-8,9-epoxide (Guengerich genomic instability in hepatocellular ic mechanisms may contribute to the
et al., 1998). Depurination of this gua- carcinomas (Kaplanski et al., 1997) carcinogenicity and toxicity of TCE
nine adduct creates an apurinic site. by inducing mitotic recombination at other sites, including the liver, the
Alternatively, the N7-guanine adduct and loss of heterozygosity. The lung, and the haematopoietic sys-
may convert to the more stable ring- concurrent presence of hepatitis B tem. In addition to genotoxicity, epi-
opened aflatoxin B1–formamidopy- virus, which causes chronic active genetic alterations, oxidative stress,
rimidine adduct (Groopman et al., hepatitis and cirrhosis, increases the cytotoxicity, and altered rates of
1981). Differences in the mutational incidence of hepatocellular carcino- cell division or apoptosis may be in-
specificity of an apurinic site-con- mas caused by aflatoxins in humans volved in tumour induction in the liver
taining genome (derived from depu- (IARC, 2002). or lung. The immunotoxicity of TCE
rination of aflatoxin B1–N7-guanine) may be involved in the development
Trichloroethylene
compared with that of a genome with of haematopoietic cancers. However,
the aflatoxin B1–N7-guanine adduct Data from human studies suggest the data are inadequate for reliable
itself, where mutations also occurred that exposure to TCE increases the conclusions to be drawn about caus-
at the base 5′-adjacent to the site of frequency of CA in peripheral lym- al relationships between non-geno-
the adducted guanine, suggest that phocytes (Tabrez and Ahmad, 2009) toxic mechanisms and TCE-induced
intercalation of the aflatoxin moiety and leads to mutations in the von tumours in humans or laboratory
on the 5′ side of the modified guanine Hippel–Lindau tumour suppressor animals (IARC, 2014). From toxicity
perturbs both the modified and the gene VHL in renal cell carcinoma and carcinogenicity studies in hu-
complementary DNA strands, caus- (Brüning et al., 1997; Brauch et al., mans and laboratory animals, there
ing interference with 5′ base pairing 1999), but these findings have been is strong evidence for the kidney as a
(Gopalakrishnan et al., 1990; Bailey reported in only a limited number target tissue for TCE-induced tumour
et al., 1996). Thus, mutations result- of studies. TCE exposure induced formation. The database supporting
ing from aflatoxin B1–N7-guanine MN both in vitro (Wang et al., 2001; the non-genotoxic mechanism of
adducts may not be due only to Robbiano et al., 2004; Hu et al., kidney carcinogenesis is moderate.
depurination. 2008) and in vivo (Hrelia et al., 1994; However, the strong evidence of ge-
A specific AGG → AGT transver- Kligerman et al., 1994; Robbiano notoxicity of DCVC, the kidney me-
sion mutation at codon 249 of the et al., 2004). Although TCE itself tabolite of TCE, supports the over-
TP53 tumour suppressor gene in appears to be incapable of inducing all conclusion that the evidence for

12
a genotoxic mechanism of kidney of various parts of the long arm of interaction between the parent elec-
carcinogenesis is strong. The evi- chromosome 5 or 7, or complete trophile or one or more electrophilic

CHAPTER 1
PART 1
dence for the liver as a target tissue loss of these chromosomes, gain of metabolites and nucleophilic DNA,
for TCE, from cancer assays and tox- the entire chromosome 8, and an leading to point mutations and induc-
icity findings in laboratory animals, is increased frequency of transloca- tion of CA. These effects have been
strong. The evidence for non-geno- tions between chromosomes 8 and observed in humans, in animals, and
toxic and/or genotoxic mechanisms 21 in peripheral lymphocytes of ex- in in vitro systems. In addition, pro-
of liver carcinogenesis is moderate. posed workers (Smith et al., 1998; duction of reactive oxygen species,
The available data suggest multiple Zhang et al., 1999, 2002). Benzene inhibition of DNA synthesis or repair,
non-genotoxic mechanisms and the and its quinone metabolites are in- and cytotoxicity/cell proliferation
potential for genotoxic mechanisms hibitors of topoisomerase II, leading could complement DNA modification
from the TCE metabolites dichloro- to increased frequencies of DNA to enhance DNA damage. Tumour
acetate and chloral hydrate. cleavage complexes and DNA dou- outcome can result from certain
ble-strand breaks; this effect can re- DNA adducts leading to mutations
Benzene
sult in the formation of chromosome and dysregulation initially described
Benzene induced CA, SCE, and MN translocations and inversions (Hutt with reference to proto-oncogenes
in bone marrow cells of exposed and Kalf, 1996; Lindsey et al., 2004, and tumour suppressor genes. For
mice, CA in bone marrow cells of 2005; Deweese and Osheroff, 2009). benzene, chromosomal transloca-
exposed rats, and CA and mutations Other potential pathways involved in tions, in combination with haemato-
in human cells in vitro. CA in human benzene-induced acute myeloid leu- toxicity or immunosuppression, are
peripheral lymphocytes have long kaemia include mutagenesis (pos- associated with increased risk of
been associated with occupational sibly through generation of reactive haematopoietic cancer in humans.
exposure to benzene (Forni, 1979; oxygen species), epigenetic changes The extent to which other process-
IARC, 1982; Eastmond, 1993; Zhang due to altered methylation status, de- es (inflammation, oxidative stress,
et al., 2002; Holecková et al., 2004). creased immunosurveillance (Cho, immunosuppression, epigenetic al-
As noted above, metabolism of ben- 2008; Li et al., 2009), haematotoxicity terations, and immortalization) might
zene produces several electrophilic and alterations in stem cell pool size contribute to the carcinogenicity of
agents (benzene oxide, in equilibrium (Rothman et al., 1997), and inhibition this class of chemicals in general is
with its tautomer oxepin, muconalde- of gap-junction intercellular commu- limited by the availability of few or no
hyde, benzoquinone, and benzene nication (Rivedal and Witz, 2005). published studies that address these
dihydrodiol epoxide) that can react Thus, multiple mechanisms are likely effects.
with DNA or proteins. DNA binding to be involved in benzene-induced
Polymorphisms and
and adduct formation may not be leukaemogenesis. Benzene produc-
susceptibility
the major steps in the development es multiple cytogenetic abnormalities
of benzene-induced leukaemias in human lymphocytes (Tough and Susceptibility to the carcinogenic
(Whysner et al., 2004). Although the Brown, 1965; Picciano, 1979; Smith effects of organic compounds may
mechanisms of benzene-induced and Zhang, 1998; Zhang et al., 2002) derive from acquired characteristics,
carcinogenesis and the potential and induces specific chromosomal such as altered expression of certain
relative roles of each of these me- changes associated with non-Hodg- enzymes, or from genetic factors,
tabolites are not fully known, there is kin lymphoma in human lympho- such as enzyme polymorphisms.
strong support for the involvement of cytes (Zhang et al., 2007). Induction Polymorphisms of enzymes involved
clastogenic and aneugenic effects, of DNA double-strand breaks and in the metabolism of organic com-
such as formation of CA, MN, and chromosomal rearrangements in pounds are likely to be responsible
DNA strand breaks. lymphoid cells in combination with for individual differences in activa-
Exposure to benzene has been immunosuppression by benzene tion and detoxification reactions that
associated with chromosomal might be the cause of lymphoma. control tissue levels of electrophilic
changes that are commonly ob- The carcinogenicity of the group intermediates. The enzymes that
served in acute myeloid leukaemia, of electrophilic chemicals dis- catalyse epoxide formation and elim-
including those comprising loss cussed above is likely to be due to ination are polymorphic in human

Part 1 • Chapter 1. Electrophilic agents 13


populations, and some isozymes derived epoxides or the in vivo muta- 20% of Caucasians and almost 50%
may be induced by a variety of en- genicity of 1,3-butadiene in occupa- of Asians) has a homozygous de-
vironmental and pharmaceutical tionally exposed workers (Wiencke letion (GSTT1-null genotype) (Bolt
agents. For example, factors that et al., 1995; Abdel-Rahman et al., and Thier, 2006). As expected, these
explain differences in the response 2003). The extent to which these individuals show a significantly ele-
to aflatoxin between human individ- enzyme polymorphisms influence vated level of hydroxyethyl valine in
uals and between animal species the carcinogenicity of 1,3-butadiene their haemoglobin, due to the pres-
and strains include the proportion is not known. The genotoxic effects ence of endogenous ethylene oxide
of aflatoxin metabolized to the exo- of 1,3-butadiene can be modulated (Thier et al., 2001). Nevertheless, the
8,9-epoxide (mainly by CYP450 by alterations in key determinants influence of this genetic trait on the
enzymes) relative to other, much of its metabolism; this suggests that formation of this type of adduct as a
less toxic metabolites, and the prev- markers of individual susceptibility result of exposure to exogenous eth-
alence of pathways that lead to the can be identified. For example, mice ylene oxide in the workplace is less
formation of non-toxic conjugates that lack a functional microsomal clear.
with reduced mutagenicity and cyto- EH (mEH) gene were more sus- In the cytoplasm of erythrocytes
toxicity (Guengerich et al., 1998). ceptible than wild-type mice to the obtained from 36 individuals, ethyl-
Similarly, the expression of en- mutagenic effects of 1,3-butadiene ene oxide was eliminated 3–6 times
zymes involved in aflatoxin metabol- or diepoxybutane (Wickliffe et al., as fast in samples from so-called
ism can be modulated with che- 2003). EH activity varies consider- conjugators (defined by a standard-
mopreventive agents, resulting in ably among humans. 1,3-Butadiene- ized conjugation reaction of methyl
inhibition of DNA adduct formation exposed workers with the genotype bromide and GSH; 75% of the popu-
and hepatocarcinogenesis, as has for low-activity EH were reported to lation) as in samples from individuals
been demonstrated in rats. Oltipraz be more susceptible to 1,3-butadi- who lack this GST-specific activity
is a chemopreventive agent that in- ene-induced genotoxicity (assessed (the remaining 25%). In whole-blood
creases GSH conjugation and inhib- by HPRT mutant frequency in lym- samples incubated with ethylene ox-
its the activity of some CYP450 en- phocytes) than individuals with the ide, an increase in the frequency of
zymes (e.g. CYP1A2). Results from more common EH genotype (Abdel- SCE was observed in lymphocytes
clinical trials with oltipraz in China Rahman et al., 2001, 2003). No sig- from the non-conjugators but not in
are consistent with experimental nificant effects were observed for lymphocytes from the conjugators
data in showing that after dietary induction of HPRT mutations or SCE (Hallier et al., 1993).
exposure to aflatoxins, modulation in individuals with GSTM1 or GSTT1 The carcinogenicity and toxic-
of the metabolism of aflatoxins with polymorphisms (Abdel-Rahman ity of TCE, particularly in the liver
oltipraz can lead to reduced levels of et al., 2001). MN frequencies were and kidney, are associated with its
DNA adducts (IARC, 2002; Kensler higher among 1,3-butadiene-ex- metabolism. There are inter-individ-
et al., 2005). posed workers in China with poly- ual differences, both in humans and
Increased susceptibility to the morphisms in GSTM1 and/or GSTT1 in rodents, in the formation of TCE
toxic effects of benzene has been compared with workers with the metabolites that are thought to be
linked to genetic polymorphisms that wild-type genes (Cheng et al., 2013). responsible for the toxic and carcino-
increase the rate of metabolism of These differences in response are genic effects of TCE in the kidney and
benzene to active intermediates or consistent with the known important liver. The susceptibility to adverse
decrease the rate of detoxification of roles of EH and GST in the detoxi- health effects of TCE may be influ-
these active intermediates (Rothman fication of 1,3-butadiene epoxides in enced by genetics, sex, life stage,
et al., 1997; Xu et al., 1998; Kim et al., tissues in which these intermediates and other conditions that influence
2004). are produced. the extent and nature of the metabol-
Enzyme polymorphisms also af- Ethylene oxide is a substrate of ism of this chemical. Polymorphisms
fect the metabolism of 1,3-butadi- the GST isozyme T1 (Hayes et al., in metabolism genes in both ox-
ene. Genetic polymorphisms in GST 2005). This detoxifying enzyme is idative (e.g. CYP2E1, ADH, and
and microsomal EH affect the in polymorphic, and a relatively large ALDH) and GSH conjugation (e.g.
vitro mutagenicity of 1,3-butadiene- proportion of the population (about GSTs) pathways have been studied

14
in connection with susceptibility to pigmentosum cells (human fibro- pancytopenia) may have increased
TCE toxicity and carcinogenicity. blasts) compared with repair-profi- susceptibility to leukaemia from

CHAPTER 1
PART 1
Polymorphisms in genes for organic cient cells; the location of mutations formaldehyde.
anion transporters (OAT1 and OAT3) was affected by repair proficiency A common polymorphism in
in the kidney may also influence the (Levy et al., 1992). the DNA repair gene XRCC1 is
rates of uptake and the extent of cellu- Polymorphisms in genes involved a biomarker of susceptibility to
lar accumulation of DCVG or DCVC. TP53-induced mutations in work-
in repair of DNA double-strand
With respect to life-stage susceptibil- ers exposed to vinyl chloride (Li
breaks (WRN [Werner syndrome],
ity, data are available to support the et al., 2003). In workers exposed to
TP53, BLM [Bloom syndrome],
influence of differences in exposure 1,3-butadiene, MN frequencies were
RAD51, and BRCA1) can modify
(e.g. transplacental transfer or ex- higher in peripheral lymphocytes of
susceptibility to benzene-induced
posure through breast milk in early individuals with polymorphisms in
life stages) or life stage-specific dif- haematotoxicity in exposed workers
XRCC1 compared with individuals
ferences in toxicokinetics. Lifestyle (Shen et al., 2006; Lan et al., 2009;
carrying the wild-type repair gene
factors (e.g. consumption of alcohol- Ren et al., 2009).
(Wang et al., 2010).
ic beverages) may also affect TCE Mice deficient in nucleotide exci-
In summary, genetic polymor-
metabolism, and nutrition or obesity sion repair were more susceptible
phisms and variability in expression
may affect internal concentrations of than wild-type mice to the mutagenic
of enzymes due to induction or inhi-
TCE and its metabolites. effects of 1,3-butadiene or its reac-
bition of constitutive enzyme levels
Genes involved in DNA repair act tive metabolites epoxybutene and di-
can have considerable impact on the
to maintain the integrity of the ge- epoxybutane (Wickliffe et al., 2007).
carcinogenic process. Determining
nome by removing lesions (i.e. ad- Chicken cells deficient in the
the existence and functional role of
ducts) that – if left unrepaired – could Fanconi anaemia complemen-
genetic polymorphisms in cancer eti-
result in mutations or chromosomal tation groups/breast cancer A
damage. Individuals with defects in ology is an active area of research in
(FANC/BRCA) pathway are hyper- molecular epidemiology.
genes that encode DNA repair en-
sensitive (with reduced survival) to
zymes are at elevated risk for cer-
formaldehyde at levels measured in
tain cancers (Poulsen et al., 1993).
human plasma (Ridpath et al., 2007).
Heterozygous carriers may also
This observation is consistent with
have increased susceptibility, be-
cause of suboptimal levels of repair. an essential role for this pathway
The reactive aflatoxin B1 me- in the repair of DNA–protein cross-
tabolite exo-8,9-epoxide induced links caused by formaldehyde, and
a higher mutation frequency in a suggests that patients with Fanconi
shuttle vector plasmid transfected anaemia (a genetic disorder that
into DNA repair-deficient xeroderma is characterized by progressive

Part 1 • Chapter 1. Electrophilic agents 15


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Part 1 • Chapter 1. Electrophilic agents 21


CHAPTER 2
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 2.

Aromatic amines and


aristolochic acids
Frederick A. Beland and M. Matilde Marques

Carcinogenicity in humans uation for these dyes was raised to can be attributed to 4-aminobiphe-
Group 1 based on this mechanistic nyl, o-toluidine, 2-naphthylamine, or
Exposure to 4-aminobiphenyl, o-tolu­ information, although at present the other aromatic amines. Hair dyes
idine, 2-naphthylamine, and benzi­ corresponding epidemiological data are an additional source of expo-
dine (Fig. 2.1) has been consisten- are considered to provide inade- sure to 4-aminobiphenyl and o-tolu-
tly associated with the induction quate evidence for the carcinogeni- idine (IARC, 2010, 2012a; Lizier and
of cancer of the urinary bladder in city of these dyes in humans (IARC, Boldrin Zanoni, 2012).
humans. This association is based 2010, 2012a). Exposure to phenacetin (Fig. 2.1),
upon occupational exposures, pri- Cigarette smoke contains 4-amino­ through its use as an analgesic,
marily of workers in the rubber and biphenyl, o-toluidine, and 2-naphthyl- causes cancer of the kidney and
dye industries (IARC, 2010, 2012a). amine, and tobacco smoking caus- ureter in humans (IARC, 2012c).
Similarly, occupational exposure to es cancer of the bladder in humans Chlornaphazine (Fig. 2.1), a chemo­
4,4′-methylenebis(2-chloroaniline) (IARC, 1986, 2004, 2010, 2012a, b). therapeutic agent that has been used
(MOCA; Fig. 2.1), a curing agent for The contribution of 4-aminobiphenyl, for the treatment of Hodgkin lympho-
polyurethane pre-polymers, causes o-toluidine, and 2-naphthylamine ma and for the control of polycythae-
cancer of the bladder in humans, al- to the induction of smoking-related mia vera, causes cancer of the
though the epidemiological data are cancer of the bladder is confounded bladder in humans, presum­ably due
not as strong as those for the other by the presence of numerous other to metabolism to 2-naph­ thylamine
agents (IARC, 2010, 2012a). Certain carcinogens, including carcinogenic (IARC, 2012c). An additional source
azo dyes that are used in commercial aromatic amines, in tobacco smoke. of human exposure to o-toluidine
products, for example, Direct Black Cigarette smoking also causes oth- is from the anaesthetic prilocaine
38, Direct Blue 6, and Direct Brown er cancers (e.g. cancer of the lung, (Fig. 2.1) (IARC, 2010, 2012a).
95 (Fig. 2.2), are known to undergo oral cavity, and pancreas, and pos- Exposure to herbal remedies pre-
azo reduction in vivo to yield the car- sibly breast cancer), but at present pared from plant species of the ge-
cinogen benzidine. The overall eval- it is unclear whether these cancers nus Aristolochia has been causally

Part 1 • Chapter 2. Aromatic amines and aristolochic acids 23


associated with the induction of Fig. 2.1. Structures of IARC Group 1 aromatic amines, drugs that are
urothelial cancer in humans (IARC, metabolized to Group 1 aromatic amines, and aristolochic acids. MOCA,
2002, 2012c). The induction of 4,4′-methylenebis(2-chloroaniline).
urothelial tumours has been attributed
to the presence of aristolochic acid I
and aristolochic acid II (Fig. 2.1).

Metabolism and DNA adduct


formation

4-Aminobiphenyl, o-toluidine, 2-naph-


thylamine, and MOCA are metabolized
to electrophilic N-hydroxyarylamines
by hepatic cytochrome P450 en-
zymes (IARC, 2010, 2012a, c). The
N-hydroxyarylamines undergo ac-
id-catalysed reactions with DNA
to form a variety of DNA adducts.
With the exception of MOCA, C8-
substituted deoxyguanosine adducts
are typically the major products,
along with smaller amounts of N2 -
substituted deoxyguanosine and
N6 -substituted deoxyadenosine ad-
ducts (Fig. 2.3); with MOCA, only
C8-substituted deoxyadenosine ad-
ducts have been detected (IARC,
2010, 2012a). These DNA adducts
can also be formed from reactive
esters of N-hydroxyarylamines (e.g.
N-sulfoxyarylamines and N-aceto­xy­
arylamines). Benzidine, which has
two amino groups, also forms a C8-
substituted deoxyguanosine adduct
via a pathway involving an initial ronides can undergo acid-cata- adduct detected in each instance
N-acetylation followed by N-hydroxyl­ lysed hydrolysis to release the was a C8-substituted deoxyguano-
ation of the remaining amino function N-hydroxyarylamines, which can sine adduct. The importance of uri-
(Fig. 2.3). enter the bladder epithelium and re- nary acidity for the hydrolysis of the
The carcinogenic activity of ar- act with DNA either directly or after N-hydroxyarylamine N-glucuronides
omatic amines in the bladder in esterification. DNA adducts derived and perhaps for the reaction of the
humans has been attributed to an from 4-aminobiphenyl, o-toluidine, N-hydroxyarylamines with urothe-
initial N-hydroxylation, catalysed benzidine, and MOCA have been lial DNA has been demonstrated
by hepatic cytochrome P450 en- detected in bladder tissue or exfoli- by the positive correlation between
zymes, followed by transport of ated bladder cells from exposed in- urinary acidity and the levels of ben-
the N-hydroxyarylamines to the dividuals (IARC, 2010, 2012a; Böhm zidine DNA adducts in exfoliated
bladder as either aglycones or et al., 2011; Lee et al., 2014). With bladder cells from exposed workers
N-glucuronide conjugates (Bois et the exception of MOCA, which forms (Rothman et al., 1997). Additional
al., 1995). In the bladder lumen, only C8-substituted deoxyadenosine support for this mechanism comes
the N-hydroxyarylamine N-glucu­ adducts, the major – if not the only – from the observation of a positive

24
correlation between urinary acidity Fig. 2.2. Structures of benzidine-derived azo dyes.

CHAPTER 2
and the incidence of bladder cancer

PART 1
in smokers (Alguacil et al., 2011).
The major metabolic activation
pathway for aristolochic acid I and
aristolochic acid II involves nitro
reduction, followed by cyclization
to give N-hydroxyaristolactams,
which – in contrast to other
N-hydroxyarylamides – do not ap-
pear to require additional activa-
tion to react with DNA (Stiborová
et al., 2011, 2013). Nonetheless,
N-hydroxyaristolactams have been
shown to serve as substrates for
human sulfotransferases, particu-
larly sulfotransferase family cy-
tosolic 1B member 1 (SULT1B1),
forming highly reactive N-sulfoxy
derivatives (Sidorenko et al., 2014).
The major adducts resulting from
the N-hydroxyaristolactams are N2 -
substituted deoxyguanosines and N6 -
substituted deoxyadenosines (Fig.
2.4) (IARC, 2002, 2012c).
DNA adducts derived from aris-
Alterations in the TP53 higher-grade tumours (grades 2 or 3,
tolochic acids have been detected
tumour suppressor gene in i.e. moderately or poorly differentiat-
in renal tissue from patients who humans ed) and only at G:C base pairs.
had been exposed to aristolochic Mutant p53 protein has also been
acid-containing herbal products and Mutations in the TP53 tumour sup- detected in workers exposed oc-
from individuals who had consumed pressor gene have been found in cupationally to benzidine (Xiang et
wheat grains contaminated with approximately 50% of all bladder al., 2007). The occurrence and the
Aristolochia (IARC, 2002, 2012c; cancers in humans (Petitjean et al., amount of mutant protein were pos-
Chen et al., 2012; Jelaković et al., 2007), with G:C base substitution itively correlated with the level of
2012; Schmeiser et al., 2012, 2014; mutations occurring to a greater benzidine exposure and the extent
Yun et al., 2013, 2014). Typically, extent than A:T base substitution of neoplastic changes in exfoliated
the major lesion detected is an N - 6 mutations. urothelial cells.
deoxyadenosine adduct derived TP53 gene mutations have been Urothelial tumours arising from
from aristolochic acid I, accompa- detected in bladder cancer patients exposure to aristolochic acids have
nied by smaller amounts of a similar exposed occupationally to 4-amino­ been consistently shown to carry mu-
adduct derived from aristolochic acid biphenyl, 2-naphthylamine, and/or tations in the TP53 tumour suppres-
II and an N2 -deoxyguanosine adduct benzidine (Sørlie et al., 1998). The sor gene, of which the most common
derived from aristolochic acid I. mutations occurred exclusively in mutation is an A  →  T transversion

Part 1 • Chapter 2. Aromatic amines and aristolochic acids 25


Fig. 2.3. Structures of representative DNA adducts obtained from Group 1 Support for this mechanism comes
aromatic amines. dR, deoxyribose. from the observation that the DNA
lesions detected in the bladders of
dogs treated with 4-aminobiphenyl or
MOCA appear to be C8-substituted
deoxyguanosine and deoxyaden-
osine adducts that are identical to
the DNA adducts detected in blad-
der tissues or exfoliated bladder
cells from humans exposed to these
carcinogens (IARC, 2010, 2012a).
2-Naphthylamine DNA adducts de-
tected in the bladders of dogs ex-
posed to 2-naphthylamine are en-
tirely consistent with a mechanism
involving the formation of N-hydroxy-
2-naphthylamine (IARC, 2010).
In contrast to what is observed in
humans, benzidine in not a bladder
carcinogen in dogs. This lack of car-
cinogenicity has been attributed to
the inability of dogs to N-acetylate
aromatic amines (IARC, 2010). With
most aromatic amines, N-acetylation
is considered to be a detoxifica-
tion event; however, with benzi-
dine, N-acetylation appears to be
required to give N-acetylbenzidine,
which undergoes a subsequent
mutation (reviewed in IARC, 2002, Tumour sites and the
N-hydroxylation of the second amino
2012c; Hollstein et al., 2013; see also mechanism of tumour
function. This metabolic pathway oc-
Schmeiser et al., 2012; Chen et al., induction in experimental
curs in humans but not in dogs.
2013; Hoang et al., 2013; Poon et animals
4-Aminobiphenyl, o-toluidine,
al., 2013; Aydin et al., 2014). These 2-naph­ thylamine, benzidine, ben-
4-Aminobiphenyl, 2-naphthylamine,
mutations have been demonstrated zidine-based dyes, and MOCA in-
and MOCA are bladder carcinogens
in tumour tissue from patients who duce hepatocellular tumours in mice
in dogs (IARC, 2010, 2012a). Bladder
had consumed herbal preparations (IARC, 2010, 2012a). DNA adducts
tumours also occur in mice treat-
containing aristolochic acids and in derived from 4-aminobiphenyl and
ed with 4-aminobiphenyl. As with
urothelial cancer tissues of patients benzidine have been examined in liv-
humans, the induction of bladder
er tissue from exposed mice, and the
from regions with a high incidence of tumours in dogs is thought to result
major DNA lesions detected in each
endemic (Balkan) nephropathy due from hepatic N-hydroxylation, trans- instance were C8-substituted deoxy­
to consumption of grains contaminat- port of the N-hydroxyarylamines guanosine adducts, consistent with
ed with Aristolochia. The presence to the bladder as either aglycones formation of an N-hydroxyarylamine
of A  →  T transversion mutations is or N-hydroxyarylamine N-glucu­ intermediate (IARC, 2010). In rats,
consistent with the observation that ronides, and subsequent hydro- DNA adducts derived from benzi-
the major lesion detected in patients lysis of the N-hydroxyarylamine dine-based dyes and MOCA have
is an N -deoxyadenosine adduct de-
6
N-glucuronides in the bladder lumen been examined in the liver, which
rived from aristolochic acid I. to release the N-hydroxyarylamines. is also a target tissue for these

26
Fig. 2.4. Structures of DNA adducts derived from aristolochic acids through kidney have been reported to occur
N-hydroxyaristolactam intermediates. dR, deoxyribose. sporadically. Mice treated with mix-

CHAPTER 2
PART 1
tures of aristolochic acids I and II
develop tumours of the forestomach,
kidney, and lung. In rabbits, mixtures
of aristolochic acids I and II admin-
istered intraperitoneally are associat-
ed with tumours of the kidney, ureter,
and peritoneal cavity.
DNA adducts derived from aris-
tolochic acid I and aristolochic acid II
have been detected in target tissues
in mice (forestomach, kidney, and
lung), rats (forestomach and kidney),
and rabbits (kidney) (IARC, 2002,
2012c; Debelle et al., 2003; Gillerot
et al., 2003; Dong et al., 2006; Mei
et al., 2006; Shibutani et al., 2007;
Chan et al., 2008; Rosenquist et al.,
2010; Shibutani et al., 2010; Baudoux
et al., 2012; McDaniel et al., 2012;
Wang et al., 2012a; Yun et al., 2013,
2014). Typically, three DNA adducts
are detected: an N6 -deoxyadenosine
adduct derived from aristolochic acid
I, an N6 -deoxyadenosine adduct de-
rived from aristolochic acid II, and an
N2 -deoxyguanosine adduct derived
from aristolochic acid I.

carcinogens, and again the major C8-substituted deoxyguanosine ad­ Oncogene alterations in
DNA adducts detected in each in- ducts have also been detected in experimental animals
stance were consistent with forma- the bladder DNA of mice treated
tion of an N-hydroxyarylamine inter- with 4-aminobiphenyl (Poirier et al., Transversion mutations at codon 61
mediate (IARC, 2010). 1995). These adducts presumably of the H-Ras oncogene (CAA → AAA)
In mice, there appears to arise from hepatic N-hydroxylation have been observed in the livers of
be a balance between hepatic and possibly O-acetylation of mice treated with 4-aminobiphenyl
N-acetylation, which is considered N-hydroxy-4-aminobiphenyl in the (IARC, 2010, 2012a). G  →  T trans-
to be a detoxification step, and he- bladder epithelium. version mutations in the cII trans-
patic N-hydroxylation, which is con- The carcinogenicity of aristolochic gene have been detected in the liv-
sidered to be an activation step. acids has been assessed in rats and ers and bladders of transgenic mice
Should N-hydroxylation occur, the to a lesser extent in mice and rab- treated with 4-amino­biphenyl (Wang
N-hydroxyarylamines can be further bits, primarily by oral dosing (IARC, et al., 2012b; Yoon et al., 2012). The
activated by hepatic O-acetylation to 2002, 2012c). Aristolochic acid I and occurrence of these mutations at
yield O-acetoxyarylamines, which mixtures of aristolochic acids I and G:C base pairs is consistent with
can give rise to the DNA adducts II consistently induce tumours of the the observation that the major DNA
detected in liver tissue (IARC, 2010). forestomach in rats. Tumours of the adduct detected in target tissues

Part 1 • Chapter 2. Aromatic amines and aristolochic acids 27


after exposure to 4-aminobiphenyl Summary with DNA or be further activated by
is a C8-substituted deoxyguanosine O-esterification to give rise to DNA
adduct. In humans, exposure to aromatic adducts, predominantly at deoxy-
Transversion mutations at co- amines and aristolochic acids that guanosine (primarily with aromatic
don 61 of the H-Ras oncogene are IARC Group 1 carcinogens has amines) and deoxyadenosine (pri-
(CAA  →  CTA) have been detected been associated with induction of marily with aristolochic acids), in tu-
in tumours from rats and mice fed tumours of the urinary tract. With ar- mour target tissues of humans and
mixtures of aristolochic acids I and omatic amines, the primary tumour experimental animals.
II and/or aristolochic acid I (IARC, site is the bladder; with aristolochic Mutations of the TP53 tumour
2002, 2012c; Wang et al., 2011, acids, the primary site for tumour for- suppressor gene consistent with the
2012a). A  →  T transversion muta- mation is the kidney. Experimental major DNA adducts derived from ar-
tions have also been detected in animals treated with aromatic omatic amines and aristolochic acids
the cII transgene of rats and the cII amines or aristolochic acids develop have been detected in tumours from

and lacZ transgenes of mice treated tumours of the urinary tract; tumours exposed humans. Similarly, muta-

with mixtures of aristolochic acids I also arise in other tissues, primarily tions of the H-Ras oncogene con-
the liver. sistent with the major DNA adducts
and II, and in the gpt transgene of
Aromatic amines and aristolo­ derived from aromatic amines and
mice treated with aristolochic acid I
aristolochic acids have been found
or aristolochic acid II (IARC, 2012c; chic acids that are IARC Group 1
in target tissues of experimental
McDaniel et al., 2012; Xing et al., carcinogens are metabolized by
animals.
2012). The occurrence of these mu- amine oxidation (in the case of ar-
tations at A:T base pairs is consistent omatic amines) or nitro reduction Disclaimer
with the observation that the major (in the case of aristolochic acids) to
DNA lesions detected in target tis- N-hydroxyarylamine metabolites in The views expressed in this chapter
sues after exposure to aristolochic both humans and experimental an- do not necessarily represent those
acids are N -substituted deoxyaden-
6 imals. These N-hydroxyarylamine of the United States Food and Drug
osine adducts. intermediates can react directly Administration.

28
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1814–22. http://dx.doi.org/10.1093/carcin/ org/10.1093/carcin/bgu101 PMID:24776219
bgu095 PMID:24743514

30
CHAPTER 3
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 3.

Arsenic and metals


Michael P. Waalkes

Arsenic and arsenic compounds as a contaminant of drinking-water adult offspring in C3H/HeNCr mice
together with various metals, spe- (IARC, 2004). The most prevalent (two studies) and CD1 mice (IARC,
cifically including beryllium and source of human exposure to ar- 2012). In addition, in a study of CD1
beryllium compounds, cadmium senic is now drinking-water, where mice with “whole-life” exposure to
and cadmium compounds, chromi- it is found primarily as the inorgan- multiple levels of sodium arsenite in
um(VI) compounds, and nickel and ic forms of arsenite and arsenate. drinking-water from 2 weeks before
nickel compounds, were re-evalu- Inorganic arsenic can be metabolized breeding (male and female mice),
ated in IARC Monographs Volume by most mammals to form trivalent during gestation and lactation (fe-
100C (IARC, 2012) as carcinogenic and pentavalent methylated metab- male mice), and after weaning until
to humans (Group 1). The most re- olites through specific methyltrans- age 2 years (offspring, both sexes),
cent earlier evaluations appeared ferases, with S-adenosylmethionine bronchiolo-alveolar carcinoma oc-
in Volume 84 (IARC, 2004) for ar-
as the methyl donor (IARC, 2004, curred in a dose-related fashion
senic, Volume 58 (IARC, 1993) for
2012). Questions remain about the in both male and female offspring
beryllium and cadmium, and Volume
relative contribution of the inorganic (Tokar et al., 2011). In CD1 mice, in
49 (IARC, 1990) for chromium and
and the methylated arsenic species utero exposure via maternal con-
nickel.
to the overall carcinogenic potential sumption of sodium arsenite in drink-
Arsenic and arsenic of exposure to arsenic. Arsenic and ing-water during gestation with or
compounds arsenic compounds in drinking-water without subsequent dimethylarsinic
are human carcinogens; there is suf- acid (DMA(V)) in drinking-water of
Carcinogenicity ficient evidence in humans for can- the offspring throughout adulthood
Early on, exposure to inorganic arse- cer of the lung, urinary bladder, and induced bronchiolo-alveolar carcino-
nic via drinking-water or oral use of skin, as well as limited evidence for ma in animals that received prenatal
arsenic-based drugs was considered cancer of the kidney, liver, and pros- arsenite alone, DMA(V) alone, or the
carcinogenic to the skin in humans, tate (IARC, 2004, 2012). combination of prenatal arsenite and
and exposure to inorganic arsenic In rodents, transplacental arsenic DMA(V) (Tokar et al., 2012a). In CD1
via inhalation in occupational set- exposure via maternal consumption mice, in utero exposure to arsenic via
tings was evaluated as carcinogenic of sodium arsenite in drinking-wa- maternal consumption of monometh-
to the lung in humans (IARC, 1973). ter during gestation induced bron- ylarsinous acid (MMA(III)) in drink-
Arsenic was most recently assessed chiolo-alveolar carcinoma in the ing-water produced bronchiolo-alveolar

Part 1 • Chapter 3. Arsenic and metals 31


carcinoma in male offspring as cal hyperplasia) of the lung epitheli- In multiple studies in rodents, in-
adults, but not in a dose-related um. The role of the separate moieties organic arsenic or DMA(V) given in
fashion (Tokar et al., 2012b). Multiple of the inhaled compound (i.e. gallium drinking-water or by the transplacen-
intratracheal instillations of inorgan- and arsenic) in the carcinogenic re- tal route had initiating, promoting, or
ic arsenic produced lung tumours sponse could not be defined by this co-carcinogenic activity in the skin,
in hamsters (three studies) (IARC, one study in rodents, and it was con- kidney, and urinary bladder with oth-
2012). In adult strain A/J mice, oral cluded that either moiety alone or er, non-arsenic-based compounds
sodium arsenate increased the size some combination of both could be (IARC, 2004, 2012). Multiple studies
and multiplicity of lung tumours in humans have found oral arsenic
active (IARC, 2006).
(male mice), and oral DMA(V) in- exposure to be carcinogenic to the
There is limited evidence that the
creased the incidence and multiplici- skin and urinary bladder, and there
liver is a target site for the carcino-
ty of lung tumours (IARC, 2012). Oral is limited evidence that the kidney
genic effects of arsenic and arsenic
exposure to DMA(V) increased the is a target site in humans (IARC,
compounds in humans (IARC, 2012).
incidence of lung tumours in Ogg−/− 2004, 2012). It is difficult to assess
In rodents, multiple studies showed
mice, which cannot repair certain the relevance to humans of rodent
that in utero exposure to arsenic via
types of oxidative DNA damage, but studies that use multiple agents, one
maternal consumption of sodium
not in Ogg+/+ mice (IARC, 2012). of which is an arsenic compound of
Multiple studies in humans have arsenite in drinking-water during concern.
found oral arsenic exposure to be gesta­
tion induced hepatocellular Overall, the target sites for which
carcinogenic to the urinary bladder, carcinoma in the adult offspring of there is sufficient evidence in humans
typically producing transitional cell C3H/HeNCr mice (two studies) and for the carcinogenicity of arsenic and
carcinoma (IARC, 2004, 2012). Most CD1 mice (IARC, 2012). In addition, arsenic compounds include the uri-
oral exposure in humans would in- in a study of CD1 mice with “whole- nary bladder and the lung, and there
volve inorganic arsenic as the prima- life” exposure to multiple levels of are multiple concordant rodent stud-
ry form. Multiple studies in rodents sodium arsenite in drinking-water ies for these two sites (IARC, 2012).
show that chronic oral exposure to (see above), hepatocellular carci- The skin is also a target site in hu-
DMA(V) causes transitional cell car- noma occurred in a dose-related mans for inorganic arsenic and arse-
cinoma of the urinary bladder in adult fashion in both male and female off- nic compounds, but in rodents there
rats, but not in mice (IARC, 2012). spring (Tokar et al., 2011). In CD1 is insufficient evidence that inorganic
DMA(V) exposure can be from the mice, in utero exposure via maternal arsenic or arsenic compounds acting
drinking-water or the feed. Exposure consumption of sodium arsenite in alone can cause cancer of the skin
to inorganic arsenic has not been drinking-water during gestation with (IARC, 2012). There is limited evi-
shown to be carcinogenic to the uri- dence that the liver is a target site for
or without subsequent DMA(V) in
nary bladder in rodents; the reasons the carcinogenic effects of arsenic
the drinking-water of the offspring
for this are not clear. and arsenic compounds in humans,
throughout adulthood induced hepa­
Gallium arsenide is considered and sufficient evidence that the liv-
tocellular carcinoma in animals that
carcinogenic to humans, based er is a target site in rodents (IARC,
received prenatal arsenite alone
largely on one robust study in ro- 2012). There is limited evidence that
or the combination of prenatal ar-
dents together with ancillary evi- the kidney is a target site in humans
senite and DMA(V). The combined
dence (IARC, 2006). Chronic inhala­ (IARC, 2012), and one recent study
treatment produced hepatocellular
tion of gallium arsenide induced lung in mice provided evidence that can-
bronchiolo-alveolar adenoma or carcinoma at a significantly higher cer of the kidney can be induced by
carcinoma in a dose-related fashion rate than prenatal arsenite alone or a combination of inorganic arsenic
in female F344 rats, but not in male DMA(V) alone (Tokar et al., 2012a). (prenatal) and DMA(V) in adulthood
rats or male or female B6C3F1 mice In CD1 mice, in utero exposure to (Tokar et al., 2012a). There is limited
(IARC, 2006, 2012). Male rats ex- arsenic via maternal consumption of evidence that the prostate is a tar-
posed to gallium arsenide did show MMA(III) in drinking-water produced get site in humans, and there are no
dose-related increases in the num- hepatocellular carcinoma in male off- studies in rodents showing increased
ber of pre-neoplastic lesions (atypi- spring as adults (Tokar et al., 2012b). incidence of prostate cancer after

32
exposure to inorganic arsenic or same tumour type induced in rats plification, altered DNA methylation
arsenic compounds (IARC, 2012). by chronic oral exposure to DMA(V) (epigenetic effects), or aneuploidy

CHAPTER 3
PART 1
Inorganic arsenic can cause lung (IARC, 2012). Some researchers may cause alterations in gene ex-
cancer in humans after inhalation or believe that the rat is a poor model pression that lead to genomic in-
ingestion, but there are no studies for studying arsenic toxicology in hu- stability and cellular transformation.
showing development of lung cancer mans, because the toxicokinetics of The metabolism of inorganic arsenic
in rodents after inhalation exposure arsenic are dramatically different as by methylation may contribute to its
(IARC, 1973, 2012). In fact, an ade- a result of sequestration of arsenic in epigenetic effects, because the ar-
quate inhalation study in rodents with the blood of rats (Carter et al., 2003; senic methylation pathway overlaps
inorganic arsenic has never been Aposhian et al., 2004). However, for with DNA methylation by consump-
performed, presumably because the DMA(V) and cancer of the urinary tion of S-adenosylmethionine as the
agent had already been declared a bladder, there is clear site concor- common methyl donor (Brocato and
human carcinogen and rodent re- dance between humans and rats. For Costa, 2013). However, it is notewor-
search resources were directed bladder tumours induced by DMA(V) thy that inorganic arsenic can cause
elsewhere. in the rat, the mechanism may in- malignant transformation in cells that
volve sustained cytotoxicity, pos- do not methylate the metalloid, indi-
Mechanisms of
sibly from oxidative stress (Kitchin cating that neither methylation nor a
carcinogenesis
and Conolly, 2010), followed by cell methylated metabolite are required
Although a unifying mechanis- proliferation and genomic instability. for a cell to acquire a malignant phe-
tic hypothesis for arsenic-induced Specific methylated forms of arsenic notype after exposure to inorganic
carcinogenesis may seem reason- may be involved in the sustained cy- arsenic (Kojima et al., 2009). Thus,
able, it is important to emphasize totoxicity (Cohen et al., 2007). cell-specific, complex, multifaceted
that given the multitude of toxic Inorganic arsenic and methyl- mechanisms are likely to be opera-
events at the subcellular level seen ated arsenic metabolites generally tive with arsenic (Kitchin and Conolly,
with inorganic arsenic and arsenic show weak activity as mutagens. 2010; Tokar et al., 2010).
compounds (e.g. oxidative stress, Low-dose exposure to inorganic ar- Another important issue with
altered DNA repair, altered DNA senic can increase the number of mechanistic implications is the
methylation, gene amplification, and mutations resulting from genomic strong evidence that cancer can
altered growth factors), it is likely that instability, perhaps through produc­ develop long after elevated arsenic
multiple mechanisms are operative tion of reactive oxygen species, exposure ends. For instance, a re-
in arsenic-induced carcinogenesis and cells that methylate inorganic cent study on a human population
(Kitchin and Conolly, 2010; Tokar arsenic show much more oxidative in Chile measured rates of cancer
et al., 2010; Hartwig, 2013). These DNA damage than cells that poorly of the bladder and lung in individ-
multiple mechanisms are probably methylate the metalloid during in vi­ uals who were highly exposed to
linked, at least in part, to the quali- tro malignant transformation (Kojima inorganic arsenic in drinking-water
ties of specific target tissue (e.g. high et al., 2009). MMA(III) may be one of during 1958–1970 but drank low-ar-
oxygen tension in the lung might fa- the most deleterious arsenic methyl- senic water thereafter. These sub-
vour oxidative stress, in contrast with ation products, although the number jects still showed very high risks of
the situation in the bladder) (Kitchin of tumour end-point studies is very cancer even 40 years after the high
and Conolly, 2010). The target tis- limited (only one study). The main exposures ended (Steinmaus et al.,
sue-specific toxicokinetics of arsenic cascade of mechanisms leading to 2013). Similarly, the mouse transpla-
are likely to be key and may dictate carcinogenesis for inorganic arsenic cental model demonstrated that brief
that multiple toxic events combine and arsenic compounds after expo- exposure to inorganic arsenic may
into a target-specific carcinogenic sure to low concentrations could in- result in tumours in adulthood (IARC,
mechanism (Kitchin and Conolly, clude the rapid induction of oxidative 2012). Given the time lag between
2010). DNA damage and inhibition of DNA arsenic exposure and development
In humans, arsenic causes tran- repair, followed by changes in DNA of cancer, the operative mechanisms
sitional cell carcinoma of the urinary methylation patterns, aneuploidy, would appear not to require concur-
bladder (IARC, 2012), which is the and gene amplification. Gene am- rent high tissue levels of arsenic.

Part 1 • Chapter 3. Arsenic and metals 33


In discerning mechanisms of ar- inorganic carcinogens, beryllium of cadmium chloride and cadmium
senic-induced carcinogenesis by produces oxidative stress, which sulfide both induced primarily bron-
use of in vitro model systems, the can lead to damage in DNA or oth- chiolo-alveolar carcinoma.
consideration that arsenic adverse- er key biomolecules and then pro- Multiple studies in rodents have
ly alters many cellular physiological duce gene activation and apoptosis. established that various water-sol-
functions is critical; studies have The cytotoxicity of beryllium in the uble and insoluble cadmium com-
frequently been carried out with lev- lung may result in compensatory pounds can produce soft tissue
els of arsenic that would be highly cell proliferation, along with chron- sarcomas after repository injections
unrealistic in vivo. Another common ic inflammation. The inflammatory (IARC, 1993, 2012). Studies that
issue with in vitro arsenic studies is processes induced by beryllium produced injection-site tumours
that many use short time frames, and could contribute to the formation of have provided some evidence of the
very early responses to arsenic do reactive oxygen species, precipitate carcinogenic potential of cadmium,
not necessarily reflect in vivo expo- cell turnover, and activate or disrupt but there is no concordance with
sures or take into account the adap- pulmonary cell signalling pathways. any specific target site in humans.
tive capacities towards arsenic that Beryllium can decrease DNA repair Prostatic proliferative lesions can be
are generally observed in vivo. and recombination. The impairment produced in rats after oral adminis-
of DNA repair by beryllium togeth- tration or subcutaneous injection of
Beryllium and beryllium er with increased mitotic signalling cadmium chloride.
compounds
may cooperate to induce error-prone Overall, cadmium and cadmium
cell proliferation (Beyersmann and compounds are carcinogenic to hu-
Carcinogenicity
Hartwig, 2008). The mechanisms of mans and target the lung after inha­
There is sufficient evidence in hu- beryllium-induced carcinogenesis lation (IARC, 2012). Lung cancers
mans that beryllium and beryllium are probably complex, multifacet- have been repeatedly produced in
compounds cause lung cancer ed, and interactive, as with other rodents by either inhalation or in-
(IARC, 2012). In rats, inhalation of metals that are human carcinogens tratracheal instillations of cadmium
beryllium metal, beryllium sulfate, (Beyersmann and Hartwig, 2008; compounds. There is limited evi-
or beryl ore dust produced bronchi- Kitchin and Conolly, 2010; Tokar dence in humans for prostate and
olo-alveolar carcinoma, and intratra- et al., 2010; Brocato and Costa, renal carcinogenesis with cadmium
cheal instillation of beryllium metal, 2013; Koedrith et al., 2013). or cadmium compounds. In rodents,
beryllium hydroxide, or beryllium prostatic proliferative lesions can be
oxide produced bronchiolo-alveolar Cadmium and cadmium induced by cadmium, but there is no
carcinoma. compounds concordant evidence in rodents for
Overall, for inhaled beryllium and the kidney.
Carcinogenicity
beryllium compounds, the lung is the
one identified cancer target site in Mechanisms of
There is sufficient evidence in hu-
carcinogenesis
humans, a response that has been mans that cadmium and cadmium
repeatedly duplicated in rodent mod- compounds cause lung cancer, with Cadmium-induced carcinogenesis
els (IARC, 2012). positive associations between rel- may be attributable to various mech­
evant exposure and cancer of the anisms (Beyersmann and Hartwig,
Mechanisms of
kidney and prostate (IARC, 2012). In 2008; Brocato and Costa, 2013;
carcinogenesis
rodents, there is sufficient evidence Hartwig, 2013; Koedrith et al.,
Multiple, related mechanisms are for the carcinogenicity of cadmium 2013). Direct interaction of cadmi-
likely to be operative in beryllium-in- and cadmium compounds (IARC, um with DNA appears to be limited,
duced carcinogenesis (IARC, 2012). 2012). In rats, inhalation of cadmium and cadmium is a weak mutagen.
Although beryllium is inactive or chloride, cadmium oxide, cadmium Cadmium can perturb DNA repair
weakly positive as a mutagen, chro- sulfide, or cadmium sulfate pro- and affect tumour suppressor pro-
mosomal aberrations and aneu­ duced bronchiolo-alveolar carcino- teins, potentially causing genomic
ploidy can occur in vivo in rodents ma or squamous cell carcinoma of instability and chromosomal damage.
at non-toxic doses. Like many other the lung, and intratracheal instillation Altered DNA methylation patterns

34
and disrupted signal transduction 2012), and therefore the combination lated scarring (confluent fibrosis) and
processes have been observed after of data from male and female ani- chronic inflammatory changes indic-

CHAPTER 3
PART 1
exposure to cadmium; these factors mals may be problematic. Male rats ative of high tissue burdens of the
could potentially contribute to aber- chronically exposed by inhalation to test agent and local toxicity.
rant cell growth, but their role in cad- sodium dichromate developed lung Many studies have demonstrat-
mium-induced carcinogenesis is un- tumours in one study, although the ed carcinogenic activity for various
clear. Cadmium can induce reactive Working Group for that Monograph chromium(VI) compounds in ro-
oxygen species, but this would be an cautioned about small group sizes dents, such as production of soft
indirect effect and its precise role in in that study (IARC, 1990). Nasal tissue sarcomas after repository in-
cadmium-induced carcinogenesis papilloma occurred after chronic jections (IARC, 1990, 2012). Studies
is not completely defined. Specific inhalation of chromium trioxide in that produced injection-site tumours
mechanisms of lung carcinogenesis female mice in one study (IARC, have provided some evidence of the
after exposure to cadmium have not 1990). Various compounds of chro- carcinogenic potential of chromium
been elucidated fully. As with the oth- mium(VI), including calcium chro- (VI), but there is no concordance
er inorganic human carcinogens, the mate, strontium chromate, and zinc with any specific target site in hu-
mechanisms of cadmium-induced chromate, produced local squamous mans. Sodium dichromate dihydrate
carcinogenesis are probably multi- cell carcinoma in rats when the test in drinking-water caused adeno-
faceted (Beyersmann and Hartwig, agent was first mixed with cholester- carcinoma of the small intestine in
2008; Brocato and Costa, 2013; ol and then used to fill or coat stain- mice and squamous cell carcinoma
Koedrith et al., 2013). less steel wire baskets that were of the oral mucosa and tongue in
subsequently surgically implanted rats (National Toxicology Program,
Chromium(VI) compounds via tracheotomy into the bronchus 2008), but these tissues are not
(IARC, 1990). The coated basket considered cancer target sites in hu-
Carcinogenicity
acts as a point of release for the mans. Given the response and the
There is sufficient evidence in hu- test agent. Implantation of a basket rarity of these tumours in rodents
mans for the carcinogenicity of chro- containing cholesterol alone caused and the potential for oral exposure of
mium(VI) compounds, which cause notable squamous metaplasia (7% humans to chromium(VI), these sites
cancer of the lung (IARC, 1990, of controls) and bronchial inflamma- deserve additional focus in epidemi-
2012). Most of the epidemiological tion (89% of controls), indicating that ological studies.
data come from occupational set- chronic, not agent-related, irritation is The lung is a target site for the
tings that would involve inhalation as involved with this model even without carcinogenic effects of chromi­
the primary route of exposure. Also, a test agent. Although the tumours um(VI) compounds in humans
positive associations have been ob- induced by this technique of expo- (IARC, 1990, 2012). From inhalation
served between exposure to chro- sure via implantation of a wire basket studies in rats and mice, there is
mium(VI) compounds and cancer of were histologically defined as bron- sufficient evidence that chromi-
the nose and nasal sinuses (IARC, chiolar squamous cell carcinoma, um(VI) induces lung tumours, al-
2012). In rodents, there is sufficient their relevance to human lung can- though all the available studies are
evidence for the carcinogenicity of cer induced by inhalation of chromi- considered to have some limita-
chromium(VI) compounds (IARC, um(VI), or in fact by any other agent, tions (IARC, 1990). Lung tumours
1990, 2012). Calcium chromate in- has not been rigorously validated. produced by surgically implanting
duced lung tumours (adenomas) in Repeated weekly intratracheal instil- stainless steel wire baskets con-
mice, but only when data from male lations of calcium chromate or sodi- taining chromium(VI) compounds
and female groups were combined um dichromate in male and female in cholesterol into the bronchus
(Nettesheim et al., 1971). The sex rats produced bronchiolo-alveolar of rats, like injection-site tumours,
of a test animal is considered by carcinoma and squamous cell carci- probably reflect carcinogenic activ-
IARC as a quantitative aspect of a noma in one study (Steinhoff et al., ity, but the relevance of this expo-
tumour end-point study that poten- 1986). The authors noted that the sure technique in modelling tumours
tially affects the outcome in terms of chromium(VI)-induced tumours co- produced in humans by inhalation of
chemically induced tumours (IARC, existed with extensive treatment-re- chromium(VI) compounds requires

Part 1 • Chapter 3. Arsenic and metals 35


further validation. Repeated in- reductants, various toxic intermedi- lung, whereas intratracheal instilla-
tratracheal instillations of certain ates, including radicals of chromium, tion of nickel oxide, nickel subsulfide,
chromium(VI) compounds can oxygen, and sulfur, are likely to be or metallic nickel caused squamous
produce malignant lung tumours generated, and they can react with cell carcinoma of the lung. In sev-
(Steinhoff et al., 1986). There is key biomolecules relevant to carcino- eral well-performed experiments,
limited evidence that the nose or the genesis (IARC, 2012; Hartwig, the inhalation of various nickel com-
nasal sinuses are target sites for the 2013; Proctor et al., 2014). Some pounds, both water-soluble and in-
carcinogenic effects of chromium(VI) chromium(VI) reductants undergo soluble, including metallic nickel (in
in humans (IARC, 2012), and one Fenton-type reactions to produce rats), nickel sulfate (in rats and mice),
study in mice showed nasal papillo- hydroxyl radicals, which attack DNA. and nickel subsulfide (in mice) did
mas induced by inha­lation of chromi- Chromium(VI) can stimulate forma- not cause lung tumours. Oral expo-
um(VI) (IARC, 2012). tion of superoxide and nitric oxide sure to nickel sulfate did not cause
Generally speaking, chromium(VI) in vitro. Chromium(VI) metabolites tumours in rats or mice.
compounds do show concordance can be directly genotoxic, and the Various water-soluble and insol-
between humans and rodents for the metal also causes inflammation and uble nickel compounds and metal-
established target site in humans, stimulates tumour growth pathways lic nickel produced various types of
the lung, on the basis of: (i) two pos- in cell culture systems. Aneuploidy sarcomas in rats, mice, or hamsters
itive inhalation studies in rodents, has been observed after exposure when administered by repository
both with noted limitations (IARC, to chromium(VI). Significant DNA injections (subcutaneous, intramus-
1990); (ii) lung tumours induced in methylation changes could be a con- cular, intraperitoneal, etc.) (IARC,
rodents by repeated intratracheal in- tributing factor in chromium(VI)-in- 2012). Studies that produced in-
stillations; and (iii) activity in a rodent duced carcinogenesis, particularly in jection-site tumours have provided
model with surgical implantation of the lung (Brocato and Costa, 2013). some evidence of the carcinogenic
wire baskets containing chromi­ As with other inorganic human car- potential of nickel and nickel com-
um(VI) compounds into the bronchus cinogens, it is likely that multiple, pounds, but there is no concor-
to produce lung tumours. However, probably interactive, mechanisms dance with any specific target site in
additional state-of-the-art studies in are operative in chromium(VI)-in- humans.
experimental animals with inhaled duced carcinogenesis, including There are multiple studies in
chromium(VI) compounds appear to DNA damage, oxidative stress, and rodents that recorded increased
be needed. A high-quality, contem- aneuploidy, which lead to the acqui- pheochromocytoma of the adrenal
porary tumour end-point inhalation sition of a malignant phenotype. medulla after inhalation of nickel
study in rodents would add great- compounds, including metallic nickel
ly to the understanding of chromi- Nickel and nickel compounds and nickel subsulfide (IARC, 2012).
um(VI)-induced carcinogenesis and These studies might imply the sys-
Carcinogenicity
could be designed to significantly aid temic bioavailability of inhaled nickel
in elucidation of the mode of action of There is sufficient evidence that compounds, although there are no
this compound (Proctor et al., 2014). mixtures of nickel compounds and concordant data concerning the ad-
Such a study is lacking, presumably metallic nickel cause cancers of renal gland as a target of nickel-in-
because the agent had already been the lung, nose and nasal cavity, duced carcinogenesis in humans.
declared a human carcinogen and and paranasal sinuses in humans Overall, for nickel and nickel com-
rodent research resources were di- (IARC, 2012). Epidemiological stud- pounds, target site concordance ex-
rected elsewhere. ies provided evidence for induction ists between the human lung and the
of lung cancer by specific nickel rodent lung for various nickel com-
Mechanisms of
compounds, including water-soluble pounds and metallic nickel. There
carcinogenesis
and insoluble substances. In rats, are no data in rodents on cancers of
In terms of general mechanisms, inhalation of nickel oxide, nickel sub- the nose, nasal cavity, and parana-
during in vitro conversion of chromi- sulfide, or nickel carbonyl caused sal sinuses that would be concordant
um(VI) to chromium(III) by cellular bronchiolo-alveolar carcinoma of the with data in humans.

36
Mechanisms of carcinogens, it is likely that multiple, for the metals that are human car-
carcinogenesis cinogens (i.e. beryllium, cadmium,

CHAPTER 3
potentially interdependent, complex

PART 1
mechanisms are operative in nick- chromium(VI), and nickel) (IARC,
The nickel ion Ni(II) is considered to
el-induced carcinogenesis. 2012). However, this scenario is not
be the ultimate carcinogenic species likely to be entirely true for the met-
in nickel-induced carcinogenesis Comparative mechanisms alloid arsenic and its compounds. Of
(Beyersmann and Hartwig, 2008; of the inorganic human all the inorganic human carcinogens,
IARC, 2012). Water-soluble and carcinogens inorganic arsenic alone can undergo
poorly water-soluble nickel com- conjugative biotransformation with-
pounds both enter the cell and reach Among arsenic compounds and met- in the host. Methylation of inorganic
the nucleus, although the soluble als that are human carcinogens, only arsenic generally produces mon-
compounds do this by ion channels chromium(VI) seems to have the omethylarsenic (MMA) forms and
and transporters, whereas the poorly ability to interact with DNA directly, then dimethylarsenic forms, but this
soluble compounds are taken up by at least when it undergoes intracel- process is not complete in most peo-
phagocytosis. After phagocytosis, lular reduction, and to act as a direct ple (Melak et al., 2014). Incomplete
genotoxin. Chromium(VI) does show methylation can result in the forma-
particulate nickel compounds grad-
human-to-rodent target site con- tion of very toxic, monomethylated
ually release nickel ions, making
arsenic metabolites, like MMA(III).
them available for interaction with cordance with respect to the lung,
Recent data in humans indicate
key biomolecules. An increased lev- although up-to-date inhalation stud-
that an increased MMA level, as a
el of nickel in the nucleus is evident ies in rodents would greatly aid in
percentage of total urinary arsenic,
after exposure to water-soluble or defining the mechanisms of chromi-
strongly correlates with cancer of
insoluble nickel compounds. Nickel um(VI)-induced carcinogenesis (see
the lung and bladder in a population
compounds are weakly mutagenic above; Proctor et al., 2014). With the in northern Chile exposed to envi-
in mammalian cells but induce DNA inorganic human carcinogens other ronmental inorganic arsenic (Melak
damage, chromosomal aberrations, than chromium(VI), direct induction et al., 2014). This indicates that arse-
and micronuclei in vitro and in vivo. of DNA damage is not a key mechan- nic metabolites generated by incom-
Both water-soluble and insoluble ism. All the metals that are human plete methylation are associated with
nickel compounds induce malignant carcinogens seem to be able to increased carcinogenic risk after ex-
cell transformation in vitro. However, cause oxidative stress (Beyersmann posure to inorganic arsenic.
delayed mutagenicity and chromo- and Hartwig, 2008; Hartwig, 2013), Stimulation of inflammation is also
somal instability have been observed mostly by indirect means. This may common among the group of inor-
long after treatment of cells with nick- ganic human carcinogens. The role
contribute to their carcinogenic po-
of inflammation in carcinogenesis
el. Nickel compounds induce epige- tential because the resulting oxi-
may be secondary, through provi-
netic changes, including alteration in dative species could attack DNA.
sion of oxidants or radical species
DNA methylation patterns and mod- Arsenic, cadmium, chromium, and
produced by oxidation.
ification of histones (Brocato and nickel can have epigenetic effects on Definitive mechanisms have not
Costa, 2013). An inflammatory com- DNA that alter critical gene expres- been established for any inorganic
ponent is also thought to contribute sion and promote the acquisition of human carcinogen. These agents
to nickel-induced carcinogenesis. a malignant phenotype (Brocato and are best considered to be multi-
Direct effects of nickel on DNA are Costa, 2013). faceted, interrelated, and complex
probably limited, but oxidative stress With respect to carcinogenesis carcinogens. For the inorganic car-
and oxidative DNA damage have induced by inorganic chemicals, it cinogens with multiple target sites,
been found after nickel exposure. is generally accepted that the ionic there is a strong possibility that the
As with the other inorganic human species is the most active species mechanism is target site-specific.

Part 1 • Chapter 3. Arsenic and metals 37


References
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Sampayo-Reyes A, Wollenberg ML (2004). sulfate, gallium arsenide, indium phosphide Newell RF, Hellman A (1971). Effect of
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38
CHAPTER 4
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 4.

Smokeless tobacco
and its constituents
Stephen S. Hecht

Introduction appropriate here than the term “con- the risk of head and neck squamous
cordance”, which connotes a one-to- cell carcinoma (including cancers
Smokeless tobacco is defined one agreement, with no conflicting of the oral cavity, larynx, and phar-
as follows in Volume 89 of the data. ynx) (Zhou et al., 2013). This section
IARC Monographs (IARC, 2007): considers coherence between these
“Smokeless tobacco is used with­ Coherence: carcinogenicity conclusions and studies of the car-
out burning the product, and can be of smokeless tobacco in cinogenicity of smokeless tobacco in
used orally or nasally. Oral smoke­ humans versus experimental experimental animals.
less tobacco products are placed animals The use of smokeless tobacco by
in the mouth, against the cheek or
humans is a voluntary practice en-
behind the lip and sucked (dipped) Evaluations of smokeless tobac-
gaged in by hundreds of millions of
or chewed. Tobacco pastes or pow- co use by the IARC Monographs
people worldwide. There are great
ders are used in a similar manner concluded that this practice is car-
variations in use of smokeless to-
and applied to the gums or teeth. cinogenic to humans (Group 1),
bacco: in Sweden, fine-cut tobac-
Fine tobacco mixtures are usually caus­ing cancers of the oral cavity,
co, called snus, is placed between
inhaled and absorbed in the nasal oe­sophagus, and pancreas (IARC, the upper lip and teeth; in North
passages.” 1985, 2007, 2012). A meta-analysis America, fine-cut tobacco, frequent-
This chapter considers carcino­
of epidemiological data also con- ly in teabag-like sachets, is placed
genicity studies, data on constitu-
cluded that use of smokeless tobac- between the cheek and gums; and
ents, and mechanistic investigations
co signif­icantly increased the risk of in South-East Asia and other parts
on smokeless tobacco, to evaluate
these cancers (Boffetta et al., 2008). of the world, there are vast arrays
over­all coherence between observa-
A recent population-based case– of different practices (IARC, 2007).
tions in humans and in experimental
animals. Because of the differences control study, which was carried out Processed and fermented tobacco
between human use of smokeless in New England, USA, and was not of varying types and blends are the
tobacco and the exposure condi- included in the above-mentioned common ingredients in all of these
tions in studies in experimental an- evaluations, demonstrated a statisti- practices. Nicotine, perhaps along
imals, the term “coherence”, which cally significant association between with other tobacco alkaloids and con-
means logical consistency, is more ever use of smokeless tobacco and stituents, is the addictive substance

Part 1 • Chapter 4. Smokeless tobacco and its constituents 39


that drives the continuing use of these and not administered snuff (Park et of 1–10 µg per gram of product
products (DHHS, 1988; Stolerman al., 1986). This observation has not (IARC, 2007). No smokeless tobac-
and Jarvis, 1995; Benowitz, 1999; been replicated, and its relevance co product analysed for these com-
IARC, 2007). to human use of smokeless tobac- pounds has ever been reported to be
With reference to the use of co is unclear. Overall, there is some free of them. Nitrosamine carcino-
smoke­ less tobacco by humans, it coherence between studies in ex- genesis was discovered by the pio-
has not yet been possible to devel- perimental animals on the carcino- neering work of Magee and Barnes
genicity of smokeless tobacco and on dimethylnitrosamine (Magee and
op an experimental model in which
Barnes, 1956). Subsequently, multi-
laboratory animals voluntarily and cancer of the oral cavity in humans
ple studies by numerous investiga-
habitually consume these products as induced by use of smokeless to-
tors demonstrated that more than
the way they are used by humans. bacco; the conclusion of the IARC
200 nitrosamines are carcinogenic
Various approaches have been ex- Monographs that there is sufficient
in laboratory animals, frequently in-
plored, including addition of tobacco evidence in experimental animals
ducing tumours in an organ-specific
to the diet, oral treatment of animals for the carcinogenicity of smokeless
and systemic fashion, and in many
with tobacco extracts, exposure of tobacco followed from this evidence cases after treatment of animals
animals to powdered tobacco by (IARC, 2012). However, the results with very low doses (Preussmann
inhalation, placement of tobacco in are somewhat inconsistent and are and Stewart, 1984; Gray et al., 1991;
the cheek pouch of hamsters, and limited by the requirement of surgery Peto et al., 1991; Lijinsky, 1992).
surgical modification of the oral cav- and other unnatural approaches in More than 30 different animal spe-
ity. However, none of these methods an attempt to replicate in laboratory cies develop cancer after treatment
faithfully replicate the human habit, animals the voluntary use of smoke­ with various nitrosamines (Bogovski
and they have not always produced less tobacco by humans. and Bogovski, 1981). Nitrosamines
statistically significant results in car- are genotoxic carcinogens that ab-
Coherence: carcinogenicity solutely require metabolism to exert
cinogenicity studies. The most con-
of smokeless tobacco their carcinogenic effects (Hecht,
sistent findings in animal carcinogen-
in humans versus 1998b). NNN and NNK, the most
icity studies of smokeless tobacco carcinogenicity of smokeless carcinogenic of the tobacco-specific
have been reported in a model in tobacco constituents in nitrosamines, are typical members of
which an artificial lip canal is created experimental animals the nitrosamine class of carcinogens
by surgery on rats. Several studies (Hecht, 1998a). Amounts of NNN
of this type produced tumours of the There is remarkable coherence be-
and NNK in many different types of
oral cavity, including squamous cell tween the carcinogenic activity in rats
smokeless tobacco products have
carcinomas, and their incidence was of tobacco-specific nitrosamines,
been summarized based on the re-
significantly increased compared which are constituents of smokeless
sults of thousands of analyses; lev-
tobacco, and observations in hu-
with controls in some experiments els of NNN generally exceed those
mans who use smoke­less tobacco.
(IARC, 2007). Also, in one study, in- of NNK (IARC, 2007).
Tobacco-specific nitrosamines – N′-
sertion of snuff into the cheek pouch Multiple carcinogenicity studies
nitrosonornicotine (NNN), 4-(methyl-
of hamsters infected with herpes of NNN have been reported (Hecht,
nitrosamino) -1- (3 -pyridyl) -1-bu-
simplex virus type 1 (HSV-1) or type tanone (NNK), N′-nitrosoanabasine 1998a). A recent investigation ex-
2 (HSV-2) significantly increased (NAB), and N′-nitrosoanatabine plored the carcinogenicity in rats of
the incidence of squamous cell car- (NAT) – are the most prevalent (S)-NNN, the enantiomer of NNN
cinoma compared with that in ani- strong carcinogens in smokeless to- that is most prevalent in tobac-
mals infected with HSV-1 or HSV-2 bacco, generally found in the range co products, comprising 57–67%

O N O OH N O

S' N N
2 N N CH3 CH3 N N
N O N O N O N O
N N N N N N
(S) -NN N (R)- NN N N NK N NA L N AB N AT

40
of total NNN in smokeless tobac- death from oesophageal tumours with Syrian golden hamsters and
co and cigarette tobacco (Balbo before oral cavity tumours could be mice are generally less coherent

CHAPTER 4
PART 1
et al., 2013; Stepanov et al., 2013). observed. with the epidemiology of smokeless
(S)-NNN was administered in the Based on consumption of half a tobacco use than are the studies in
drinking-water (15 ppm) to a group tin (17 g) per day of a popular smoke­ rats (IARC, 2007).
of 24 male Fischer 344 (F-344) rats. less tobacco product (Hecht et al., Swabbing the oral cavity and lips
Two other groups of rats were given 2008a) containing about 3 µg per of rats with a mixture of NNN and
either (R)-NNN (15 ppm) or race- gram of NNN (Hecht et al., 2011) NNK for 131 weeks produced 9 oral
mic NNN (30 ppm). The rats in the and an extraction efficiency of 60% cavity tumours in 8 of 30 rats, which
groups treated with (S)-NNN or race- (Hecht et al., 2008b), human expo- was statistically significant, but the
mic NNN began losing weight after sure would be about 34 µg per day of result was not nearly as strong as
1 year of treatment and had died or NNN, or 20 µg per day of (S)-NNN; in that noted earlier, in part because
were humanely killed by 17 months. 30 years of use, this would amount to the dose of racemic NNN in the
All rats treated with (S)-NNN had tu- about 220 mg (3 mg/kg body weight) swabbing study was about 40% of
mours of the oral cavity. A total of 91 of (S)-NNN. This compares to a dose that described for the drinking-water
such tumours were observed in 20 of 150 mg (375 mg/kg body weight) study mentioned above (Hecht et al.,
rats that were necropsied, including of (S)-NNN in the drinking-water 1986). NNK by itself did not induce
tumours of the tongue, larynx, phar- study described above. It is unclear oral cavity tumours when swabbed
ynx, oral mucosa, and soft palate. whether a body weight correction is in the oral cavity of rats or hamsters
Some of the oral cavity tumours were relevant, considering that smoke­ (Hecht, 1998a). An interesting and
large. The rats treated with (S)-NNN less tobacco is concentrated in the unexplored observation in the swab-
also had 122 oesophageal tumours. oral cavity and is frequently held at bing study was that an extract of fine-
In contrast, (R)-NNN was only weak- one site. cut moist snuff of the type used orally
ly tumorigenic. A highly significant Whereas administration of NNN inhibited the oral cavity carcinogeni-
carcinogenic response similar to that in the drinking-water to F-344 rats city of NNN and NNK.
resulting from exposure to (S)-NNN produces tumours of the oral cavity Although the carcinogenicity
was also observed in the rats treated and the oesophagus, subcutaneous studies of NNN administered oral-
with racemic NNN. The induction of injection of NNN causes mainly tu- ly to rats are in many respects re-
tumours of the oral mucosa, tongue, mours of the nasal mucosa, with ma- markably consistent with the results
larynx, and pharynx as well as oe- lignant tumours arising predominant- of epidemiological studies of can-
sophageal tumours in all rats treat- ly in the olfactory epithelium (Hecht, cers of the oral cavity and the oe-
ed with (S)-NNN or racemic NNN is 1998a). Treatment of mink with NNN sophagus in humans, they did not
remarkably consistent with the epi- by subcutaneous injection also pro- produce any pancreatic tumours.
demiological studies of smokeless duced malignant nasal tumours In another example of coherence,
tobacco use summarized above. (Koppang et al., 1992; Koppang et NNK and its metabolite 4-(methyl-
Although this was the first study to al., 1997; IARC, 2007). nitrosamino)-1-(3-pyridyl)-1-butanol
investigate the carcinogenicity of Carcinogenicity studies of NNN (NNAL) both produced significantly
(S)-NNN, previous studies of race- with Syrian golden hamsters have increased incidences of exocrine
mic NNN administered in the drink- involved subcutaneous injection pancreatic tumours when admin-
ing-water to rats uniformly produced of NNN or swabbing of the cheek istered in the drinking-water to male
high yields of oesophageal tumours, pouch. Tumours of the trachea and F-344 rats at doses of 1 ppm (NNK)
and oral cavity tumours were occa- nasal cavity were observed upon or 5 ppm (NNAL) (Rivenson et al.,
sionally observed (Hecht, 1998a; subcutaneous injection; the cheek 1988). It should be noted, however,
IARC, 2007). The doses of NNN pouch was generally unresponsive that the lung was clearly the main
given in the earlier studies probably (Hecht, 1998a). Treatment of various target organ for NNK and NNAL
either were too low to observe a high strains of mice with NNN by oral or in these studies – with significant
incidence of oral cavity tumours in intraperitoneal administration has increases in lung cancer for both
addition to oesophageal tumours, resulted mainly in pulmonary adeno- agents (P < 0.01) – whereas the re-
or were so high that they caused mas (Hecht, 1998a). Thus, studies sults of epidemiological studies on

Part 1 • Chapter 4. Smokeless tobacco and its constituents 41


smokeless tobacco use and lung Furthermore, the level of NNAL with DNA also react with Hb. These
cancer are, in aggregate, inconclu- plus its glucuronides in the urine of Hb adducts, when treated with base,
sive at present (Boffetta et al., 2008). smokeless tobacco users is higher release 4-hydroxy-1-(3-pyridyl)-1-
than that in controls and is also sig- butanone (HPB) and have therefore
Coherence: mechanistic nificantly correlated with years of use been termed HPB-releasing Hb ad-
studies of carcinogenicity (Hecht et al., 2007). ducts. Their formation and persis-
of smokeless tobacco
Nitrosamines require metabolism tence in rats treated chronically with
to exert their carcinogenic effects, and either NNK or NNN has been well
Mechanistic studies can help bridge
the tobacco-specific nitrosamines documented (Hecht, 1998a; IARC,
the gap between epidemiological
NNN and NNK are no exception 2007).
investigations and experimental
(Preussmann and Stewart, 1984; Detection of NNN–DNA and
studies in laboratory animals. With
Hecht, 1998a). Many studies have NNK–DNA adducts as well as HPB-
respect to the role of tobacco-spe-
conclusively demonstrated that releasing Hb adducts would be antic-
cific nitrosamines in carcinogenesis
α-hydroxylation of these compounds ipated in smokeless tobacco users,
induced by smokeless tobacco prod-
catalysed by cytochrome P450 en- but there are no published studies
ucts, as indicated by the animal stud-
zymes leads to the formation of reac- on NNN–DNA and NNK–DNA ad-
ies described above, the first mech-
anistic question that arises concerns tive metabolites and DNA adducts, ducts in this group. However, several
uptake of constituents. Many studies and that these DNA adducts are studies have reported the presence
have demonstrated the presence crucial in the carcinogenic process. of HPB-releasing Hb adducts in hu-
of tobacco-specific nitrosamines in These studies have been reviewed mans, with the highest levels con-
the saliva of smokeless tobacco us- in detail (Hecht, 1998a, 2008; IARC, sistently seen in smokeless tobacco
ers (IARC, 2007). In one study that 2007). As an example of the impor- users (Hecht, 1998a; IARC, 2007).
quantified levels of NNK in a product tance of DNA adducts in carcino- These studies provide evidence that
before and after use, it was deter- genesis by NNN, it is worth noting NNN and NNK are metabolically
mined that approximately 59% of the that the formation of NNN–DNA ad- activated to form HPB-releasing Hb
NNK in a popular brand of smoke- ducts in the oesophagus, oral cavity, adducts in smokeless tobacco us-
less tobacco was extracted during and liver of rats treated chronically ers, although it is possible that there
use (Hecht et al., 2008b). A second with 10 ppm of (S)-NNN or (R)-NNN could be other sources of these
study of this type reported removal of in drinking-water correctly predicted adducts as well. Collectively, these
30% of the NNK and 23% of the NNN cancer induction in the oral cavity studies demonstrate coherence
from an oral snus product during use and oesophagus of rats upon treat- between mechanisms of NNN and
(Caraway and Chen, 2013). ment with these enantiomers as NNK metabolic activation in rats and
Analysis of the urine of smoke- described above (Lao et al., 2007; in smokeless tobacco users. Thus,
less tobacco users further demon- Zhang et al., 2009a). Thus, there is there is coherence in the carcino-
strates the uptake and metabolism great coherence between mechan- genicity data and in the mechanistic
of tobacco-specific nitrosamines. isms of NNN metabolism and DNA data available for these specific com-
NNN, NNAL, NAT, and NAB as well binding in rats and the correspond- pounds and the observed cancer-
as their glucuronides have all been ing carcinogen­ icity data. Less is causing effects of smokeless tobac-
detected in the urine of smokeless known about mechanisms of pan- co in humans.
tobacco users at levels similar to or creatic carcinogenesis by NNK, but There are still some noteworthy
greater than those found in the urine DNA adducts of NNK and its metab- gaps that prevent the development
of most smokers (Stepanov and olite NNAL have been characterized of a completely coherent picture of
Hecht, 2005; Hecht et al., 2007). It in the pancreas in rats (Zhang et al., NNN metabolism in laboratory ani-
has been estimated that NNAL plus 2009b). mals and humans. Multiple studies,
its glucuronides comprise 14–17% of In tandem with the formation of including some of those described
the NNK dose in people who use a DNA adducts by NNN and NNK in above, indicate that in F-344 rats,
popular smokeless tobacco product, experimental animals, the formation 2′-hydroxylation of NNN is important
and that their uptake of NNK is about of haemoglobin (Hb) adducts occurs, in the formation of DNA adducts and
6 µg per day (Hecht et al., 2008b). because intermediates that react in the expression of carcinogenicity

42
by NNN (Hecht, 1998a; IARC, 2007). by smokeless tobacco itself, both in Conclusions
It is not known which cytochrome laboratory animals and in humans

CHAPTER 4
PART 1
P450 enzyme is responsible for (IARC, 2007). The few studies that There is considerable coherence
have been reported either used between established target tissues
NNN 2′-hydroxylation in the oral
non-specific techniques or did not for the carcinogenicity of smokeless
cavity and oesophagus in rats, or in
tobacco in humans – the oral cavity,
humans. Two human cytochrome find consistent effects of smokeless
oesophagus, and pancreas – and
P450 enzymes that catalyse (S)- tobacco on DNA adduct formation.
target tissues in rats treated orally
NNN metabolism by 5′-hydroxyla- Similarly, there is at present no con-
with NNN or NNK, which are constit-
tion – cytochrome P450 2A6 and vincing published evidence that use
uents of all smokeless tobacco prod-
2A13 enzymes – do not catalyse the of smokeless tobacco produces
ucts and are present in commonly
DNA adducts in the oral cavity, oe-
2′-hydrox­ylation (Wong et al., 2005). used products at concentrations
sophagus, or pancreas in humans. higher than those of other strong car-
This raises some questions about
This represents a significant gap in cinogens. There is also coherence
the enzymology of (S)-NNN meta-
a mechanistically coherent pathway between the mechanisms by which
bolic activation in humans. Further, in
to cancer upon smokeless tobacco NNN and NNK induce cancer in rats,
studies of NNN metabolism in patas
use as observed in epidemiological via DNA adducts and their conse-
monkeys, the major pathway appears
studies. quent effects, and observations in
to be 5′-hydroxylation (Upadhyaya et
Nevertheless, many studies in humans. There is less coherence
al., 2002). More research is needed
human users of smokeless tobac- between carcinogenicity and mecha-
to determine whether these obser- nistic aspects of smokeless tobacco
co – but fewer in laboratory ani-
vations reflect a lack of coherence exposure per se in laboratory ani-
mals – demonstrate genetic effects
between rats and humans or simply that are consistent with the conse- mals and humans, in part because of
a lack of relevant data. quences of DNA adduct formation. operational difficulties in carrying out
As noted above, the formation of carcinogenicity studies, and perhaps
Higher frequencies of micronuclei in
DNA adducts is critical in the car- because the right questions have
buccal cells of smokeless tobacco
cinogenic process induced by the not been addressed with respect to
users have been reported in multiple
mechanisms.
agents discussed here. In contrast to studies (Proia et al., 2006). Mutations
the plethora of information available in important growth control genes, Acknowledgement
on DNA adduct formation in labora- such as TP53 and RAS, from oral
tory animals by smokeless tobac- cavity tumours of smokeless tobacco This research on tobacco-specific
co constituents – most commonly users have also been observed fre- nitrosamines is supported by grant
NNN and NNK – there is a paucity quently and are likely to be the result CA-81301 from the United States
of studies on DNA adduct formation of DNA damage (IARC, 2007). National Cancer Institute.

Part 1 • Chapter 4. Smokeless tobacco and its constituents 43


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Psychopharmacology (Berl). 117(1):2–10, activation of structurally similar carcinogenic pyridyloxobutyl and pyridylhydroxybutyl DNA

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Part 1 • Chapter 4. Smokeless tobacco and its constituents 45


CHAPTER 5
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 5.

Tobacco smoke
and its constituents
Stephen S. Hecht and David M. DeMarini

This chapter addresses the con- Coherence: carcinogenicity the induction of lung tumours, both
cordance in studies of carcinogen- of tobacco smoke in humans benign and malignant, by cigarette
icity and mechanisms between versus experimental animals smoke.
experimental animals and humans Although these studies have
Volume 100E and previous IARC established animal models for the
for tobacco smoke and its constit-
Monographs and certain reviews on study of tobacco smoke carcino-
uents. Volume 100E of the IARC
tobacco smoke have summarized genesis and, in aggregate, support
Monographs updated the literature the literature on animal studies of the the epidemiological findings that
on tobacco smoke and the evalua- carcinogenicity of tobacco smoke smoking is a cause of cancers of the
tions of its carcinogenicity (IARC, (IARC, 1986, 2004, 2012b; Hecht, lung and larynx in humans, specif-
2012b). It concluded that there was 2005). These studies demonstrated ic problems associated with animal
sufficient evidence that tobacco that cigarette smoke can induce tu- studies of tobacco smoke expo-
smoking causes multiple types of mours of the lung and nasal cavity in sure have been recognized. Many
cancer in humans, including (to var- mice and rats and tumours of the lar- of these issues result from the fact
ying extents) cancers of the lung, ynx in hamsters. Some recent stud- that most laboratory animals are ob-
oral cavity, pharynx, oesophagus, ies not included in the evaluations ligate nose breathers and, thus, do
in Volume 100E have consistently not inhale tobacco smoke voluntari-
stomach, colorectum, liver, pancre-
established the carcinogenicity of ly and habitually in the same way in
as, nasal cavity and paranasal si-
cigarette smoke to the lung in the which humans smoke tobacco prod-
nuses, larynx, uterine cervix, ovary
A/J mouse, where it produces ade- ucts (Wynder and Hoffman, 1967).
(mucinous), urinary bladder, kidney,
noma and adenocarcinoma, as well Constant whole-body exposure of
ureter, and bone marrow (myeloid as causing emphysema (Stinn et al., rodents to cigarette smoke, often at
leukaemia). 2010, 2013). The A/J mouse, which relatively high concentrations, pro-
is highly susceptible to lung tumour duces avoidance reactions, stress,
development, appears to present weight loss, and other indicators of
a relatively reproducible system for toxicity that are widely different from

Part 1 • Chapter 5. Tobacco smoke and its constituents 47


the human responses to voluntary imals (IARC, 2010). Similarly, NNK is in urine, collected years before di-
inhalation driven by the desire for re- a powerful lung carcinogen, inducing agnosis, and oesophageal cancer,
curring small doses of nicotine. adenocarcinoma of the lung in rats, but not lung cancer, in smokers, after
There are some mechanistic dif- mice, and hamsters independent of correction for duration and intensity
ferences as well. For example, muta- the route of administration and fre- of smoking (Yuan et al., 2011). This
tions in the KRAS gene are frequent- quently at very low doses (Hecht, indicates considerable concordance
ly observed in lung adenocarcinoma 1998). Thus, PAHs and NNK are between target tissues of NNN and
in humans, as are K-ras mutations in widely considered to be causes of NNK in rats and observations of can-
lung adenocarcinoma in mice; how- lung cancer in smokers. PAHs – cer incidence in smokers (Stepanov
ever, the mutation frequency is not and their diol epoxide metabolites et al., 2014).
increased and the mutation spec- in particular – produce mutations in Carcinogenicity studies in lab-
trum is not altered in mice exposed TP53 and KRAS that are similar to oratory animals and studies in hu-
to cigarette smoke (Hutt et al., 2005; those observed in lung tumours from mans exposed occupationally have
DHHS, 2010; Stinn et al., 2013). smokers (DHHS, 2010). In recent established aromatic amines such
Taken together, there is only moder- nested case–control studies within as 4-aminobiphenyl and 2-naphthyl­
ate concordance between the carci- prospective cohorts, biomarkers of amine as human bladder carcino-
nogenic and mechanistic effects of PAH and NNK exposure were asso- gens (IARC, 1987, 2012a). These
tobacco smoke evident in laboratory ciated with risk of lung cancer, after and other aromatic amines are com-
animals and epidemiological obser- correction for duration and intensity ponents of mainstream cigarette
vations in humans (Witschi, 2007). of smoking (Hecht et al., 2013). smoke (Xie et al., 2013). There is
1,3-Butadiene is another com- also considerable mechanistic evi-
Concordance: carcinogenicity pound likely to be involved in the dence from studies of haemoglobin
of tobacco smoke in humans
etiology of lung cancer in smokers. It adducts consistent with the propos-
versus carcinogenicity of
is found in relatively high concentra- al that 4-aminobiphenyl is respon-
tobacco smoke constituents
in experimental animals tions in tobacco smoke and is a pow- sible for bladder cancer in smokers
erful lung carcinogen in mice, but not (Castelao et al., 2001; IARC, 2012a).
There is considerable concordance in rats (IARC, 2008). Benzene is a leukaemogen in hu-
between the known carcinogenic Other tobacco smoke compounds mans, and it occurs in considerable
properties of many tobacco smoke with the lung as a target tissue/organ quantities in cigarette smoke (IARC,
constituents and the multiple target in some animal studies include iso- 1987, 2012a). The uptake of benzene
tissues of tobacco smoke as demon- prene, ethylene oxide, ethyl carba- by smokers has been demonstrated
strated in epidemiological studies mate, benzene, and various metals conclusively by biomarker studies
(IARC, 2012b). (Hecht, 2011). (Hecht et al., 2010). Thus, it is like-
It should be noted that carcino- The oral cavity, pharynx, and ly that benzene is responsible for
genicity assays of pure compounds oesophagus of rats are established leukaemia in smokers, although it
generally do not suffer from the target tissues of the tobacco-specif- does not cause leukaemia in rodents
above-mentioned operational dif- ic nitrosamine N′-nitrosonornicotine (IARC, 2012a).
ficulties with respect to tobacco (NNN), and in particular its (S) enan- Multiple tobacco smoke carcino-
smoke (Witschi, 2007). tiomer (Hecht, 1998; Balbo et al., gens have produced tumours of the
With respect to lung cancer, 2013). NNN is found in the smoke of upper respiratory tract. Tumours of
multiple polycyclic aromatic hy- all tobacco products (IARC, 2007). the larynx, nose, and trachea as well
drocarbons (PAHs) and the tobac- N-nitrosodiethylamine is another as of the pharynx and oesophagus
co-specific nitrosamine 4-(methylni- tobacco smoke constituent that in- were induced in inhalation studies
trosamino)-1-(3-pyridyl)-1-butanone duces oesophageal tumours in rats, with benzo[a]pyrene, an archetyp-
(NNK) are found in the smoke of although its levels in smoke are con- al PAH, in hamsters (IARC, 2010).
all cigarettes tested to date (IARC, siderably lower than those of NNN. Tumours of the nose have also been
2004). There is abundant evidence One nested case–control study observed in rats treated with tobac-
attesting to the ability of PAHs to found a strong relationship between co-specific nitrosamines (Hecht,
induce lung cancer in laboratory an- levels of NNN and its glucuronides 1998; Balbo et al., 2013). Inhalation

48
studies with formaldehyde and ac- lead to the eventual development of and cause DNA replication errors
etaldehyde produced nasal tumours lung cancer (IARC, 2004; DHHS, that may lead to mutations. If these

CHAPTER 5
PART 1
(IARC, 1985, 2006, 2012a). 2010, 2014). This scheme is for lung mutations occur in important regions
Tobacco smoke contains com- cancer because it is for this disease of critical growth control genes, such
pounds that are carcinogenic to that the most data are available. as the oncogene KRAS or the tu-
the colorectum in rats, most nota- The central pathway of Fig. 5.1 mour suppressor gene TP53, cellular
bly certain heterocyclic aromatic in particular shows great coherence growth processes become severely
amines (IARC, 2004, 2012b; Hecht, with established genotoxic mecha- dysregulated, resulting in uncon-
2012). With respect to induction nisms by which many carcinogens, trolled cell proliferation and cancer.
of liver cancer by tobacco smoke, including most of the more than 70 There are convincing data from
there is coherence with furan and established carcinogens in cigarette studies of smokers and lung cancer
N-nitrosodimethylamine, which are smoke, drive the process of cancer patients that illustrate coherence of
liver carcinogens in rats (Peto et al., induction. Thus, exhaustive mecha- these observations in humans with
1991; NTP, 1993), whereas NNK nistic studies carried out both in vitro the results observed in the plethora
and its major metabolite 4-(methyl- and in laboratory animals since the of mechanistic studies noted above.
nitrosamino)-1-(3-pyridyl)-1-butanol middle of the 20th century and con- Carcinogen uptake by smokers has
(NNAL) induce pancreatic cancer in tinuing today provide solid evidence been unequivocally demonstrated
rats (Hecht, 1998). that most carcinogens, either direct- by biomarker studies that compare
Collectively, there is considerable ly or after metabolism catalysed by levels of carcinogens and their me-
concordance between established multiple cytochrome P450 enzymes, tabolites in the urine of smokers
sites of tobacco smoke carcinogen- react with nucleophilic sites in DNA and non-smokers (Hecht et al.,
icity in humans and target tissues to form covalent binding products 2010). These studies leave no doubt
in experimental animals of individ- called DNA adducts. that exposure to multiple carcino-
ual carcinogens present in tobacco There are cellular repair systems gens, including tobacco-specific ni-
smoke.
that have evolved to repair these trosamines, PAHs, aromatic amines,
DNA adducts and restore the nor- and various volatile compounds in-
Concordance: overall
mechanism of cancer mal structure of DNA. These repair cluding benzene and 1,3-butadiene,
induction in humans versus systems are crucial because cer- is significantly higher for smokers
laboratory studies tain rare DNA repair-deficiency syn- than for non-smokers.
dromes, such as xeroderma pigmen- These and related studies also
Fig. 5.1 presents a widely accepted tosum, lead to a high susceptibility to show that virtually all of these car-
mechanistic framework describing cancer development. Thus, DNA ad- cinogens are metabolized by cy-
the events that occur in smokers and ducts, if left unrepaired, can persist tochrome P450 enzymes, resulting

Fig. 5.1. Mechanistic framework describing events involved in lung carcinogenesis in smokers. Adapted with
permission from DHHS (2010).

Part 1 • Chapter 5. Tobacco smoke and its constituents 49


in the formation of highly reactive in 163 of the 188 tumours, including genes important in cancer. Overall,
metabolites that react with DNA to 915 point mutations, 12 dinucleotide these results are coherent with the
produce adducts. The induction of mutations, 29 insertions, and 57 de- induction of multiple mutations in
the cytochrome P450 1A1 enzyme in letions. The analysis identified 26 critical growth control genes by met-
the lungs of smokers via activation of genes mutated at significantly ele- abolically activated carcinogens in
the aryl hydrocarbon receptor, result- vated frequencies, including TP53, cigarette smoke, although it should
ing in the conversion of benzo[a]py­ KRAS, CDKN2A, and STK11, con- be recognized that other processes
rene and related compounds to their sistent with other studies and with downstream of carcinogen exposure
DNA-reactive forms, is a frequently the known involvement of these probably also contribute to the muta-
observed and consistent finding in genes in growth control. Mutations tion load.
the literature on the effects of ciga- were found most frequently in TP53 In aggregate, these studies pres-
rette smoking (DHHS, 2010). and KRAS (Ding et al., 2008). ent a coherent mechanism based on
Many studies have demonstrated Another study examined a small
multiple studies, including chemical
the presence of multiple DNA ad- cell lung cancer cell line. More than
analyses, measurements of muta-
ducts in the lungs of smokers, gen- 22 000 somatic substitutions were
tion induction, and tests in labora-
erally at higher levels than those in identified, among which were 134 in
tory animals as well as biochemical
non-smokers. Although there is still coding exons. G  →  T transversions
and molecular biological evaluations
room for further elaboration of the were the most common (34%), fol-
of human tissues, blood, and urine.
specific DNA adducts involved in lowed by G  →  A transitions (21%)
The data are consistent and convinc-
this process, there can be little doubt and A  →  G transitions (19%), sim-
ing with respect to the central track
about the higher levels of DNA dam- ilar to earlier data in many studies
of Fig. 5.1.
age in the lung tissue of smokers (Pleasance et al., 2010).
compared with non-smokers (IARC, Another investigation focused It is clear that other processes
2004; Phillips and Venitt, 2012). on a non-small cell lung cancer are involved. Certain compounds in
Consistent with these data are the from a patient aged 51 years who tobacco smoke, or their metabolites,
common findings of mutagenicity in had smoked 25 cigarettes per day may interact directly with cellular re-
urine of smokers and sister chroma- for 15 years before excision of the ceptors. This can lead to activation
tid exchange in peripheral lympho- tumour, which was histologically of protein kinases, growth recep-
cytes of smokers (IARC, 2004). characterized as an adenocarcino- tors, and other molecules that can
Multiple recent studies with cur- ma. Single nucleotide variants were contribute to carcinogenesis (Chen
rently available DNA sequencing common, mostly at GC base pairs, et al., 2011). It is well established that
methods have demonstrated that frequently G  →  T transversions. tobacco smoke contains inflammato-
DNA adducts in the lungs of smokers Approximately 17.7 mutations per ry substances, resulting in enhanced
result in mutations. megabase were observed, for a total pneumocyte proliferation, activation of
Greenman et al. (2007) stud- of more than 50 000 single nucleo- nuclear factor kappa-light-chain-en-
ied mutations in the coding exons tide variants. At least eight genes
hancer of activated B cells (NF-κB),
of multiple protein kinase genes in in the EGFR-RAS-RAF-MEK-ERK
and tumour promotion (Takahashi
lung cancer and other cancers. Lung pathway were either mutated or am-
et al., 2010). There are also co-car-
cancers were among those with the plified (Lee et al., 2010).
cinogens that undoubtedly con-
most somatic mutations (4.21 per These results are fully consis-
tribute to the overall mechanism
megabase); the authors attributed tent with those reported earlier
of tobacco smoke carcinogenesis.
this to frequent exposure to exoge- (DHHS, 2010) and with data in the
nous mutagens (Greenman et al., Catalogue of Somatic Mutations in Furthermore, cigarette smoke induc-
2007). Cancer (COSMIC) database (http:// es oxidative damage, altered gene
In another study, 188 primary lung www.sanger.ac.uk/genetics/CGP/ promoter methylation, dysregulation
adenocarcinomas were sequenced. cosmic/) and the IARC TP53 data- of gene expression by microRNAs,
Analysis of 247 megabases of tu- base (http://www-p53.iarc.fr/), which and chronic cell injury and cytotoxic-
mour DNA sequence identified 1013 store and display somatic mutations ity with regenerative proliferation as
non-synonymous somatic mutations in TP53 and KRAS as well as other an amplifying factor, all of which can

50
contribute to the overall carcinogen- Disclaimer the contents reflect the views of the
ic effect (IARC, 2012b; Milara and agency, nor does mention of trade

CHAPTER 5
We thank J.A. Ross and C.E. Wood for

PART 1
Cortijo, 2012; Momi et al., 2014). names or commercial products con-
their helpful comments on this manu- stitute endorsement or recommen-
In summary, cigarette smoking
script. This article was reviewed by the dation for use.
represents a potent combination of
National Health and Environmental
biological effects associated with
Effects Research Laboratory, United
carcinogenesis, coherent with land-
States Environmental Protection
mark publications dating back more
Agency and approved for publica-
than 60 years (Hecht and Szabo,
tion. Approval does not signify that
2014).

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52
CHAPTER 6
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 6.

Anticancer agents: qualitative


and quantitative aspects
Kari Hemminki and Harri Vainio

Introduction of potency based on bioassay data various human exposures, com-


has been generated from values of parisons of potency assessments
Typically, regulation of human expo- TD50, i.e. the chronic dose rate (in with data from animals have been
sure to carcinogenic compounds is mg/kg body weight [bw]/day) that is hampered.
based mainly on qualitative consid- estimated to reduce the proportion An additional source of quantita-
erations for compounds that cause of tumour-free animals by 50% (Gold tive data on cancer risks is the group
cancer in experimental animals. et al., 2005). For humans, exposures of patients who are survivors of first
This approach is based on the old to ionizing radiation, occupational primary cancers after treatment
paradigm of using animal models to carcinogens, and tobacco smoke with anticancer therapy and who
understand human physiology and have been the primary sources of are monitored for treatment-relat-
pathology; in the regulatory setting, quantitative data on cancer risks, ed risks of second primary cancers
no alternatives to this paradigm have including considerations of dose, (Travis, 2006; Travis et al., 2006).
been available specifically in relation duration of exposure, and latency Active research on second can-
to cancer (Maronpot et al., 2004). period (i.e. time from exposure to cers has been carried out since the
Quantitative aspects of carcino- occurrence of cancer) (Breslow and 1980s, but because of the increas-
genesis, including estimates of car- Day, 1987; Moolgavkar et al., 1999; ing numbers of patients enrolled and
cinogenic potency in animals and in Brenner et al., 2003; Pierce and the extended periods of follow-up,
humans, would have both regula- Vaeth, 2003; Preston et al., 2003, the more recent studies provide
tory and scientific implications. For 2004; IARC, 2004). However, be- the most comprehensive evidence
animals, a systematic assessment cause of the complexities of these on the magnitude of the effects

Part 1 • Chapter 6. Anticancer agents: qualitative and quantitative aspects 53


Table 6.1. Tumour sites and histological types of cancer induced in humans and in rodents after exposure to
anticancer agents

Agent Tumour sites and histological types

Humans Rats Mice

Cyclophosphamide AML Lymphoma Lymphoma


Bladder cancer Leukaemia Acute lymphocytic leukaemia
Mammary adenoma Mammary carcinoma
Transitional cell carcinoma of the bladder Lung cancer
Neurogenic sarcoma Liver cancer
Chlorambucil AML Lymphoma Lymphoma
Myeloid leukaemia Myeloid leukaemia
Mammary carcinoma
Melphalan AML Retroperitoneal sarcoma Lymphoma
Thiotepa Leukaemia Lymphocytic leukaemia Lymphoma
Uterine sarcoma Lymphocytic leukaemia
Squamous cell cancers of the skin and the ear
canal
AML, acute myeloid leukaemia.
Source: Compiled from IARC (2012).

(Hijiya et al., 2007; Hodgson et al., (Hemminki et al., 2008). Anticancer Of the anticancer agents included in
2007; Maule et al., 2007; Hemminki agents are also used in some other Volume 100A, the current selection
et al., 2008; Swerdlow et al., 2011). cases, such as for certain autoim- does not include busulfan, 1-(2-chlo-
Although these studies present valu- mune diseases, but even if a single roethyl) -3 -(4-methylcyclohexyl) -
able data on many exposure-relat- anticancer agent is given, other med- 1-nitrosourea (methyl-CCNU), treo-
ed aspects, relevant treatments are ication and the inherent cancer risk sulfan, and some mixtures of anti-
seldom based on single agents or of some autoimmune conditions may cancer agents for which evidence is
single modalities, and individual car- limit the applicability of the results. sufficient in humans but evidence is
cinogens can rarely be singled out. In this chapter, data on antican- limited or lacking in animals.
Nevertheless, striking new data from cer agents from Volume 100A of
these studies show increased risks the IARC Monographs are used to Therapeutic applications and
of almost all site-specific cancers make qualitative comparisons be- trends in use
that emerge during the follow-up tween cancers induced in humans
The four anticancer agents dis-
period. Such data challenge the “ca- and in experimental animals, with
cussed here were first used in clin-
nonical” site-specificity of carcino- reference to the possible underlying
ical practice in the 1960s, but since
genesis. In their review of human mechanisms. Furthermore, quanti-
then the clinical indications have
carcinogens, Cogliano et al. indicate tative comparisons of carcinogens
been narrowed and their therapeutic
that no agents classified as carci- with respect to potency in humans
use has declined, with the possible
nogenic to humans (Group 1) are and in experimental animals are
exception of cyclophosphamide.
identified as causing prostate can- discussed. This review is limited to
Cyclophosphamide may be used
cer (Cogliano et al., 2011). However, anticancer agents for which the evi- alone for the treatment of several
some evidence is available. The risk dence of carcinogenicity was consid- types of cancer, but most often it is
of prostate cancer is significantly in- ered to be sufficient both in humans administered in combination with
creased in survivors of non-Hodgkin and in experimental animals: cyclo- other drugs. Diseases for which cy-
lymphoma after chemotherapy, for phosphamide, chlorambucil, mel- clophosphamide is the recognized
those diagnosed at age 40–49 years phalan, and thiotepa (IARC, 2012). treatment include breast cancer,

54
Table 6.2. Values of TD50 (the chronic dose rate estimated to reduce the proportion of tumour-free animals by 50%)
for anticancer agents in rodents

CHAPTER 6
PART 1
Agent TD50 (mg/kg bw/day)
 

All tumours combined Haematopoietic malignancies

Rats Mice Rats Mice

Cyclophosphamide 2.2a 2.8 3.4b 7.9


Chlorambucil 0.7 0.1 1.6 0.6
Melphalan 0.6 0.1 0.9 0.5
Thiotepa 0.04 0.07 0.2 0.2
bw, body weight.
a Bladder tumours in rats: TD = 21 mg/kg bw/day (Gold et al., 1987).
50
b From Gold et al. (1987).

Source: Adapted with permission from Kaldor et al. (1988), copyright 1988, with permission from Elsevier.

lymphoma, leukaemia, sarcoma, and Tumour sites Carcinogenic potency


ovarian cancer. Cyclophosphamide
is also used for treatment of diseases Tumour sites and histological types Gold et al. have systematically an-
other than cancer, such as nephro- of cancer induced in humans and in alysed the carcinogenic potency of
tic syndrome and many autoimmune rodents by the four anticancer drugs compounds tested in animal exper-
diseases, including Wegener granu- are listed in Table 6.1. In humans, iments in the context of the United
lomatosis, rheumatoid arthritis, lupus cyclophosphamide causes acute States National Toxicology Program
erythematosus, mycosis fungoides, myeloid leukaemia and bladder (Gold et al., 1987, 2005). These data
and several forms of vasculitis. cancer of undefined histology. were used by Kaldor et al. to quanti-
The current clinical use for chlo- Lymphoma, leukaemia, and mam- fy the carcinogenic potency of anti-
rambucil mainly involves treatment mary carcinoma have been detected cancer agents (Kaldor et al., 1988).
of chronic lymphocytic leukaemia. in rats and mice after administration With respect to potency, it should be
Chlorambucil may also be used for of cyclophosphamide. In rats, transi- noted that low daily doses producing
treatment of non-Hodgkin lympho-
tional cell carcinoma of the bladder cancer (i.e. low TD50 values) indicate
ma, Waldenström macroglobulin-
and neurogenic sarcoma have been high carcinogenic potency.
aemia, polycythaemia vera, troph-
reported. In mice, cancers of the Data on rats and mice from
oblastic neoplasms, and ovarian
lung and liver have been detected. Kaldor et al. (1988) are collected in
cancer. Chlorambucil has also been
Chlorambucil causes acute myeloid Table 6.2. For ease of analysis, re-
applied as an immunosuppressive
leukaemia in humans. Lymphoma sults for male and female rodents
drug for various autoimmune and
and leukaemia have been detected were averaged. When information
inflammatory conditions.
in rats and mice, as well as mammary was lacking in the paper by Kaldor et
The use of melphalan has de-
clined for treatment of most can- carcinoma in rats. Melphalan causes al., data were taken from other sourc-
cers, but since about 2000 it has acute myeloid leukaemia in humans, es, as indicated. The TD50 values
been given in high doses to patients retroperitoneal sarcoma in rats, and for all tumours combined are lower
with myeloma in combination with lymphoma in mice. Thiotepa causes than those for haematopoietic ma-
autologous stem cell transplantation. leukaemia in humans and lympho- lignancies. According to these data,
Thiotepa has previously been cytic leukaemia in rats and mice. It thiotepa is the most potent carcino-
used in the palliation of a wide varie- has been reported to induce uterine gen, with TD50 values of 0.04 mg/kg
ty of neoplastic diseases. It may still sarcoma and squamous cell cancers bw/day in rats and 0.07 mg/kg bw/
be prescribed in intravesical chemo- of the skin and the ear canal in rats day in mice for all tumours com-
therapy for bladder cancer. and lymphoma in mice. bined. Chlorambucil and melphalan

Part 1 • Chapter 6. Anticancer agents: qualitative and quantitative aspects 55


Table 6.3. Estimated carcinogenic potency (10-year cumulative incidence [%] divided by total dose in grams) in
humans of anticancer agents

Agent Leukaemia Bladder cancer


   

From Kaldor et al. (1988) Calculateda

Low dose High dose

Cyclophosphamide 0.28 – 0.04 0.02b, 0.1c


Chlorambucil 4.2, 1.8 16.5 1.4 –
Melphalan 18.7, 3.3 14.1 11.5 –
Thiotepa – 55.3 3.2 –
a Calculated from Kaldor et al. (1990).
b Calculated from Kaldor et al. (1995).
c Calculated from Travis et al. (1995).

are equally potent as carcinogens, for primary ovarian cancer (Kaldor per 10 years for the second study re-
whereas cyclophosphamide is weak- et al., 1990). The potency accord- sulted in potency values of 0.02 and
er by approximately an order of mag- ing to Kaldor et al. was calculated 0.1, respectively (Table 6.3). Thus,
nitude. After treatment of rats with from the cumulative baseline inci- the potency of cyclophosphamide
cyclophosphamide, bladder cancer dence of leukaemia in Sweden of determined in relation to bladder
was detected with a TD50 of 21 mg/kg 0.2 per 10 years, multiplied by the cancer was lower than its potency in
bw/day, i.e. an order of magnitude relative risk given in the relevant pa- the haematopoietic system. A similar
lower than the value for all tumours per; the product was then divided outcome was evident in the rodent
combined (Gold et al., 1987). by the median doses cited for the studies (Table 6.2).
The measure of potency used low dose and the high dose (Kaldor
Mechanisms of action
by Kaldor et al. was the 10-year et al., 1988). The low and high doses
cumulative incidence of leukaemia differed widely, and the calculated
Cyclophosphamide is activated
(a percentage) divided by the total potency values were clearly higher
through a cytochrome P450-
administered dose in grams; thus, a for the low dose than for the high
mediated reaction to yield phos-
large number indicates high poten- dose. Thiotepa and melphalan were
phoramide mustard and acrolein,
cy (Kaldor et al., 1988). These data the most potent drugs, followed by both of which can bind to DNA.
are shown in Table 6.3. Information chlorambucil and the much weaker Phosphoramide mustard undergoes
was lacking for thiotepa, but of the cyclophosphamide. The potency of rapid dephosphoramidation, which in
remaining compounds melphalan cyclophosphamide to induce bladder neutral in vitro conditions proceeds
was the most potent, with values cancer was also calculated, accord- with a half-life of 8 minutes, result-
of 18.7 and 3.3 from two separate ing to the data from two studies; in ing in the formation of nornitrogen
studies. Chlorambucil showed an the first study, bladder cancer was mustard (Hemminki, 1985). Because
intermediate potency, which was an diagnosed in women after treatment most of the metabolic activation of
order of magnitude higher than that for ovarian cancer (Kaldor et al., cyclophosphamide takes place in the
of cyclophosphamide. 1995), and in the second study, blad- liver, it seems likely that a consider-
More recent data were added to der cancer was diagnosed in sur- able proportion of DNA binding in
Table 6.3 from studies in which the vivors of non-Hodgkin lymphoma peripheral tissues is in fact mediated
anticancer agent was the principal (Travis et al., 1995). The use of a by nornitrogen mustard (Hemminki,
drug used and no radiotherapy was cumulative baseline incidence of 1985). As summarized in Volume
applied. Kaldor et al. published a bladder cancer in women of 0.4 per 100A of the IARC Monographs, cy-
multinational study of secondary leu- 10 years for the data of the first study clophosphamide has several end-
kaemias in women after treatment and a sex-adjusted incidence of 0.9 points indicative of genotoxic effects

56
in humans, including DNA damage as Conclusions not more research activity to follow
measured by the comet assay, muta- up other patient groups who receive

CHAPTER 6
PART 1
tions at the HPRT locus, and sister For the anticancer drugs cyclophos- anticancer agents, such as patients
chromatid exchange. Historically, cy- phamide, chlorambucil, melphalan, with autoimmune diseases. The cur-
clophosphamide has been included and thiotepa, the data summarized in rent potency data for four anticancer
this chapter show that the target sites drugs suggest that the TD50 values
in several genetic structure–activity
for which there is sufficient evidence for rats and mice are reasonably
studies (Vogel et al., 1996, 1998).
of carcinogenicity are generally sim- homogeneous and consistent. As a
Chlorambucil and melphalan are
ilar in rodents and humans, particu- carcinogen, cyclophosphamide was
direct-acting derivatives of nitrogen
larly for bladder cancer induced by the least potent and thiotepa was
mustard, and thiotepa is a direct-act- the most potent agent in any of the
cyclophosphamide.
ing trifunctional derivative of aziri- rodent models analysed. In humans,
Anticancer agents allow unique
dine. These compounds bind to DNA cyclophosphamide was the least
comparisons of carcinogenic poten-
and give a positive response in a potent and thiotepa and melphalan
cy among species, because the dos-
wide spectrum of assays for genomic were the most potent compounds to
es administered to humans and an-
injury, including tests for cytogenetic induce secondary cancers.
imals are known. Cancer treatment
damage, specifically as indicated by has become increasingly multimod- Acknowledgements
chromosomal aberrations and sis- al and involves the use of multiple
ter chromatid exchange in patients. drugs; this makes it difficult to single This research was supported by the
These drugs have also been in- out individual agents. Also, survival Deutsche Krebshilfe, the Swedish
cluded in several genetic structure– rates have risen and the probabili- Council for Working Life and Social
activity studies (Vogel et al., 1996, ty of detecting second tumours has Research, and the EU FP7 grant
1998). increased. It is unclear why there is number 260715.

Part 1 • Chapter 6. Anticancer agents: qualitative and quantitative aspects 57


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58
CHAPTER 7
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 7.

Polycyclic aromatic
hydrocarbons and associated
occupational exposures Charles William Jameson

Properties of PAHs this makes them more readily uptake or degradation, which means
available for biological uptake and that their persistence in the environ-
Most polycyclic aromatic hydrocar- degradation (Mackay and Callcott, ment is increased (Johnsen et al.,
bons (PAHs) with potential biolog- 1998; Choi et al., 2010). Furthermore, 2005; Haritash and Kaushik, 2009).
ical activity have been determined two- to four-ring PAHs volatilize The properties that influence the
to have a molecular structure that sufficiently to appear in the atmo- biological activity of PAHs include
ranges in size from two to six fused sphere predominantly in gaseous their vapour pressure, their adsorp-
aromatic rings (IARC, 2010). The form, although the physical state of tion on surfaces of solid carrier par-
physicochemical properties of four-ring PAHs can depend on tem- ticles, their absorption into liquid
these PAHs that are critical to their perature (Atkinson and Arey, 1994; carriers, their lipid–water partition
biological activity vary greatly, be- Srogi, 2007). coefficient in tissues, and their limits
cause their molecular weights cover In contrast, PAHs with five or of solubility in the lipid and aqueous
a vast range. more rings have low solubility in wa- phases of tissues. These properties
Aqueous solubility of PAHs de- ter and low volatility. They therefore are linked with the metabolic activa-
creases approximately logarith- occur predominantly in solid form, tion of PAHs, as well as their deposi-
mically with increasing molecular bound to particulates in polluted air, tion and disposition.
mass (Johnsen et al., 2005). Two- soil, or sediment (Choi et al., 2010). PAHs share a similar mecha-
ring PAHs, and, to a lesser extent, In the solid state, these compounds nism of carcinogenic action in both
three-ring PAHs, dissolve in water; are less accessible for biological humans and experimental animals.

Part 1 • Chapter 7. Polycyclic aromatic hydrocarbons and associated occupational exposures 59


This includes metabolic conversion PAH body burdens among exposed critical for determining the over-
to oxides and dihydrodiols, which in workers that are considerably higher all evaluation for each one (IARC,
turn are oxidized to diol epoxides. than those in the general population. 2010).
These oxides and diol epoxides are There is growing awareness that The remaining 45 PAHs reviewed
the ultimate DNA-reactive metabo- by IARC were acenaphthene, ace-
uptake of PAHs through the skin is
lites of PAHs. The oxides form stable pyrene (3,4-dihydrocyclopenta[cd]
substantial (Jongeneelen, 2001).
DNA adducts, and the diol epoxides pyrene), anthanthrene, anthracene,
Dermal uptake has been shown to
form stable adducts but also unsta- 11H-benz[bc]aceanthrylene, benz[l]
contribute to the internal exposure aceanthrylene, benzo[b]chrysene,
ble adducts (so-called depurinating
adducts) with DNA through formation of workers to PAHs; a study in the benzo[g]chrysene, benzo[a]fluoran-
of electrophilic carbonium ions. creosote industry found that the total thene, benzo[ghi]fluoranthene, benzo
internal dose of PAHs did not neces- [a]fluorene, benzo[b]fluorene, ben-
Occupational exposure to sarily correlate with levels of inhala- zo[c]fluorene, benzo[ghi]perylene,
PAHs benzo[e]pyrene, coronene, 4H-cyclo­-
tion exposure alone, and that dermal
penta[def]chrysene, 5,6-cyclopen-
exposure contributed significantly
Occupational exposure to PAHs oc- teno-1,2-benzanthracene, dibenz
curs predominantly through inhala- (Vanrooij et al., 1992).
[a,c]anthracene, dibenz[a,j]anthra-
tion and dermal contact. Industrial cene, dibenzo[a,e]fluoranthene,
Classification of PAHs
processes that involve the pyroly- 13H-dibenzo[a,g]fluorene, dibenzo
sis or combustion of coal and the [h,rst]pentaphene, dibenzo[a,e]py-
The IARC Monographs Programme
production and use of coal-derived rene, dibenzo[e,l]pyrene, 1,2-dihy-
products, including coal tar and has reviewed experimental data for
droaceanthrylene, 1,4-dimethylphen­
coal tar-derived products, are major 60 individual PAHs (IARC, 2010).
anthrene, fluoranthene, fluorene,
sources of occupational exposure to Of these 60 PAHs, one, benzo[a]
1-methylchrysene, 2-methylchry-
PAHs. Workers at coal-tar produc- pyrene, is classified as carcino- sene, 3-methylchrysene, 4-methyl­
tion plants, coking plants, bitumen genic to humans (Group 1). Other chrysene, 6-methylchrysene,
production plants, coal-gasification PAHs reviewed by IARC include 2-methyl­fluoranthene, 3-methylflu-
sites, smokehouses, aluminium pro- cyclopenta[cd]pyrene, dibenz[a,h] oranthene, 1-methylphenanthrene,
duction plants, coal-tarring facilities, anthracene, and dibenzo[a,l] naphtho[1,2-b]fluoranthene, naph-
and municipal waste incinerators are tho[2,1-a]fluoranthene, naphtho
pyrene, which are classified as
exposed to PAHs. Exposure may [2,3-e]pyrene, perylene, phenan-
probably carcinogenic to humans
also result from inhalation of engine threne, picene, pyrene, and triphen-
(Group 2A), and benz[ j]aceanthry-
exhaust and from use of products ylene. These compounds were de-
that contain PAHs in a variety of lene, benz[a]anthracene, benzo[b]
termined to be not classifiable as
other industries, such as mining, oil fluoranthene, benzo[ 
j]fluoranthene,
to their carcinogenicity to humans
refining, metalworking, chemical pro- benzo[k]fluoranthene, benzo[c] (Group 3), because of limited or in-
duction, transportation, and the elec- phen­anthre­ne, chrysene, dibenzo adequate experimental evidence
trical industry (Vanrooij et al., 1992). [a,h]pyrene, dibenzo[a,i]pyrene, in- (IARC, 2010).
Studies in Germany measured deno[1,2,3-cd]pyrene, and 5-methyl- As noted above, benzo[a]pyrene
concentrations of PAHs in the chrysene, which are classified as is the only PAH classified by IARC
breathing zone of chimney sweeps in Group 1. A review of the data
possibly carcinogenic to humans
during “black work”; the PAHs in available for this PAH indicates that
(Group 2B). It should be noted that in
the air samples varied depending the complete sequence of steps in
the evaluations of benz[ j]aceanthry-
on the type of fuel burned (oil, oil/ the metabolic activation pathway
lene, benzo[c]phenanthrene, benzo
solid, or solid) (Knecht et al., 1989). of benzo[a]pyrene to mutagenic
Concentrations of PAHs in coal-tar [a]pyrene, cyclopenta[cd]pyrene, and carcinogenic diol epoxides has
products may range from less than dibenzo[a,h]anthracene, and diben- been demonstrated in humans, in
1% to 70% or more (ATSDR, 2002). zo[a,l]pyrene, the mechanistic data human tissues, and in experimental
Occupational exposure can lead to available for these compounds were animals. After exposure, humans

60
Table 7.1. Group 1 agents associated with dermal exposures to polycyclic aromatic hydrocarbons (PAHs) that cause
squamous cell carcinoma of the skin in humans and in rodents

CHAPTER 7
PART 1
Agent Target organ

Humans Mice Rats

Benzo[a]pyrene No data Skin Skin


Chimney sweep soots Skin, including scrotum Skin No data
Coal tar Skin, including scrotum Skin No data
Coal-tar pitch Skin, including scrotum Skin No data
Mineral oils, untreated and mildly treated Skin, including scrotum Skin No data
Shale oils Skin, including scrotum Skin No data

metabolically activate benzo[a]py- mechanistic evidence from studies potential chemical interactions and
rene to benzo[a]pyrene diol epox- in exposed humans and in experi- the presence of other carcinogenic
ides that form DNA adducts. The mentally exposed animals, and from substances.
anti-benzo[a]pyrene-7,8-diol-9,10- in vitro studies in human and animal Certain occupations associated
oxide–deoxyguanosine adduct has with high exposure to PAHs have
tissues and cells, provides biologi-
been measured in populations (e.g. been classified by IARC as carci-
cal plausibility to support the overall
coke-oven workers and chimney nogenic to humans (Group 1); these
sweeps) exposed to PAH mixtures classification of benzo[a]pyrene as
include coal gasification, coke pro-
that contain benzo[a]pyrene. The carcinogenic to humans (Group 1) duction, coal-tar distillation, chimney
reactive anti-benzo[a]pyrene-7,8-di- (IARC, 2010, 2012). sweeping, paving and roofing with
ol-9,10-oxide induces mutations in coal-tar pitch, and work involving
rodent and human cells. Mutations Studies of cancer in humans mineral oils, shale-oil production,
(G  →  T transversions) in the K-ras and aluminium production. In most
proto-oncogene in lung tumours from There are no epidemiological stud-
cases the classification is based on
mice treated with benzo[a]pyrene are ies on human exposure to individu- epidemiological studies of increased
associated with anti-benzo[a]pyrene- al PAHs, because these chemicals cancer incidence without reference
7,8-diol-9,10-oxide–deoxyguanosine never occur in isolation in the envi- to supporting evidence from bio-
adducts. Similar mutations in the ronment but are present as compo- assays in experimental animals.
KRAS proto-oncogene and muta- The roles of individual PAHs in the
nents of complex chemical mixtures.
tions in the tumour suppressor gene genesis of cancer observed in these
PAHs are very widespread environ-
TP53 were found in lung tumours occupations could not be defined
mental contaminants, because they
from non-smokers exposed to PAH- (IARC, 2010).
rich products of coal combustion that are formed during incomplete com-
are known to contain benzo[a]pyrene bustion of materials such as coal, Tumour site concordance
(as well as many other PAHs). In an oil, gas, wood, or waste, or during
in vitro study, the codons in TP53 pyrolysis of other organic materials, There are six IARC Group 1 agents
that are most frequently mutated in such as tobacco. Data on the carci- that cause non-melanoma tumours
lung cancer in humans were shown nogenicity of PAHs to humans are of the skin (Rice, 2005). Five of
to be targets for DNA adduct forma- these are related to occupations
available primarily from studies in
tion and mutation induced by ben- where PAH exposures are high and
occupational settings where workers
zo[a]pyrene. The strong and exten- are believed to be the causative
sive experimental evidence for the are exposed to mixtures containing agents (Table 7.1). There is a precise
carcinogenicity of benzo[a]pyrene PAHs. It is difficult to ascertain the correlation between carcinogeni-
in many animal species, support- carcinogenicity of the component city to human skin and carcinogen-
ed by the consistent and coherent PAHs in these mixtures, because of icity to mouse skin for these five

Part 1 • Chapter 7. Polycyclic aromatic hydrocarbons and associated occupational exposures 61


Table 7.2. Group 1 carcinogens associated with inhalation exposures to polycyclic aromatic hydrocarbons (PAHs)
that cause lung cancer in humans and in rodents

Agent Target organ Route/target organ

Humans Mice Rats

Benzo[a]pyrene No data Intraperitoneal injection of and Intratracheal and intrapulmonary


oral exposure to soot extracts/ instillation of soot extracts/lung
lung
Chimney sweep soots Lung No data Intratracheal instillation of soot extracts/
lung
Coal-tar vapours from Lung Inhalation/lung Inhalation/lung
coke ovens
Soots and vapours from Lung, bladder No data No data
aluminium production

PAH-associated exposures when by an appropriate route (Table 7.2). abolically active cell types include
the complex mixtures isolated from Both mice and rats developed lung pulmonary macrophages as well as
the occupational environment are tumours after inhalation of coal-tar epithelial cells.
applied topically. vapours from coke ovens. Soot ex- Benzo[a]pyrene is the only PAH
In 1775, Pott made the pioneering tracts caused lung tumours in rats that has been classified by IARC as
observation that cancer of the scro- after intratracheal instillation. There carcinogenic to humans (Group 1).
tum in chimney sweeps was an occu- appears to be a good correlation be-
As indicated above, the basis for
pational disease resulting from direct tween lung cancer in humans and in
this classification is the extensive
contact with soot (Pott, 1775). All five rodents for these two mixtures when
knowledge of the mechanism of car-
established PAH-based chemical studied by an appropriate route in
cinogenic action of benzo[a]pyrene
carcinogens for human skin to which mice and rats. All these complex
exposures occur by direct dermal mixtures have genotoxic activity, in humans and experimental ani-
contact are complex mixtures: coal which is recognized to underlie their mals. None of the many remaining
tar, coal-tar pitch, untreated and carcinogenic activity in the lung. In PAHs shown to be carcinogens in
mildly treated mineral oils, shale oils, summary, many of the individual animals have been classified as an
and soots. Because these mixtures PAHs in these complex mixtures that IARC Group 1 carcinogen, most like-
contain PAHs, all have a genotoxic have been classified by IARC as ei- ly because much less mechanistic
component to their mode of action in ther probably carcinogenic or possi- information is available for these
rodents. Most of the individual PAHs bly carcinogenic to humans are also agents than for benzo[a]pyrene.
classified by IARC as either probably genotoxic and have been shown to These other PAHs are classified as
carcinogenic to humans (Group 2A) cause lung tumours in rodents when either probably carcinogenic to hu-
or possibly carcinogenic to humans administered by an appropriate route mans (Group 2A) or possibly carci-
(Group 2B) (listed above) are geno- (IARC, 2010).
nogenic to humans (Group 2B). Most
toxic and have been shown to cause The various tissue compartments
marked human occupational expo-
skin cancer and/or be initiators of of the respiratory tract are meta-
sure to these compounds involves
skin cancer in rodents (IARC, 1983, bolically active towards exogenous
complex mixtures that contain more
2010). chemicals in both humans and ex-
Soots and vapours from coke pro- perimental animals and are clear- than one of these PAHs and that of-
duction, aluminium production, and ly capable of transforming many ten contain other, non-PAH carcin-
related industries also cause lung metabolism-dependent chemicals, ogens. Therefore, the carcinogenic
cancer in humans, but only extracts including carcinogenic PAHs, to activity of these mixtures cannot
of soot and vapours from coke pro- their chemically reactive metabo- confidently be ascribed to any one of
duction have been tested in rodents lites (Rice, 2005). In the lung, met- their individual components.

62
References

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PART 1
Atkinson R, Arey J (1994). Atmospheric IARC (2010). Some non-heterocyclic Mackay D, Callcott D (1998). Partitioning
chemistry of gas-phase polycyclic aromatic polycyclic aromatic hydrocarbons and and physical chemical properties of PAHs.
hydrocarbons: formation of atmospheric some related exposures. IARC Monogr In: Neilson A, editor. PAHs and related
mutagens. Environ Health Perspect. Eval Carcinog Risks Hum. 92:1–853. compounds. The handbook of environmental
102(Suppl 4):117–26. http://dx.doi. Available from: http://publications.iarc.fr/110 chemistry, Volume 3. Berlin, Germany:
org/10.1289/ehp.94102s4117 PMID:7821285 PMID:21141735 Springer-Verlag; pp. 325–45. http://dx.doi.
org/10.1007/978-3-540-49697-7_8
ATSDR (2002). Toxicological profile for wood IARC (2012). Chemical agents and related
creosote, coal tar creosote, coal tar, coal occupations. IARC Monogr Eval Carcinog Pott P (1775). Chirurgical observations.
tar pitch, and coal tar pitch volatiles. Atlanta Risks Hum. 100F:1–599. Available from: London, United Kingdom: Hawes, Clarke &
(GA), USA: Agency for Toxic Substances and http://publications.iarc.fr/123 PMID:23189753 Collins; p. 63.
Disease Registry. Available from: http://www.
atsdr.cdc.gov/toxprofiles/tp85.pdf. Johnsen AR, Wick LY, Harms H (2005). Rice JM (2005). Human relevance of animal
Principles of microbial PAH-degradation neoplasms: site concordance between
Choi H, Harrison R, Komulainen H, Delgado in soil. Environ Pollut. 133(1):71–84. http:// humans and experimental animals for
Saborit JM (2010). Polycyclic aromatic dx.doi.org/10.1016/j.envpol. 20 0 4.0 4.015 cancers caused by exposures to chemical
hydrocarbons. In: WHO guidelines for indoor PMID:15327858 carcinogens, review prepared for the
air quality: selected pollutants. Geneva, International Programme on Chemical
Switzerland: World Health Organization. Jongeneelen FJ (2001). Benchmark guideline Safety. Geneva, Switzerland: World Health
Available from: http://www.ncbi.nlm.nih.gov/ for urinary 1-hydroxypyrene as biomarker of Organization.
books/NBK138709/. occupational exposure to polycyclic aromatic
hydrocarbons. Ann Occup Hyg. 45(1):3–13. Srogi K (2007). Monitoring of environmental
Haritash AK, Kaushik CP (2009). ht t p: //dx.doi.o rg /10.10 9 3 /annhyg /4 5.1. 3 exposure to polycyclic aromatic hydrocarbons:
Biodegradation aspects of polycyclic PMID:11137694 a review. Environ Chem Lett. 5(4):169–95.
aromatic hydrocarbons (PAHs): a review. http://dx.doi.org/10.1007/s10311-007-0095-0
J Hazard Mater. 169(1–3):1–15. http:// Knecht U, Bolm-Audorff U, Woitowitz HJ
dx.doi.org/10.1016/j.jhazmat.2009.03.137 (1989). Atmospheric concentrations of Vanrooij JG, Bodelier-Bade MM, De Looff AJ,
PMID:19442441 polycyclic aromatic hydrocarbons during Dijkmans AP, Jongeneelen FJ (1992). Dermal
chimney sweeping. Br J Ind Med. 46(7):479– exposure to polycyclic aromatic hydrocarbons
IARC (1983). Polynuclear aromatic 82. PMID:2765421 among primary aluminium workers. Med Lav.
compounds, Part 1, chemical, environmental 83(5):519–29. PMID:1297067
and experimental data. IARC Monogr Eval
Carcinog Risk Chem Hum. 32:1–453.
Available from: http://publications.iarc.fr/50
PMID:6586639

Part 1 • Chapter 7. Polycyclic aromatic hydrocarbons and associated occupational exposures 63


CHAPTER 8
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 8.

Benzene and
haematological cancers
Bernard D. Goldstein and Martyn T. Smith

Introduction disorders in humans, as distinct from logical evidence also strongly sup-
AML. Whereas epidemiological evi- ports this notion (Bassig et al., 2015;
Haematological cancers present an dence establishes that benzene is a Linet et al., 2015).
example of seeming discordance cause of human AML (IARC, 1987),
between epidemiological data and
long-term studies in experimental Studies in experimental
animal data, which is apparently re- animals
animals exposed to benzene gener-
solved by mechanistic information.
Known causes of haematological ally have not indicated an increased In 1974, IARC concluded that studies
cancers in humans are ionizing ra- risk of AML. In contrast, increased in experimental animals did not show
diation, chemotherapeutic agents, incidence of lymphoma has been evidence of carcinogenesis from
infectious agents such as human readily evident in such studies, but exposure to benzene (IARC, 1974).
immunodeficiency virus (HIV), and the corresponding epidemiological Subsequently, results from two long-
occupational exposures to chemical evidence can be debated. However, term studies of experimental animals
agents such as formaldehyde and
recent mechanistic data, as well as exposed to benzene that did report
benzene. These agents are recog-
information that has led to a reclas- cancer were published by a research
nized as causes of acute myeloid
leukaemia (AML), and all have been sification of haematological neo- group at New York University (NYU)
implicated in the causation of other plasms, are consistent with benzene in the USA (Snyder et al., 1980, cit-
forms of haematological cancers. being recognized as a cause of hu- ed in IARC, 1982), who exposed
This chapter focuses on the issue man lymphoproliferative disorders C57BL/6J and AKR/J mice to ben-
of whether benzene can be consid- (Smith et al., 2007, Goldstein, 2010; zene by inhalation, and by Maltoni
ered to cause lymphoproliferative Smith, 2010). Recent epidemio- et al. (1989) in Bologna, Italy, who

65
Part 1 • Chapter 8. Benzene and haematological cancers
exposed Sprague-Dawley rats by munity long before cohort-based NHL and other lymphoprolifera-
gavage. Both studies reported an epidemiological studies provided tive disorders (Bassig et al., 2015;
increased incidence of lymphoma unequivocal evidence. Despite sev- Linet et al., 2015).
as well as a variety of other tumours. eral studies evaluating AML in shoe
There are now at least seven stud- Changing diagnostic criteria
workers in Italy (Vigliani and Saita,
ies, including long-term animal bio- for haematological disorders
1964) and leather workers in Turkey
assays by the United States National
Toxicology Program and by others, (Aksoy, 1989), the relationship be- The classification of haematologi-
showing an increased incidence tween benzene and AML required cal disorders continues to evolve,
of lymphomas of various types a conventional cohort study in a including relatively recent modifica-
(Huff et al., 1989; Farris et al., 1993; well-defined worker population tions that have major implications
Huff, 2007; National Toxicology (Infante et al., 1977) before causality for studying and understanding
Program, 2007; Kawasaki et al., was fully accepted. causality. These reclassifications are
2009). Numerous epidemiological stud- largely based on advances in under-
In contrast, evidence that AML re- standing the pathological and molec-
ies evaluating the relationship be-
sults from exposure to benzene has ular basis for haematological diseas-
tween exposure to benzene and
been difficult to obtain in experimen- es, and on the development of assays
different forms of lymphoma have
tal animals, and has generally been
that permit differentiation among the
lacking in the same studies that read- yielded inconsistent results. Some
various haematological cell types.
ily showed an increase in the inci- studies suggest a relationship (e.g.
Myeloid leukaemias are no longer
dence of lymphatic cancers. In other Arnetz et al., 1991; Hayes et al.,
divided simply into AML and chron-
NYU studies, 3 of 40 CD1 mice were 1997; Fabbro-Peray et al., 2001;
ic myeloid leukaemia (CML). The
found to have myeloproliferative dis- Kristensen et al., 2008; Wong et al.,
French–American–British classifi-
orders, and 1 of 40 Sprague-Dawley 2010), whereas others do not (e.g.
rats was found to have AML; the re- cation has eight distinct subtypes of
Raabe et al., 1998; Divine et al., AML (Bennett et al., 1989). The more
sults are notable only because these
1999; Bloemen et al., 2004). recent World Health Organization
disorders are rare in both species
Recent meta-analyses suggest an (WHO) classification makes greater
(Goldstein et al., 1982). The NYU
group also noted an 8-fold increase association of exposure to benzene use of cytogenetic findings to sub-
in the number of early precursor cells with NHL, multiple myeloma, chron- classify AML (Swerdlow et al., 2008).
in the bone marrow of CD1 mice ex- ic lymphocytic leukaemia (CLL), and Myelodysplasia has also been sub-
posed to benzene (Snyder et al., acute lymphoblastic leukaemia (ALL) divided into various types, enabling
1981). An increase in the number of (Infante, 2006; Smith et al., 2007; recognition of the different pathways
haematopoietic progenitor cells was Steinmaus et al., 2008; Vlaanderen and rates of transformation to AML
also noted by Cronkite et al. (1989), et al., 2011). However, at the Working (Bennett et al., 1989; Swerdlow et al.,
who also observed AML in CBA/Ca 2008).
Group meeting for Volume 100F
mice exposed to benzene. More re- Similarly, the newer approaches
of the IARC Monographs, the epi-
cent inhalation studies by Kawasaki to classifying NHL have divided this
et al. (2009) found an increase in demiological evidence relating ex-
entity into more than 40 subtypes,
the incidence of AML in leukae- posure to benzene to NHL or mul-
while at the same time moving pre-
mia-prone C3H/He mice exposed tiple myeloma was not deemed to
viously separate diseases into this
to benzene, particularly if they were establish causality. As discussed
general classification. NHL now
Trp53-deficient. Thymic lymphomas below, recent changes in diagnostic
includes CLL, ALL, and multiple
and non-Hodgkin lymphoma (NHL) criteria complicate epidemiological
myeloma, as well as the multitude
were also noted. evaluation of lymphoproliferative dis- of lymphocytic disorders that were
orders, as does the recognition that formerly included within the NHL
Epidemiological studies
the criteria will continue to evolve. diagnostic rubric (Swerdlow et al.,
The causal relationship between Recently published epidemiological 2008). It should be noted that both
exposure to benzene and AML studies also conclude that expo- CLL and ALL are no longer stand-
was accepted by the medical com- sure to benzene is associated with alone diagnoses (Swerdlow et al.,

66
2008). This is not surprising, be- its effects through metabolism to one gens to give additional emphasis to
cause clinicians have long known or more haematotoxic metabolites. the role of mechanistic understand-

CHAPTER 8
PART 1
that there was little distinction in clin- Like other well-established causes ing. IARC now allows a chemical
ical course, prognosis, and response of AML, including ionizing radiation to be classified as carcinogenic to
to chemotherapy between CLL and and chemotherapeutic agents, ben- humans (Group  1) “when evidence
lymphomas with similar mature lym- zene has the ability to cause aplas- of carcinogenicity in humans is less
phocytes involving other organs tic anaemia at high doses and a than sufficient but there is sufficient
but not blood, or between ALL and pancytopenic effect at lower doses, evidence of carcinogenicity in exper-
more aggressive lymphomas with indicating toxicity to the pluripotent imental animals and strong evidence
similar primitive lymphocytes in- haematopoietic stem cell population in exposed humans that the agent
volving other organs but not blood. responsible for the production of red acts through a relevant mechanism
However, the ability to use molecular blood cells, white blood cells, and of carcinogenicity” (IARC, 2006).
markers to build on this clinical in- platelets. In the case of benzene and
sight permits reclassification. Lymphocytes are affected by ben- lymphoproliferative cancers, there
zene, as is evident from a decreased is clearly sufficient evidence in ex-
Evidence for an overlap lymphocyte count in exposed work- perimental animals and strong
between myeloid and evidence in humans of a relevant
ers (Goldstein, 1988; Rothman et al.,
lymphoid cancers in humans
1996), and from the severe loss of mechanism of carcinogenicity. This
function of lymphatic organs in ex- evidence includes the recognition
There are two lines of evidence
perimental animals as a result of that the stem cell known to be at
that myeloid and lymphoid haema-
tological cancers are closely relat- longer-term high-level exposure. risk for benzene-induced AML is
ed: (i) evidence that has led to the Particularly pertinent to the mech- also responsible for lymphoprolifer-
general acceptance that there is a anistic considerations with respect ation, the well-known vulnerability
common haematological stem cell, to whether benzene is a cause of of human lymphocytes to benzene
and (ii) clinical evidence based on lymphatic tumours is the finding in toxicity, and the demonstration of
the use of newer biological markers many studies of genotoxic effects in increased chromosomal abnormali-
that show the overlap between the circulating lymphocytes of exposed ties in the circulating lymphocytes of
two types, which were previously humans or experimental animals workers exposed to benzene. It also
considered to be separate. The clin- (Vigliani and Forni, 1969; Forni, includes the promiscuous DNA dam-
ical evidence includes recognition of 1979; Zhang et al., 1996, 2002, age caused by benzene metabolites
the following: perhaps 10% of new 2005, 2011; Navasumrit et al., 2005). and the observation of aberrations in
cases of acute leukaemia have both multiple chromosomes among work-
These effects are seen in bone mar-
lymphoblastic and myeloid char- ers who have been heavily exposed
row precursor cells and in circulat-
acteristics; leukaemia that occurs to benzene or who have haematolog-
ing lymphocytes and include overt
in individuals with Down syndrome ical cancers attributable to benzene.
chromosomal abnormalities, trans-
can be lymphoid or myeloid, as can These mechanistic findings bridging
locations, deletions, and aneuploidy
leukaemia resulting from chemo- lymphoproliferative and myelopro-
of several different chromosomes
therapy for cancer; and blast trans-
(Zhang et al., 2007, 2011, 2012). The liferative cancers appear to explain
formation of CML can be lymphoid
evidence is consistent with exposure the seeming lack of congruence
rather than the more usual myeloid
to benzene potentially affecting mul- between epidemiological data and
(Gassmann et al., 1997; Calabretta
tiple sites within the genetic material animal data for myeloid and lymphoid
and Perrotti, 2004; Lee et al., 2009;
of pluripotent stem cells. cancers observed with benzene, and
Xavier et al., 2009).
with other agents.
Mechanism of benzene- Conclusions
induced haematotoxicity
Both IARC and the United States
Benzene, which has been a known National Toxicology Program have
cause of human bone marrow toxic- in recent years changed their ap-
ity since the 19th century, produces proach to classification of carcino-

Part 1 • Chapter 8. Benzene and haematological cancers 67


References
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Part 1 • Chapter 8. Benzene and haematological cancers 69


CHAPTER 9
PART 1
part 1.
concordance between cancer in humans and in experimental
animals

chapter 9.

Human tumour viruses


Paul F. Lambert and Lawrence Banks

Among the biological agents re- because species specificity limits the causes lymphoproliferative diseas-
viewed in Volume 100B of the IARC feasibility of this approach for most of es in New World monkeys and in
Monographs (IARC, 2012) are sever- these viruses. One exception is hu- humanized SCID mice, the use of
al oncogenic viruses that are strictly man T-cell lymphotropic virus type 1 surrogate hosts has not proven very
species-specific, causing cancer in (HTLV-1): in addition to its ability to useful for defining tumour site con-
humans only. For this reason, the infect humans, this virus can infect cordance between humans and ex-
question about tumour site concor- several other species – including perimental animals.
dance between humans and experi- rabbits, rats, and monkeys – and it Animal models for human tumour
mental animals is not easy to answer does induce adult T-cell leukaemia/ viruses that make use of animal virus-
for these agents, because cancer bi- lymphoma (ATLL), albeit in monkeys es are scarce. In fact, although many
oassays in animals are often lacking, only. animal viruses that infect non-human
and hence a proper comparison be- For other human tumour virus- primate species are related to the hu-
tween data in humans and in experi- es, the use of humanized severe man tumour viruses, the incidence of
mental animals is not obvious. In this combined immunodeficiency (SCID) cancer is low in these species (as it
chapter, some aspects of this issue mice, in which the human target is in humans), which renders cancer
are discussed. cell for the virus is placed in a mu- studies costly and difficult. Moreover,
The use of animals as surrogate rine host, can provide a platform for animal models for tumour viruses in
hosts for the study of human tu- in vivo infection. However, except non-primate species often do not
mour viruses is often problematic, for Epstein–Barr virus (EBV), which accurately reflect the mechanism of

Part 1 • Chapter 9. Human tumour viruses 71


the disease caused by the cognate cells from human cord blood. These in vitro, and their expression in these
human tumour virus. For instance, mice are able to reconstitute most lymphomas in monkeys and humans
woodchuck hepatitis virus induces major components of the human provides strong support for a direct
hepatocellular carcinoma (HCC) haematolymphoid system including oncogenic role of EBV in vivo.
that is histopathologically very simi- T cells, B cells, natural killer cells, LMP1 of EBV can transform ro-
lar to that caused by hepatitis B vi- macrophages, and dendritic cells, dent fibroblasts and is expressed
rus (HBV) in humans, but it does so and this humanized mouse model in most of the human cancers as-
through a different mechanism. can simulate key aspects of EBV in- sociated with EBV infection. Three
Transgenic mouse models pro- fection. Inoculation with a high dose strains of LMP1 transgenic mice
vide a powerful tool in mechanistic of EBV caused a B-cell lymphopro- were established that express LMP1
studies on the role of individual viral liferative disorder in these mice, under the control of the immunoglob-
genes in cancer. Indeed, for sever- with histopathological findings and ulin heavy chain promoter/enhancer.
al of the human tumour viruses de- latent EBV gene expression similar Mice of all three strains developed
scribed in Volume 100B of the IARC to those in immunocompromised pa- lymphoma, the incidence of which
Monographs, transgenic mouse tients. Inoculation with a low dose of increased considerably with age: af-
studies provide important mechanis- EBV resulted in apparently asymp- ter 18 months, 42% of the transgen-
tic evidence. However, such trans- tomatic persistent infection. The ic mice had developed lymphoma.
genic models are inadequate for number of activated CD8-positive T LMP1 was strongly expressed in the
understanding the cancer etiology in cells increased considerably in the lymphoma tissues but was hardly
the context of natural viral infection. peripheral blood of the infected mice, expressed in normal lymphoid tis-
For several of the human tu- and various assays demonstrated an sues. These results show that LMP1,
mour viruses classified by IARC in EBV-specific T-cell response. In ad- without the expression of other EBV
Group 1 (carcinogenic to humans), dition, immunoglobulin M (IgM) anti- genes, is oncogenic in vivo, and indi-
a number of studies with surrogate bodies specific to the EBV-encoded cate that the LMP1 protein is a major
hosts, with cognate animal viruses, protein BFRF3 were detected in se- contributing factor to the develop-
and with transgenic mouse models rum from infected animals (Yajima ment of EBV-associated lymphomas
are reviewed below. et al., 2008). (Kulwichit et al., 1998).
Inoculation of cotton-top tama- The role of LMP1 was also stud-
Epstein–Barr virus (EBV) rins – a type of New World monkey ied in the epidermis of LMP1 trans-
– with EBV induced multiple EBV genic mice. Epidermal cells that
Human peripheral blood leukocytes genome-positive malignant large- carried the transgene showed a
injected into SCID mice increase cell lymphomas that closely resem- 2–3-fold increase in the mitotic in-
in number and survive for at least bled the EBV genome-positive B-cell dex and an increased expression
6 months. These mice secrete hu- lymphomas observed in human allo- of proliferative cytokeratin markers.
man immunoglobulin and show a graft recipients (Young et al., 1989a). This is direct evidence that LMP1
specific human antibody response The tumours in tamarins expressed induces proliferation in otherwise
after immunization. The major cell EBV nuclear antigen 1 (EBNA1) and normal epithelial cells in vivo. When
populations present in peripheral EBNA2, EBNA leader protein, and treated topically with 7,12-dimethyl­
blood leukocytes are found in the the latent membrane protein (LMP). benz[a]anthracene, LMP1 transgen-
lymphoid tissue and blood of the The expression of EBNA2 and ic mice developed small papillomas
SCID mice recipients, although rel- LMP in these lymphomas in tama- more rapidly and in larger numbers
ative proportions may differ. Mice rins strengthens their resemblance than did non-transgenic controls.
injected with peripheral blood leuko- to lymphomas observed in human Furthermore, LMP1 could replace
cytes from EBV-seropositive donors transplant recipients, because 12-O-tetradecanoylphorbol-13-ace­
often develop EBV-positive B-cell these tumours in humans are also tate in promoting tumour formation,
lymphomas (Mosier et al., 1988). EBNA2-positive and LMP-positive but it inhibited expansion and did not
In a later study, a highly immuno- (Young et al., 1989b). Both proteins stimulate progression of the pap-
deficient mouse strain (NOG) was are important effector molecules of illomas to carcinomas, or to more
injected with haematopoietic stem EBV-induced B-cell transformation malignant spindle cell carcinomas.

72
These data show that early in the MYC, LMP2A accelerates the de- ic neoplasms and a higher incidence
carcinogenic process, LMP1 acts as velopment of B-cell lymphomas in of HCCs. No hepatocellular adeno-

CHAPTER 9
PART 1
a tumour promoter after chemical ini- a transgenic mouse model (Bieging mas or HCCs were seen in non-in-
tiation, but it may also block expan- et al., 2009; Bultema et al., 2009). A fected woodchucks that received
sion or progression of benign lesions more recent study has also shown AFB1 (but pre-neoplastic hepatic foci
(Curran et al., 2001). that LMP1 and LMP2 can cooperate were seen), and no pre-neoplastic or
A further study on LMP1 trans- in the induction of epithelial squa- neoplastic lesions were found in un-
genic mice showed that they have mous cell carcinomas (SCCs) when treated controls. These results pro-
a higher incidence of lymphoma co-expressed under the control of a vide strong evidence of a synergistic
and that the progression to lympho- keratin 14 (K14) promoter in trans- hepatocarcinogenic effect of viral
ma correlates with higher expres- genic mouse models (Shair et al., infection and dietary AFB1 intake
sion levels of LMP1, compared with 2012). (Bannasch et al., 1995).
non-transgenic controls. Although Establishment of long-term latent Transgenic mice that contain the
LMP1 is expressed in all B lympho- infections with EBV was possible in BglIIA fragment of HBV under the
cytes of the transgenic mice, lym- a humanized mouse model that was transcriptional control of the mouse
phoma develops in a specific sub- challenged either with wild-type EBV albumin promoter overexpress the
set, the B-1a lymphocytes, which is or with a replication-defective virus. HBV large envelope polypeptide
a population predisposed to clonal B-cell lymphomas developed in both and accumulate toxic quantities of
expansion with age. The malignant cases, but at a higher frequency after hepatitis B surface antigen (HBsAg)
lymphocytes show constitutively infection with the wild-type virus, in- in their hepatocytes. As a result,
active Stat3 signalling, have de- dicating a potential role for lytic virus the mice develop severe, prolonged
creased levels of p27, and display infection in the development of ma- hepatocellular injury associated with
activated Akt and nuclear factor kap- lignancy (Ma et al., 2011). an inflammatory response, followed
pa-light-chain-enhancer of activated For EBV, the overall concordance by chronic hepatocellular regener-
B cells (NF-κB) pathways, properties between the animal models and hu- ation, transcriptional deregulation,
that are associated with promoting mans with respect to the types of tu- dysplasia, aneuploidy, hepatocellular
growth and survival of B lympho- mour and the identity and function of adenoma, and eventually HCC. The
cytes. The transgenic lymphomas the major oncogenes and oncogene incidence of HCC depends on the
mirror multiple aspects of EBV- products is high. frequency, severity, and age of onset
induced tumours; this suggests that of the liver cell injury, which itself de-
these properties of LMP1 are major Hepatitis B virus (HBV) pends on the intrahepatic concentra-
factors in cancer development (Shair tion of HBsAg and is influenced by
et al., 2007). Interactive viral–chemical hepato- genetic background and sex. Thus,
Transcripts of LMP2A can be ex- carcinogenesis was studied in wood- the excessive expression of a sin-
pressed in resting virus-carrying B chucks (Marmota monax) inoculated gle structural viral gene is sufficient
lymphocytes in healthy individuals as newborns with woodchuck hep- to cause malignant transformation
– the reservoir of persistently latent atitis virus, which is closely related in this model. Similar events may
EBV. In LMP2A transgenic mice, to the human HBV. Starting at age be responsible for the development
a block in surface immunoglobulin 12 months, the woodchucks were of HCC in humans after HBV infec-
rearrangement results in the gen- fed a diet containing aflatoxin B1 tion, irrespective of the mechanism
eration of B-cell receptor-negative (AFB1) (at a high dose for 4 months or mechanisms involved in the initial
cells, which normally would under- and then at a lower dose for lifetime). induction of liver cell injury (Chisari
go apoptosis. LMP2A transgenic B Carriers of woodchuck hepatitis vi- et al., 1989).
cells develop and survive without a rus with or without treatment with the Transgenic mice overexpressing
B-cell receptor. These data indicate carcinogen AFB1 developed a high the HBV large envelope polypeptide
that LMP2A imparts developmental incidence of pre-neoplastic hepatic suffer from hepatic injury as a re-
and survival signals to B cells in vivo foci, hepatocellular adenomas, and sult of accumulation of HBsAg (see
(Merchant et al., 2000). Furthermore, HCCs, but AFB1 treatment resulted in the previous study). When treated
when co-expressed with human a much earlier appearance of hepat- with the hepatocarcinogens AFB1

Part 1 • Chapter 9. Human tumour viruses 73


or diethylnitrosamine, these mice cooperation between HBx and K-ras in liver tissue homogenates as the
showed more rapid and more exten- mutation in the development of HCC non-transgenic controls. In contrast,
sive formation of nodules, prolifera- in transgenic mice (e.g. Ye et al., the level of phosphatidylcholine hy-
tion of oval cells, and development of 2014). A truncated form of HBx that droperoxides was increased nearly
adenomas and primary HCCs than is commonly found in human HCCs 2-fold in transgenic mice after age
did non-exposed transgenic mice. also exhibits tumour-forming ac- 16 months. In addition, catalase
This suggests that the chronic liver tivity in association with exposure activity was increased and the con-
damage and repair caused by over- to diethylnitrosamine in transgen- centrations of total and reduced
expression of the HBV large enve- ic mouse models of HCC (Quetier glutathione were decreased. These
lope polypeptide act synergistically et al., 2015). mice show steatosis without inflam-
with chemical hepatocarcinogens to For HBV, the overall concordance mation early in life, and finally devel-
produce liver neoplasia (Sell et al., between the animal models and hu- op HCC from age 16 months. The
1991). mans with respect to the types of tu- HCV core protein thus alters the ox-
To explain the synergistic hepato- mour is high. idant–antioxidant status in the liver
carcinogenic effect of viral infection in the absence of inflammation and
and dietary AFB1 intake, it was sug- Hepatitis C virus (HCV) may thereby contribute to or facilitate
gested that infection with HBV and Role of the HCV core protein the development of HCC after HCV
associated liver injury might alter the in HCV-induced steatosis infection (Moriya et al., 2001).
expression of carcinogen-metaboliz-
ing enzymes. This hypothesis was Several histological features in the Transgenic mice carrying the
tested in the HBV transgenic mouse liver are characteristic of chronic hep- complete HCV polyprotein
model described in the previous atitis C: bile duct damage, lymphoid
study. In these mice, the expres- follicles, and steatosis (fatty change). In a study to determine whether
It has been suggested that the core expression of HCV proteins alters
sion levels of the cytochrome P450
hepatic morphology or function in
isozymes Cyp2a5 and Cyp3a – both protein of HCV functions as a tran-
the absence of inflammation, trans-
involved in AFB1 metabolism – were scriptional regulator that induces
genic C57BL/6 mice carrying the
examined. Increases in the expres- phenotypic changes in hepatocytes.
complete viral polyprotein (FL-N
sion of and alterations in the distribu- Two independent strains of trans-
transgene) or viral structural proteins
tion of Cyp2a5 were age-dependent genic mice carrying the HCV core
(S-N transgene) were compared with
in these mice and were associated gene developed progressive hepatic
non-transgenic littermates for al-
with the extent of liver injury. Cyp3a steatosis, confirming that the core
tered liver morphology and function.
expression was also increased, but protein plays a direct role in the de-
No inflammation was seen in the liv-
this was less clearly related to age. velopment of steatosis, which char-
ers of transgenic mice, but mice ex-
These data show that expression of acterizes hepatitis C. These mice
pressing either transgene developed
specific cytochrome P450 isozymes express the core protein in the liver
age-related hepatic steatosis. The
is altered in association with over- at concentrations similar to those in numbers of apoptotic or proliferat-
expression of the HBV large enve- the liver of patients with chronic hep- ing hepatocytes were not increased
lope polypeptide and the ensuing atitis C. This transgenic mouse sys- significantly. Hepatocellular adeno-
liver injury in this mouse model. This tem may be a good animal model for ma or HCC developed in older male
may have general relevance to hu- the study of pathogenesis in human mice expressing the FL-N or the S-N
man HCC, the etiology of which is HCV infection (Moriya et al., 1997). transgene, but the incidence was
associated with a diverse range of In a further study by the same increased significantly only in FL-N
liver-damaging agents (Kirby et al., group, several parameters of oxida- transgenic mice. Neither of these tu-
1994). tive stress and redox homeostasis mours was observed in age-matched
The HBV X protein (HBx) is highly were measured in these transgen- non-transgenic mice. Expression of
multifunctional. In transgenic mouse ic mice. At age 3–12 months, the viral proteins gave rise to common
models, the expression of HBx pro- mice showed similar concentrations pathological features of hepatitis C
motes HCC (Yu et al., 1999). More of phosphatidylcholine hydroperox- in the absence of a specific antiviral
recent studies have also shown ides and phosphatidylethanolamine immune response, which suggests a

74
metabolic or genetic host suscepti- breakdown of mitochondrial out- This issue may be resolved with the
bility for HCV-associated HCC (Lerat er membranes. HCC developed in development of more physiological

CHAPTER 9
PART 1
et al., 2002). about 35% of Pparα+/+:HCVcp mice models that permit chronic and pro-
In a subsequent study by the by age 24 months, but no tumours ductive HCV replication. A recently
same group, the mechanisms un- were seen in the other genotypes. developed model holds promise in
derlying HCV-induced defects in These phenomena were closely this regard. Genetically engineered
lipid metabolism were studied in associated with sustained PPARα mice expressing two host restriction
transgenic mice that expressed the activation: in Pparα+/–:HCVcp mice, factors – human CD81 and occludin
full viral protein repertoire at lev- PPARα activation and the related – can be infected with HCV, and in
els corresponding to those seen changes did not occur despite the these mice sustained viraemia and
in natural human HCV infection. presence of a functional Pparα al- infectivity can be observed for more
Expression of the full-length HCV lele. Persistent activation of PPARα than 12 months after infection, with
open reading frame was associ- is essential for the pathogenesis of the expected fibrotic and cirrhotic
ated with hepatocellular steatosis, hepatic steatosis and HCC induced progression in the liver (Chen et al.,
impaired triglyceride secretion, and by HCV infection (Tanaka et al., 2014).
nuclear translocation of sterol reg- 2008).
ulatory element-binding protein 1c Kaposi sarcoma-associated
(SREBP1c), followed by increased Conclusions herpesvirus (KSHV)
transcription of lipogenic enzymes. Species specificity of KSHV
Stress markers in the endoplasmic In the absence of animal models
reticulum were expressed at similar that develop HCC in the context of After injection, KSHV can infect
levels in HCV transgenic mice and in an HCV infection, various groups non-human primates (Renne et al.,
non-transgenic controls. Transgenic have reported the use of transgenic 2004), NOD/SCID mice (Parsons
mice expressing the full-length HCV mouse models. Studies with mice et al., 2006), and humanized SCID
polyprotein have reduced plasma expressing HCV replicons, poly- mice (Dittmer et al., 1999; Foreman
triglyceride concentrations and de- proteins, or single HCV proteins as et al., 2001; Wu et al., 2006; Wang
velop hepatocellular steatosis in the transgenes, alone or in combination, et al., 2014). These infections do not
same way as do patients with HCV under the control of liver-specific result in the formation of tumours,
infection (Lerat et al., 2009). promoters have been described by but they confirm the viral tropism
several groups (Dorner et al., 2011, (with KSHV targeting B cells and en-
Effect of peroxisome 2013). There is good concordance dothelial cells) and drug susceptibili-
proliferator-activated
between the outcomes observed in ty (to ganciclovir) in vivo.
receptors
these mice and those in humans with In one report of KSHV infection
Peroxisome proliferator-activated HCV infection; however, questions in marmosets (Callithrix jacchus),
receptor alpha (PPARα) is a central remain about to what extent the re- viral replication was apparent in pe-
regulator of triglyceride homeosta- sults obtained with these experimen- ripheral blood mononuclear cells,
sis and a mediator of hepatocar- tal approaches reflect the patholog- and a Kaposi sarcoma-like lesion
cinogenesis in rodents. In a study to ical consequences of human HCV developed on one of the animals
determine the role of PPARα in HCV infection that contribute to HCC. (Chang et al., 2009). Viruses that
core protein-induced disease, dou- It remains a matter of consider- are homologous to KSHV exist in
ble transgenic mice were generated able debate whether HCV causes the bank vole (Myodes glareolus),
that carried Pparα (homozygous, HCC through a direct mechanism in as Murid herpesvirus 68 (MHV-68),
heterozygous, and null) and the which virally encoded genes contrib- and in virtually all non-human pri-
HCV core protein gene (HCVcp) as ute to carcinogenesis, or through an mates (Fleckenstein and Ensser,
transgenes. Severe steatosis was indirect mechanism, where the inju- 2007). The infection of macaques
observed only in Pparα+/+:HCVcp ry to the liver caused by HCV infec- (Macaca mulatta) with rhesus rhad-
mice, as a result of higher fat- tion and host immune responses to inovirus in the context of simian im-
ty acid uptake and decreased that infection, such as inflammation, munodeficiency virus (SIV) induces
mitochondrial β-oxidation due to contribute to the onset of cancer. B-cell lymphoma and endothelial cell

Part 1 • Chapter 9. Human tumour viruses 75


hyperplasia (Mansfield et al., 1999; which were plasmacytoma, follicular activity in non-malignant lympho-
Wong et al., 1999). Several tumour B-cell lymphoma, small lymphocytic cytes of K1 transgenic mice and
graft models of Kaposi sarcoma and lymphoma, and composite lympho- the persistence of this activity in the
primary effusion lymphoma have ma. These results imply that LANA lymphoma tumours that these mice
been established (Boshoff et al., can activate B cells and trigger the develop suggest that the KSHV K1
1998; Staudt et al., 2004; Wu et al., first step towards lymphomagenesis transgenic mouse may be a mod-
2005; An et al., 2006; Mutlu et al., (Fakhari et al., 2006). The authors of el of premalignancy (Prakash et al.,
2007; Sin et al., 2007). the study speculated that in asymp- 2002).
In humans, KSHV is associated tomatic human carriers, infected
K cyclin
with Kaposi sarcoma and primary ef- B cells remain organ-resident and
fusion lymphoma. The transforming sustain latent persistence of KSHV; KSHV encodes a cyclin D homo-
capacity of several individual KSHV this would be consistent with the low logue, K cyclin, which is thought to
proteins with respect to these two frequency of KSHV genome-positive promote viral oncogenesis. In cul-
tumour types has been studied in cells in peripheral blood lympho- tured cells, expression of K cyclin not
experimental systems, particularly in cytes of LANA-seropositive individu- only triggers cell-cycle progression
transgenic mice. als (Whitby et al., 1995). but also engages the p53 tumour
Studies in transgenic mice vFLIP suppressor pathway, which probably
restricts the oncogenic potential of K
Transgenic mice in which individual The KSHV-encoded viral Fas- cyclin (Verschuren et al., 2002). The
KSHV proteins are expressed are of- associated death domain-like IL-1- tumorigenic properties of K cyclin
ten used to replicate aspects of the converting enzyme inhibitory protein were assessed in transgenic mice in
pathogenesis of KSHV. However, (vFLIP) is expressed in latently in- which expression of K cyclin was tar-
single transgenic models have some fected cells and plays an important geted to B and T lymphocytes via the
limitations. Whereas lymphoprolif- role in survival and proliferation of Eμ promoter/enhancer. About 17%
erative lesions and lymphomas in primary effusion lymphoma cells. of K cyclin transgenic mice had de-
mice are easily classified on the One function ascribed to this protein veloped lymphoma by age 9 months,
basis of histology and marker gene is activation of NF-κB. Transgenic and all lymphomas had lost p53
expression, this is more difficult for mice expressing vFLIP display con- activity. The critical role of p53 in
endothelial cell tumours, which are stitutive activation of NF-κB path- suppressing K cyclin-induced lym-
often referred to as Kaposi sarco- ways, show an enhanced response phomagenesis was confirmed by the
ma-like lesions but can easily be to mitogenic stimuli, and have an greatly accelerated onset of B and
mistaken for fibrosarcomas. increased incidence of lymphoma. T lymphomagenesis in all K cyclin/
These results demonstrate that the p53 –/– mice compared with K cyclin/
LANA
KSHV-encoded vFLIP is an onco- p53+/– and K cyclin/p53+/+ mice, but
The KSHV latency-associated nu- protein that could contribute to the suppression of apoptosis does not
clear antigen (LANA) is consistently development of lymphoproliferative appear to be the underlying mecha-
expressed in all KSHV-associated disorders via constitutive NF-κB ac- nism, given the very high numbers of
tumour cells and was shown to bind tivation (Chugh et al., 2005). apoptotic cells observed in all Eμ-K
the tumour suppressor proteins cyclin/p53 –/– thymic lymphomas
K1 protein
p53 and pRb. The contribution of (Verschuren et al., 2004).
this antigen to lymphomagenesis in The K1 protein of KSHV is a trans-
vGPCR
vivo was investigated in transgenic membrane signalling protein. In
mice that expressed LANA under transgenic mice that express the In transgenic mice, the expression of
the control of its own B-cell-specific KSHV K1 gene under the control of viral G protein-coupled receptor (vG-
promoter. All of the mice developed the simian virus 40 (SV40) promoter, PCR) by cells of endothelial origin
splenic follicular hyperplasia due to tumours were observed that showed triggers the development of an an-
an expansion of IgM-positive, IgD- features of spindle cell (sarcomatoid) gioproliferative disease that resem-
positive B cells, and 11% developed cancers and malignant plasmablas- bles Kaposi sarcoma. It includes ex-
different types of lymphoma, among tic lymphoma. The enhanced NF-κB pression of angiogenic factors such

76
as placental growth factor, platelet- various cell lines. In particular, HPV transgenic mice treated with estro-
derived growth factor B, and induci- type 16 (HPV16) had transforming gen for 6 months developed high-

CHAPTER 9
PART 1
ble nitric oxide synthase by the vGP- potential in established rodent cells grade dysplasia and/or cervical can-
CR-expressing cells. Finally, contin- (Yasumoto et al., 1986), with the prin- cer, but K14-E6 mice only developed
ued vGPCR expression is essential cipal activity residing in the E7 onco- cervical cancer after treatment with
for progression of the Kaposi sarco- protein (Vousden et al., 1988). The estrogen for 9 months (Riley et al.,
ma-like phenotype, and downregu- E5 and E6 proteins of both HPV16 2003; Shai et al., 2007, 2008).
lation of vGPCR expression results and HPV18 also showed transform- An additional activity of HPV16 E7
in reduced expression of angiogenic ing potential in such assays (Pim that contributes to its oncogenicity
factors and regression of the lesions. et al., 1992). The E6 protein can in- is the ability to inactivate p21 (Shin
These findings implicate vGPCR as et al., 2009). In transgenic mice car-
activate p53 (Scheffner et al., 1990),
a key element in the pathogenesis rying a tetracycline-regulated HPV16
and as a result, E6 can abrogate
of Kaposi sarcoma (Jensen et al., E7 transgene, the continued expres-
cell-cycle arrest induced by a variety
2005).
of DNA-damaging agents, such as sion of E7 was found to be critical for
Further support for a role of vG-
actinomycin D. The normal response the maintenance not only of cervical
PCR in tumorigenesis has come
to DNA damage, i.e. inhibition of cancer but also of the dysplastic neo-
from studies with MHV-68. In mice,
DNA synthesis and increase in p53 plasia that is recognized as its pre-
this virus can replicate transiently
protein levels, did not occur after cursor lesion (Jabbar et al., 2009).
before entering a latent state, but
treatment with actinomycin D of ke- This dependence on continued ex-
no lymphoproliferative disorders
ratinocytes that had been immortal- pression of E7 was observed even in
arise in immunocompetent animals.
ized with HPV16 E6/E7 (Kessis et al., the context of constitutive expression
However, when a recombinant MHV-
1993). of HPV16 E6 (Jabbar et al., 2012).
68 in which the vGPCR is replaced
Like E7, the E6 protein of HPV
with the KSHV-derived vGPCR was
used to infect mice, angiogenic le-
Transgenic models for HPV- appears to contribute to cervical
associated cancers carcinogenesis through multiple ac-
sions formed that had features char-
acteristic of those seen in human tivities. This protein is known to de-
The first germline transgenic mouse grade p53 and other cellular targets
Kaposi sarcoma lesions. The differ-
model for HPV-associated cervical through its interaction with the ubiq-
ence in activity between the wild-
cancer was developed with a K14- uitin ligase E6AP. This enzyme was
type MHV-68 and the recombinant
HPV16 construct that contained the found to be critical for E6-mediated
MHV-68 was linked to differences in
activation: the KSHV-derived vGP- early genes of the virus under the oncogenesis in the cervix (Shai et al.,
CR was constitutively active, where- control of the K14 transcriptional 2010). E6 is known to bind to sever-
as the murine vGPCR in MHV-68 promoter, which directs the expres- al cellular proteins that contain PDZ
was not (Zhang et al., 2015). This sion of these genes to the stratified domains (common structural regions
study provides compelling evidence epithelium of the oral cavity and of 80–90 amino acids in proteins
for a direct role of vGPCR in the de- the lower female reproductive tract. involved in signalling). Transgenic
velopment of Kaposi sarcoma in a These mice did not develop cer- mice expressing a mutant form of
non-transgenic animal model. vical cancers spontaneously, but HPV16 E6 that is unable to bind to
For KSHV, the overall concor- treatment with estrogen, sufficient PDZ-domain proteins (Nguyen et al.,
dance between the animal mod- to induce continuous estrus, led to 2003) had a reduced susceptibility to
els and humans with respect to the a highly penetrant cervical cancer cervical cancer compared with mice
types of tumour and the identity and phenotype in the context of a pro- expressing the wild-type E6 protein
function of the major oncogenes and gressive disease much like that seen (Shai et al., 2007). It remains un-
oncogene products is high. in women, given that it was preceded clear which of the interactions with
by the onset of cervical intraepitheli- PDZ-domain proteins contribute to
Human papillomaviruses
al neoplasia (CIN) of grades 1–3. As E6-mediated carcinogenesis in vivo,
The cell-transforming capacity of hu- in women, the cancers preferential- and how E7 is involved in this pro-
man papillomavirus (HPV)-encoded ly arose in the transformation zone cess (Simonson et al., 2005; Shai
proteins has been demonstrated in (Arbeit et al., 1994, 1996). All K14-E7 et al., 2007).

Part 1 • Chapter 9. Human tumour viruses 77


The same high-risk HPV types carcinogenesis in HPV transgenic 2007), whereas inactivation of both
associated with cervical cancer are mice (Chung et al., 2013); evidence pRb and p107 could fully recapitulate
also linked to the development of for a role of stromal ERα has also the oncogenicity of E7 in HNSCC
anal cancer. In the same K14-HPV16 been obtained in the context of hu- (Shin et al., 2012).
transgenic mouse models, the man cervical cancer (den Boon et al.,
expression of the E6/E7 transgenes 2015). ERα antagonists were effec- Carcinogenic potential
gave rise to anal cancers in mice tive in eliminating cervical cancer
of epidermodysplasia
verruciformis-associated beta
treated with 7,12-dimethylbenz[a] and the precancerous lesions in this
HPV types in the skin
anthracene (Stelzer et al., 2010). As animal model (Chung and Lambert,
with the cervical lesions, the E7 on- 2009). The cervical cancers in these HPV types of the genus beta, spe-
coprotein also plays a dominant role mouse models are strictly associated cifically HPV8 and HPV38, are as-
in this case (Thomas et al., 2011). with atypical squamous metaplasia, sociated with a rare familial benign
which is believed to be the precursor disease termed epidermodyspla-
Role of estrogen in of cervical cancer in women. sia verruciformis. Patients with this
HPV-mediated cervical
carcinogenesis disease are at an increased risk of
High-risk HPV types and
SCCs of the skin at sun-exposed
cancers of the head and neck
Estrogen is an important cofactor in areas. K14-HPV8 transgenic mice
the development of cervical cancers expressing the early genes of HPV8
The same high-risk HPV types that
in HPV transgenic mouse models in the epidermis were susceptible to
are etiologically associated with ano-
(Arbeit et al., 1996). Tumours arising spontaneous development of both
genital cancers, particularly HPV16,
in HPV16 transgenic mice – carrying benign and malignant skin cancers
are also associated with a subset of
E7 or E6/E7 as transgenes – treat- (Schaper et al., 2005). Unlike what is
human head and neck squamous
seen for the mucosal HPV types, the
ed with estrogen for 9 months were cell carcinomas (HNSCCs), most
viral E2 protein seems to play a major
much larger than those observed notably of the oropharynx (e.g. ton-
role in these HPV8-induced cancers,
after 6 months of treatment. When sils), the base of the tongue, and
because expression of E2 alone also
these mice were treated with estro- the upper oesophagus. The role of
results in the development of skin
gen for 6 months and then kept with- the HPV16 proteins E6 and E7 in
cancer, a process that is accelerated
out treatment for 3 months, they had HNSCC has been evaluated in HPV
after irradiation with ultraviolet (UV)
significantly fewer, smaller, and less transgenic mice that express the two
light (Pfefferle et al., 2008).
aggressive tumours at 9 months than oncogenes E6 and E7 in the relevant
In the case of HPV38, K10-E6/
those seen in mice treated for the full tissues. These mice do not sponta- E7 transgenic mice were highly sus-
9 months; thus, tumour regression neously develop HNSCC, but when ceptible to multistage skin carcino-
was seen in the 3-month period after treated with the synthetic carcinogen genesis, specifically when treated
treatment. Estrogen therefore plays 4-nitroquinoline-N-oxide, they be- with UVB radiation or chemical car-
a critical role not only in the genesis come more susceptible to head and cinogens (Dong et al., 2005). The
of cervical cancer but also in its per- neck cancers (Strati et al., 2006). synergy between cutaneous HPV
sistence and continued development The progressive disease observed types and UV radiation in the devel-
(Brake and Lambert, 2005). in the mice treated with 4-nitroquin- opment of SCCs of the skin has also
In a later study, estrogen receptor oline-N-oxide was similar to that been studied in transgenic SKH-hr1
alpha (ERα) was found to be nec- seen in humans, and the cancers hairless mice expressing in their
essary for development of cervical that occurred were primarily high- epidermis the E6 and E7 genes of
carcinogenesis in K14-E7 transgen- grade HNSCC, as observed in HPV- HPV20, which is commonly asso-
ic mice: exogenous estrogen failed positive HNSCCs in humans. As with ciated with SCC observed in renal
to promote either dysplasia or cer- cervical cancer, E7 proved to be the transplant recipients, or of HPV27,
vical cancer in K14-E7/ERα−/− mice more potent oncogene (Jabbar et al., which is only associated with be-
(Chung et al., 2008). Interestingly, 2010), and the inactivation of pRb nign papillomas. Upon UV irradia-
expression of ERα in the cervical could not fully account for the role of tion, both HPV20 E6/E7 and HPV27
stroma was required for cervical the E7 protein (Strati and Lambert, E6/E7 transgenic mice were more

78
susceptible to tumours compared Because HIV-1 is species-specif- and multiple other cellular proteins
with non-transgenic mice, and the ic, like the oncogenic herpesviruses (Gatignol, 2007). Tat also affects the

CHAPTER 9
PART 1
HPV20 E6/E7 transgenic mice had EBV and KSHV, there are no ideal course of HIV-1-associated disease
an increased incidence of malignant animal models for HIV-1-associated indirectly, because it is secreted by
tumours. Alterations in the expres- cancers. In contrast to what is ob- infected cells and can enter non-in-
sion of both p53 and p63 were noted served with infectious agents that fected cells (Gupta and Mitra, 2007).
in the transgenic mice exposed to are directly oncogenic, the HIV-1 ge- Evidence that Tat is involved in
UV radiation (Michel et al., 2006). nome is not present in cancer cells. oncogenesis includes its ability to
For HPV, the overall concordance Therefore, any interaction between induce apoptosis in neighbouring
between the animal models and hu- virus and host is indirect. Although non-infected cells when secreted
mans with respect to the types of tu- none of the HIV-1-encoded proteins from infected cells, thereby increas-
mour caused by mucosal HPV types has been unequivocally shown to be ing the susceptibility of bystander
and the identity and function of the directly oncogenic, some are associ- CD4-positive T cells to death in-
major oncogenes and oncogene ated with immunodeficiency, thereby duced by cross-linking (Alimonti
products is high. There are important indirectly promoting cancer develop- et al., 2003). This may contribute
context-dependent differences in the ment. In addition, there is evidence to the massive depletion of CD4-
function of the E6 oncoprotein, de- that some of the HIV-1-encoded pro- positive T cells by apoptosis, lead-
pending on the anatomical site. teins may promote cancer by other ing to the severe immunodeficien-
indirect mechanisms that are not cy seen in the acquired immune
Human immunodeficiency dependent on immunodeficiency. deficiency syndrome (AIDS). Tat
virus type 1 (HIV-1) There are also reports of transgenic has also been shown to stimulate
mouse models containing HIV provi- the growth of Kaposi sarcoma cells
Infectious agents can act as indirect (Aoki and Tosato, 2007). However,
ral transgenes. In some cases these
carcinogens by causing immunosup- when cells are removed from Kaposi
animals develop lymphoproliferative
pression. This has been shown for sarcoma lesions and expanded in
disorders, including B-cell lympho-
infection with HIV-1, which strongly vitro, they lose the KSHV genome,
mas, which show characteristics
increases the incidence of several and the question remains whether
similar to those of B-cell lymphomas
human cancers. Strikingly, the ma- the “Kaposi sarcoma” cells lacking
arising in patients with HIV infection
jority of cancers associated with HIV- KSHV used in most of these studies
(Curreli et al., 2013). Virus-encoded
1 have another known infectious eti- represent a valid model for Kaposi
proteins including Nef, gp120, p17,
ology, and HIV-1 infection increases sarcoma.
and Tat have all been implicated in
their incidence considerably. Among To investigate the role of Tat in
promoting B-cell hyperproliferation,
these cancers, those associated carcinogenesis, several studies have
although the strongest association in
with the herpesviruses KSHV and been carried out in vivo, in trans-
animals comes from work on the viral
EBV are most strongly enhanced genic mice. Transgenic mice carry-
Tat protein.
by immunosuppression. The same ing a recombinant DNA sequence
cancers are also enhanced by iat- Tat protein containing the early region of the
rogenic immunosuppression, as BK virus and the HIV-1 Tat gene
shown by their increased incidence The multifunctional Tat protein is the developed skin leiomyosarcomas,
in transplant recipients, which lends only HIV-1 protein for which there is squamous cell papillomas and carci-
additional support to the notion that experimental evidence of a potential nomas, adenocarcinomas of skin ad-
HIV-1 acts as a carcinogen mainly role in Kaposi sarcoma. Tat is an im- nexal glands, and B-cell lymphomas.
through this indirect effect. The most portant regulator of viral transcription; Although the incidence of HCC was
common cancers in individuals with it recruits cellular transcription fac- low, most animals showed liver cell
HIV-1 infection are Kaposi sarco- tors to the HIV-1 promoter, strongly dysplasia of variable degree. These
ma (caused by KSHV), lymphomas stimulates HIV-1 DNA transcription, mice were also affected by skin le-
(many of which are EBV-positive), and interacts with protein–kinase sions resembling the early stages
and cervical and anogenital carcino- complexes (Cdk9/cyclin T1, Cdk2/ of Kaposi sarcoma (see also Vogel
mas associated with HPV infection. cyclin E), protein phosphatases, et al., 1988). The Tat transgene was

Part 1 • Chapter 9. Human tumour viruses 79


detected intact in all the organs of the Human T-cell lymphotropic The transforming ability of Tax was
transgenic mice, and the Tat protein virus type 1 (HTLV-1) demonstrated in the Rat-1 fibroblast
was expressed in essentially all tis- cell line in vitro in a soft agar assay,
sues and organs of these animals. HTLV-1 naturally infects humans, and in vivo in nude mice (Tanaka
These BK virus/Tat transgenic mice but the virus can be inoculated into et al., 1990). These findings clearly
may be useful in studies of the role of different animals, including rabbits, show that Tax is oncogenic.
Tat in AIDS-associated malignancies rats, mice, and New World monkeys, Several studies with animals
and of the pathogenesis of Kaposi with various effects (Lairmore et al., transgenic for Tax clearly demon-
2005). In rabbits and rats, HTLV-1 in- strated that Tax expression leads to
sarcoma (Corallini et al., 1993).
fection is persistent but does not lead the induction of tumours, confirming
Because the Tat protein stimulates
to definite diseases. that Tax is oncogenic in vivo. In Tax
cell proliferation, inhibits apoptosis,
Different monkey species are transgenic animals, the Tax protein
displays angiogenic functions, and
naturally infected with STLV-1, the was shown to be oncogenic, with
may be involved in the pathogene-
simian analogue of HTLV-1, and sev- the tumour type depending on the
sis of Kaposi sarcoma and other tu-
eral cases of adult T-cell leukaemia/ promoter used in each study. Mice
mours arising in patients with AIDS,
lymphoma (ATLL) have been de- that expressed Tax under the con-
the BK/Tat transgenic mice (see the
scribed in African green monkeys trol of the granzyme B promoter
previous study) may be predisposed (Tsujimoto et al., 1987; Akari et al., developed tumours of natural killer
to tumour formation. When Tat trans- 1998). Experimental infection with cells (Grossman et al., 1995), and
genic mice were treated with ure- HTLV-1 of squirrel monkeys (Saimiri
mice that expressed Tax via the lck
thane, the incidence of lung tumours sciureus) caused a strong reduction
promoter developed a disease that
and lymphomas was not different in the proliferation rate of the CD4-
resembles ATLL (Hasegawa et al.,
between the transgenic mice and the positive T-cell population in those in-
2006). The major difference between
controls, whereas the incidence of fected animals that were affected by a
most of these animal tumours and
pre-neoplastic lesions and tumours pathology similar to ATLL in humans
ATLL in humans is the fact that a
in the liver was significantly higher (Debacq et al., 2005). Co-infection of
subset of human ATLL cells do not
in Tat transgenic mice than in control rhesus macaques (Macaca mulatta)
express Tax.
mice. This remarkable effect of ure- with HTLV-1 and SIV type 1 (SIV-1)
More recently, different models of
thane observed in the liver may be increased the number of multilobu-
humanized mice have been used to
due to a Tat-induced predisposition, lated lymphocytes (“flower” cells) in
assess the effects of infection with
manifested as a liver cell dysplasia, the circulation; this cell type is also
HTLV-1 (Villaudy et al., 2011; Tezuka
spontaneously affecting most of the seen in patients with ATLL. SIV-1
et al., 2014). In these cases, the
Tat transgenic mice. Liver cell dys- may have the potential to upregulate
mice developed ATLL-like leukaemic
HTLV-1 and enhance expression of
plasia may exert a promoting effect symptoms, including splenomegaly
disease (Traina-Dorge et al., 2007).
by stimulating proliferation of cell and lymphoma.
So far, non-human primates repre-
clones initiated by the mutagenic ef- Cytogenetic analysis of ATLL
sent the only suitable animal model
fect of urethane. In addition, liver cell cells has shown a common break-
to study human ATLL.
dysplasia may enhance the progres- point cluster region in chromosome
The pX region of HTLV-1 encodes
sion to malignancy of the pre-neo- 10p11.2. Further analyses have
the regulatory genes Tax and Rex,
plastic lesions induced by urethane. shown that the transcription fac-
and several accessory genes. Tax,
This study suggests a role of Tat in a 40-kD phosphoprotein, is found tor 8 (TCF8) is frequently disrupted
the promotion and progression of mainly in the nucleus but also in the by several mechanisms, including
carcinogen-initiated tumours in pa- cytoplasm (Meertens et al., 2004). epigenetic silencing. Suppressed
tients with HIV-1 infection (Altavilla Interaction of Tax with several host expression of TCF8 is associat-
et al., 2004). factors results in transactivation of ed with resistance to transforming
For HIV-1, the overall concor- some genes, transrepression of growth factor beta (TGF-β). Mice
dance between the animal mod- others, modulation of the cell cy- carrying a mutation in TCF8 fre-
els and humans with respect to the cle, and dysregulation of apopto- quently developed thymic T-cell
types of tumour is low. sis (Matsuoka and Jeang, 2007). lymphoma, indicating that TCF8 is

80
a tumour suppressor gene (Hidaka Tgat, was identified. Expression and humans with respect to the
et al., 2008). There have been only of Tgat in NIH 3T3 cells resulted in types of tumour and the identity and

CHAPTER 9
PART 1
few reports of cellular oncogenes cellular transformation, indicated function of the major oncogenes and
in ATLL cells. When complementa- by anchorage-independent growth oncogene products is high.
ry DNA expression libraries derived in semi-solid medium, and tumour
from leukaemic cells of patients with formation in nude mice (Yoshizuka
ATLL were screened for the potential et al., 2004).
to transform NIH 3T3 mouse fibro- For HTLV-1, the overall concor-
blasts, a novel transforming gene, dance between the animal models

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84
part 2.
mechanisms of carcinogenesis

chapter 10.

Key characteristics of
carcinogens
Martyn T. Smith

CHAPTER 10
PART 2
Introduction which carcinogens produce cancer. differences, may explain the lack
The Workshop participants conclud- of concordance or coherence be-
The agents documented and list- ed that carcinogens commonly show tween tumour sites in humans and
ed as carcinogenic to humans one or more of 10 key characteristics experimental animals. Neither the
(Group 1) in Volume 100 of the IARC (Table 10.1). list given in Table 10.1 nor the set of
Monographs show several key char- To achieve wide dissemination modulating factors mentioned by the
acteristics that distinguish them as and assessment, these key char- Workshop participants is meant to
carcinogenic agents. Many appear to acteristics have been described in be exhaustive, but they were agreed
act via multiple mechanisms, caus- an open access journal publication upon as being established charac-
ing various biological changes in the (Smith et al., 2016) that also delin- teristics or modulating factors. It is
multistage process of carcinogene- eates their application, and hence hoped that they will assist future
sis. Others appear to act by a single IARC Monographs Working Groups
complements material presented
predominant mechanism. in evaluating additional potential
in this chapter and, to a broader
The participants in the IARC human carcinogens.
extent, this Scientific Publication.
Workshop on Tumour Site Concor- Here, these key characteristics are
Characteristic 1: Is
dance and Mechanisms of Carcino- defined, and reference is made to
electrophilic or can be
genesis, after considering previously subsequent chapters where these
metabolically activated to
published options for classification particular characteristic properties electrophiles
of carcinogens and related matters are extensively discussed.
(see Chapter 11, by Stewart, and The Workshop participants also Electrophiles are electron-seeking
Chapter 12, by DeMarini), exten- discussed several modulating fac- molecules that commonly form addi­-
sively debated the mechanisms by tors that, along with mechanistic tion products, generally referred

Part 2 • Chapter 10. Key characteristics of carcinogens 85


Table 10.1. Key characteristics of carcinogens two bases. The DNA damage is by
itself not a mutation and generally
1. Is electrophilic or can be metabolically activated to electrophiles does not alter the linear sequence
of nucleotides (or bases) in the DNA,
2. Is genotoxic
whereas a mutation is defined as a
3. Alters DNA repair or causes genomic instability change in the DNA sequence, which
4. Induces epigenetic alterations usually arises as the cell attempts to
repair the DNA damage.
5. Induces oxidative stress

6. Induces chronic inflammation Characteristic 3: Alters DNA


repair or causes genomic
7. Is immunosuppressive
instability
8. Modulates receptor-mediated effects
Normal cells try to avoid deleterious
9. Causes immortalization
mutations by replicating their ge-
10. Alters cell proliferation, cell death, or nutrient supply nomes with high accuracy. However,
the fidelity of DNA replication can
vary widely depending on the DNA
to as adducts, with cellular peroxidases (O’Brien, 2000; Hecht, polymerase involved, and this in-
macromolecules including DNA, 2012). The ability to form adducts troduces the possibility of error.
RNA, lipids, and proteins. Some with DNA and protein is a com- Indeed, most spontaneous muta-
chemical carcinogens are direct-act- mon property of these electrophilic tions are caused by polymerase
ing electrophiles, whereas others re- and metabolically activated human error (Preston et al., 2010). The na-
quire biotransformation by enzymes carcinogens. ture of the mistake, the flanking se-
in a process termed metabolic acti- quence, the presence of DNA dam-
vation (Miller, 1970). Characteristic 2: Is genotoxic age, and the ability to correct errors
Examples of direct-acting elec- all have an impact on the outcome
trophilic carcinogens are formalde- The term “genotoxic” refers to an of this process (Arana and Kunkel,
hyde, sulfur mustard, and ethylene agent that induces DNA damage, 2010). As a consequence, defects
oxide (see Chapter 1, by Bond and but this damage may or may not in processes that determine DNA
Melnick). The classic examples of necessarily be processed by the cell replication fidelity can confer strong
chemical agents that require met- into a mutation (see Chapter 12, by mutator phenotypes that result in ge-
abolic activation to become carci- DeMarini). Thus, if an agent is found nomic instability. Thus, carcinogens
nogenic are polycyclic aromatic hy- to induce DNA damage, it can be may act not only by producing DNA
drocarbons and benzene, which by called a genotoxicant or a genotox- damage directly but also by altering
themselves are relatively inert chem- in, and if it is shown that the agent the processes that control normal
ically. This lack of reactivity puzzled also induces mutations in a mutage- DNA replication.
chemists working on experimental nicity assay, it can be classified as a Similarly, the major DNA repair
carcinogenesis for many years un- mutagen. Most of the IARC Group 1 pathways – such as base excision
til the Millers discovered metabolic carcinogens are considered to be repair, nucleotide excision repair,
activation by the mixed-function ox- genotoxic, and many are mutagenic and double-strand break repair –
idase system (Conney et al., 1957). (Waters et al., 2010), although this involved in the removal of DNA ad-
It is now known that several human may not be their primary mechanism ducts and other lesions may also be
enzymes can biotransform relatively of carcinogenesis. altered by environmental exposures.
inert chemical compounds to potent DNA damage from genotoxicity Furthermore, whereas especially ex-
toxic and carcinogenic metabolites may be in the form of DNA adducts cision repair pathways are predomi-
or reactive intermediates. These or single- or double-strand breaks. nantly error-free and thus protective,
enzymes include cytochrome P450 Other types of DNA damage are ox- double-strand break repair is largely
isozymes, flavin mono-oxygenase, idized or fragmented bases or the error-prone and may contribute to
prostaglandin synthase, and various intercalation of a molecule between genomic instability.

86
Genomic instability is a well-rec- ed that their mode of action may in- Reactive oxygen species produce
ognized feature of many cancers volve disruption of epigenetic mech- at least 24 base modifications, as
(Bielas et al., 2006). Studies of cul- anisms. Because the evidence for a well as DNA–protein cross-links and
tured cells exposed to ionizing ra- truly causal role of epigenetic chang- other lesions (Berquist and Wilson,
diation have shown that instability es in cancer produced by Group 1 2012), all potentially leading to ge-
is a relatively late-occurring event agents was deemed to be limited in nomic instability. Oxidative damage
that appears several cell genera- Volume 100, for many agents their to DNA can lead to point mutations,
tions after irradiation and results impact on the epigenome was not deletions, insertions, or chromoso-
in a reduced ability to replicate the considered to be a secondary mech- mal translocations, which may cause
genotype faithfully (see Chapter 18, anism of carcinogenesis. However, activation of oncogenes and inacti-
by Hill and Ullrich). The events that it should be noted that most carcin- vation of tumour suppressor genes,
indicate genomic instability include ogens (even those considered for potentially leading to initiation of
chromosomal aberrations, gene mu- Volume 100 in 2008 and 2009) were carcinogenesis (Klaunig et al., 2011;
tations, microsatellite instability, and evaluated by IARC Working Groups Berquist and Wilson, 2012). Thus,
apoptosis. The instability phenotype before new data on their epigenetic agents that generate and promote
may play a major role in radiation-in- effects became available. Many re- oxygen radical-induced cellular inju-

CHAPTER 10
duced cancers and other forms of cent studies have demonstrated the ry may be carcinogenic.

PART 2
cancer by providing the cell and its impact of several Group 1 agents in-
progeny with a constantly elevated cluded in Volume 100 on epigenetic Characteristic 6: Induces
rate of any of the various genetic mechanisms (Koturbash et al., 2011; chronic inflammation
and epigenetic changes that may Ravegnini et al., 2015; Chappell
occur in multistage carcinogenesis et al., 2016). This rapidly evolving Chronic inflammation from persis-
(Aypar et al., 2011). area will generate new mechanistic tent infections, such as that caused
data on carcinogens in the next few by Helicobacter pylori as well as that
Characteristic 4: Induces years. produced by silica or asbestos fibres,
epigenetic alterations has been associated with several
Characteristic 5: Induces forms of cancer (see Chapter 17, by
The term “epigenetic” refers to all
oxidative stress Kane). Indeed, inflammation is an
stable changes in gene expression
“enabling characteristic” of cancer
and chromatin organization that are Many human and animal carcino- (Hanahan and Weinberg, 2011), and
independent of the DNA sequence gens are capable of influencing re- it has been hypothesized to contrib-
itself and that can be mitotically in- dox processes and redox balance ute to cancer initiation, promotion,
herited over cell divisions. Epigenetic within target cells (see Chapter 15, and progression (Trinchieri, 2012).
phenomena, including genomic im- by Bucher). An imbalance between Inflammation acts by both intrinsic
printing, X-chromosome inactivation, formation of reactive oxygen and/ and extrinsic pathways. Persistent
global reconfiguration of the DNA or nitrogen species and their detox- infection and chronic inflammation
methylome, and changes in chro-
ification is commonly referred to as disrupt local tissue homeostasis
matin compaction states and histone
oxidative stress. Reactive oxygen and alter cell signalling, leading to
modification patterns, occur during
species, which can arise from in- the recruitment and activation of in-
development and contribute to the
flammation, may contribute to geno- flammatory cells. These constitute
lineage-specific epigenome that is
mic instability and – along with other extrinsic pathways linking inflam-
maintained over the lifetime of an
free radical species – play key roles mation to cancer (Multhoff and
organism. Many of these same phe-
nomena are altered during carcino- in many of the processes identified Radons, 2012). In contrast, intrinsic
genesis (see Chapter 20, by Rice as being necessary for the conver- pathways driven by activation of pro-
and Herceg). sion of normal cells to cancer cells. to-oncogenes in pre-neoplastic and
A wide range of known and sus- Oxidative damage is considered a neoplastic cells recruit host-derived
pected carcinogens (including chem- major factor in the generation of mu- inflammatory cells that accelerate
ical, physical, and biological agents) tations in DNA, and more than 100 tumour promotion and progression
have been shown to deregulate the different oxidative DNA adducts have (Grivennikov et al., 2010). Strong
epigenome, and it has been suggest- been identified (Klaunig et al., 2011). links exist between inflammation and

Part 2 • Chapter 10. Key characteristics of carcinogens 87


the induction of oxidative stress and Characteristic 8: Modulates humans (Group 1); these include var-
genomic instability; this makes it dif- receptor-mediated effects ious types of human papillomavirus
ficult to separate out the importance (HPV), Epstein–Barr virus (EBV),
Hormonally active agents that are
of each of these mechanisms. Kaposi sarcoma-associated her-
associated with carcinogenic effects
pesvirus (KSHV), hepatitis B virus
Characteristic 7: Is typically act as ligands via nuclear
(HBV), hepatitis C virus (HCV), and
immunosuppressive receptors, and in some cases via re-
HIV-1. These viruses have evolved
ceptors located on the cell surface.
multiple molecular mechanisms to
Immunosuppression is a reduction in There are many other agents that
the capacity of the immune system disrupt specific cellular pathways to
may be carcinogenic by acting on
to respond effectively to foreign an- facilitate aberrant replication.
receptor proteins, even though some
tigens, including antigens on tumour of these also have genotoxic effects, Although oncogenic viruses be-
cells. Persistent immunosuppression for example polycyclic aromatic hy- long to different families, their strat-
presents a risk of cancer, especially drocarbons such as benzo[a]pyrene. egies in human cancer development
excess risk of lymphoma when it is Receptor activation falls into two show many similarities and involve
accompanied by continuing expo- broad categories: (i) activation of in- viral-encoded oncoproteins target-
sure to foreign antigens such as af- tracellular receptors that translocate ing the key cellular proteins that
ter organ transplantation, or when it into the nucleus and act on DNA as regulate cell growth. Recent studies
occurs in individuals who are latent- transcription factors, and (ii) acti- have shown that virus and host inter-
ly infected with an oncogenic virus vation of cell surface receptors and actions also occur at the epigenetic
(Gutierrez-Dalmau and Campistol, some intracellular receptors that ac- level (Allday, 2013). The result of
2007; Münz and Moormann, 2008; tivate signal transduction pathways, these viral effects is to immortalize
Shelton et al., 2016). resulting in biological responses (see the cells of the target tissue such that
Immunosuppression differs from Chapter 14, by Bosland). they are not subject to the Hayflick
other mechanisms of carcinogen- The predominant effect of recep- limit, the point at which cells can no
esis in that agents that cause im- tor activation is on gene transcrip- longer divide due to DNA damage or
munosuppression may not directly tion. Although some exogenous shortened telomeres.
transform normal cells into potential ligands act as agonists by compet- For example, the HPV type 16
tumour cells. Potentially neoplastic ing for binding with an endogenous (HPV16) E6 and E7 oncogenes are
cells that arise naturally, or that have ligand, others may bind but lack selectively retained and expressed
been transformed by other carcino- intrinsic activating activity for the
in cervical carcinomas, and expres-
gens acting by a mechanism such as receptor they bind to and have an
sion of E6 and E7 is sufficient to
genotoxicity or by the various mech- antagonistic effect. There are also
immortalize human cervical epitheli-
anisms of action associated with receptors for which no endogenous
al cells (Yugawa and Kiyono, 2009).
ligand has been identified, such as
oncogenic viruses, escape immune E6 and E7 proteins do not possess
the aryl hydrocarbon receptor. One
surveillance in immunosuppressed intrinsic enzymatic activities but in-
other important type of potential ef-
individuals. As a result, survival of stead function through several direct
fect of exogenous agents on recep-
these cells and their replication to and indirect interactions with cellular
tor-mediated mechanisms involves
form tumours is greatly facilitated by proteins, some of which are well-
modulation of the amount of endoge-
immunosuppression. known cellular tumour suppressors,
nous ligand available for binding and
Several Group 1 agents included including p53 and Rb.
activating its receptor, by affecting
in Volume 100 act entirely or largely
biosynthesis, bioavailability, bioac-
by immunosuppression, often in con- Characteristic 10: Alters cell
tivation, and/or degradation of the
cert with other Group 1 agents, es- proliferation, cell death, or
ligand (Rushmore and Kong, 2002).
pecially oncogenic infectious agents. nutrient supply
The Group 1 agents that act by im- Characteristic 9: Causes
munosuppression include human immortalization There are at least three scenarios
immunodeficiency virus type 1 (HIV- related to cancer and cancer mech-
1) and the immunosuppressive drugs Volume 100 of the IARC Monographs anisms in which alterations in cellu-
ciclosporin and azathioprine (IARC, identifies several human DNA and lar replication and/or cell-cycle con-
2012a, c). RNA viruses that are carcinogenic to trol have been described. The first

88
invokes the predisposition for unre- of impaired nutrient supply. The ex- an impact on this complex process
paired DNA damage to lead to can- ponentially increasing number of ne- in multiple ways and can act through
cer-initiating mutations in replicating oplastic cells may quickly outstrip the multiple mechanisms of action to
cells. The second has attempted to supply capabilities of the existing tis- induce cancer and other adverse
identify sustained replication as a sue vasculature. Neo-angiogenesis, health outcomes. As an illustration
key mechanistic event, and the third in which new blood vessels grow of this point, the evidence has been
describes the ability of a transformed into a tumour, is key to providing this evaluated for which key characteris-
cell to escape normal growth control supply of nutrients. Thus, agents tics contribute to the carcinogenicity
and to continue replication. A com- that promote or inhibit angiogenesis, of benzene, an IARC Group 1 carcin-
ponent common to all three scenar- such as arsenic, will promote or de- ogen, in humans and in experimental
ios is the evasion of apoptosis or oth- lay tumour growth (Yang et al., 2014). animals. The results are shown in
er terminal programming, including Table 10.2, where the level of evi-
autophagy, in at least a proportion Multiple mechanisms of dence for a particular characteristic is
of the cell population (Ryter et al., action of human carcinogens classified on a scale with 2 = strong
2014). evidence, 1 = moderate evidence,
Sustained cellular proliferation The number of mechanisms by

CHAPTER 10
and 0 = weak evidence. For ben-
which chemicals are known to con-

PART 2
has been argued to be a factor in zene, there is strong evidence in my
increased cancer susceptibility. As tribute to carcinogenesis can be ex-
view that metabolic activation, geno-
summarized in the United States tensive if one includes all biochemi-
toxicity, and immunosuppression are
Environmental Protection Agency cal or molecular end-points, but the
established mechanisms of carcino-
guidance assessing risk of can- mechanisms can be grouped into a
genicity in both animals and humans
cer from early-life exposures (EPA, limited number of categories (geno-
(McHale et al., 2012). There is weak
2005), more frequent cell division toxicity, immunosuppression, etc.).
or no evidence that inflammation and
during development can result in Guyton et al. described 15 types of
immortalization play a role in the car-
enhanced fixation of mutations be- key events associated with carcino-
cinogenicity of benzene. However,
cause of the reduced time available genesis, which collectively represent
moderate evidence exists for the oth-
for repair of DNA lesions, while the majority of known carcinogen-
er five key characteristics or mecha-
clonal expansion of a mutated cell ic modes of action (Guyton et al.,
nisms in humans. This suggests that
produces a larger population of mu- 2009).
there is strong or moderate evidence
tant cells. For mature organisms, The IARC Workshop participants
that eight of the key characteristics of
sustained proliferation has also been initially identified 24 mechanistic
carcinogens contribute to the carci-
postulated to increase risk of cancer, end-points, with several subcatego-
nogenicity of benzene and that they
based on the same rationale. ries in each. This was considered too
are consistently observed, for the
The mechanism by which necro- many categories, and it was deter-
most part, both in humans and in ex-
sis may enable cancer induction is mined that several of them could be
perimental animals (Table 10.2).
also part of the description of the merged. The Workshop participants
hallmarks of cancer. In contrast to then concluded that carcinogens Factors modulating human
apoptosis and autophagy, necrotic commonly show one or more of the carcinogenesis
cell death releases pro-inflammatory 10 key characteristics described
signals into the surrounding tissue above (see Table 10.1). These Lack of concordance or coherence
microenvironment, resulting in re- represent the majority of known between tumour sites in humans
cruitment of inflammatory cells of the carcinogenic mechanisms of action. and experimental animals may be
immune system that can participate It is increasingly evident that mul- explained by several modulating
in tumour promotion through their tiple biological alterations or sets of factors that, along with mechanistic
influence on cancer cell proliferation different perturbations are neces- effects, cause discordance between
and invasiveness. sary to convert a normal cell into a the findings. For example, physiolog-
In addition to cell death caused transformed cell, and ultimately a ical differences exist between ani-
by direct toxicity of an agent, cells tumour (Hanahan and Weinberg, mals and humans, including the fact
within a tumour may die as a result 2011). Carcinogens appear to have that rodents are nose-only breathers,

Part 2 • Chapter 10. Key characteristics of carcinogens 89


Table 10.2. Key characteristics of the carcinogen benzenea

Characteristic Level of evidenceb

IARC (2012b) Humans Animals

1. Is electrophilic or can be metabolically activated to electrophiles 2 2 2

2. Is genotoxic 2 2 2

3. Alters DNA repair or causes genomic instability 1 1 1

4. Induces epigenetic alterations 1 1 0

5. Induces oxidative stress 1 1 1

6. Induces chronic inflammation 0 0 0

7. Is immunosuppressive 2 2 2

8. Modulates receptor-mediated effects 1 1 1

9. Causes immortalization 0 0 0

10. Alters cell proliferation, cell death, or nutrient supply 1 1 1


a This table shows the overall weight of evidence as stated in Volume 100F of the IARC Monographs (IARC, 2012b), and the levels of
evidence from studies in humans and animals, respectively.
b 2 = strong evidence, 1 = moderate evidence, 0 = weak evidence.

whereas humans breathe through cal: they may absorb, distribute, me- the end products are not carcinogen-
both the nose and mouth. Rodents tabolize, and excrete a compound in ic. With regard to infectious agents,
do not retain their urine as humans ways that are different to those seen it is clear that a human infectious
and dogs do, perhaps explaining the in humans. There are many exam- agent, for example a human tumour
lack of carcinogenicity of aromatic ples of this kind. For instance, mice virus that is not infectious to other an-
amines to rodents (see Chapter 2, by hydrolyse 6-propylthiopurine to mer- imal species, will not produce carci-
Beland and Marques). captopurine, which has a potent car- nogenic effects in these species (see
Experimental animals may also cinogenic effect, whereas humans Chapter 9, by Lambert and Banks).
exhibit differences in the pharmaco- oxidize the drug at two positions in
kinetics or toxicokinetics of a chemi- the molecule without hydrolysis, and

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Gutierrez-Dalmau A, Campistol JM (2007). PMID:27039969
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Part 2 • Chapter 10. Key characteristics of carcinogens 91


part 2.
mechanisms of carcinogenesis

chapter 11.

Mechanisms of carcinogenesis:
from initiation and promotion
to the hallmarks

CHAPTER 11
PART 2
Bernard W. Stewart

Introduction malignancy by describing a tumour The types of biological agents and of


as “an abnormal mass of tissue, the radiation now recognized by IARC
For many decades, a corollary to growth of which exceeds and is un- as carcinogenic to humans (Group 1)
the contemporary understanding of coordinated with that of the surround- are few compared with the number
the nature of cancer and of carcino- ing tissue, and that continues to grow of chemicals in this category (IARC
genesis has been the recognition of in the same excessive manner after 2012a, b, c, d, e, f); there is a much
causative agents. Since the 1950s, cessation of the stimulus that caused larger number of chemicals for which
many agents that contribute to the it”. According to the same textbook, at least some evidence of carcino-
development of cancer have been development of tumours of the skin, genicity is available (see Volumes
categorized as initiators or promot- the alimentary canal, or the respira- 1–105 of the IARC Monographs,
ers, on the basis of studies of chem- tory tract was to be expected among available from http://publications.
ical carcinogenesis in mouse skin individuals exposed “to various iarc.fr).
(Berenblum and Shubik, 1947). noxious agents in the environment”. Research has established how
Cancer was described with ref- Causation of cancer in humans or many carcinogenic chemicals cause,
erence to causative agents. Thus, animals by certain chemicals, radia- or are likely to cause, malignant
a 1970s pathology text (Cappell tion, and biological agents was rec- transformation, but the biological
and Anderson, 1974) introduced ognized by early in the 20th century. processes involved are diverse, and

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 93
Table 11.1. A selection of proposals for the categorization of chemical carcinogensa

Mode of action Exposure context Chemistry Human relevance Agent type


of bioassay data

Genotoxic Tobacco smoke PAHs DNA binding Atmospheric


pollutants

  Direct-acting Alcoholic N-nitroso PPARα activation Pesticides


beverages compounds

  Pro-carcinogen Occupation Aromatic amines α 2u-Globulin Organic solvents


nephropathy

  Inorganic carcinogen Pollution Halogenated Urinary tract calculi Endocrine disruptors


organic
compounds

Non-genotoxic Diet Naturally Disinfection


occurring by-products
compounds

  Solid-state carcinogen Pharmaceutical Inorganic Pharmacological


drugs compounds steroids

  Hormone Exogenous
hormones

  Immunosuppressant

  Promoter

PAHs, polycyclic aromatic hydrocarbons; PPAR, peroxisome proliferator-activated receptor.


a Knowledge about chemical carcinogens is presented from a variety of perspectives apart from that of mechanism of action. The

listings indicate those used in particular publications (e.g. Searle, 1984; Tomatis et al., 1990; Vainio et al., 1992; Vainio and Hietanen,
2003; Hsu and Stedeford, 2010) as ways of ordering data, as indicated by chapter headings in many cases, and are not necessarily
comprehensive. Categories shown in bold involve or include at least one Volume 100 (Group 1) agent.

there is no generally accepted mech- categorization of chemical carcin- “hallmark” or “hallmarks” have been
anistic basis for classifying chemi- ogens on the basis of the organ af- published. These papers typically
cal carcinogens (Loeb and Harris, fected (Warshawsky and Landolph, describe signal transduction path-
2008), beyond categorization ac- 2006). ways and their therapeutic implica-
cording to genotoxicity (Weisburger Currently, the most widely rec- tions. Although the characterization
and Williams, 1981). There is no ognized description of the nature of by Hanahan and Weinberg (2011)
single comprehensive basis for cat- cancer is that presented by Hanahan of the hallmarks of cancer did not
egorization; chemical carcinogens and Weinberg in two reviews – pub- refer to chemical carcinogens or
are sometimes ordered according lished more than a decade apart – causative agents in general, recent-
to the context in which information is that identify the “hallmarks” of can- ly the hallmarks have been used to
presented, with genotoxicity ordered cer (Hanahan and Weinberg, 2000, characterize chemical carcinogens
according to mutational signatures, 2011). These papers have been so (Kleinstreuer et al., 2013).
or agents categorized in relation to influential that others refer to “the These considerations give rise to
differing classes of receptors. There hallmarks” without further qualifica- two questions: (i) whether previous-
have been many proposals for the tion, for example in the title of a re- ly used mechanism-based descrip-
categorization of chemical carcin- cent perspective on tumour metabol- tions of chemical carcinogens may
ogens according to various crite- ism (Cantor and Sabatini, 2012). be recast in relation to the hallmarks;
ria. A selection of these is shown Since 2000, about 200 cancer re- and (ii) whether, and to what extent,
in Table 11.1; others include the search papers with a title including the hallmarks provide opportunities

94
to characterize agents apart from Morphological and genetic biological macromolecules had been
currently known carcinogens as changes variously demonstrated over dec-
contributing to the development of ades, carcinogen adducts in DNA
cancer. Both of these matters are Within 20 years of the publica- were crucial.
addressed in this chapter. tions cited above, the identification Alkylation of DNA by N-nitroso
of multistage carcinogenesis with compounds was shown by Magee
Multistage carcinogenesis particular carcinogens or other exog- and Farber (1962), with tumorigene-
enous agents had become irrelevant sis attributable to the pro-mutagenic
Exogenous agents
to an understanding of cancer de- O6 -methylguanine product, which
The widely accepted paradigm of velopment. Over the same decades, mispairs with thymine. In rats, acti-
carcinogenesis as involving a mul- the context in which carcinogenesis vation of H-Ras in mammary gland
tistage process is generally recog- was best understood changed from tumours induced by N-methyl-N′-
nized to have been developed from rodents to humans. Critical to this nitrosourea was correlated with
the two-stage model of carcino- transition was the identification of H-Ras mutation at codons 12, 13,
genesis in mouse skin (Berenblum multistage carcinogenesis with alter- and 61 (Sukumar et al., 1983).

CHAPTER 11
and Shubik, 1947), which typically ations in gene structure or expres- However, although this insight had

PART 2
involves a polycyclic aromatic hydro- sion rather than with the impact of been gained, it was clear that the eti-
carbon (PAH) and a phorbol ester exogenous agents. ology of some types of cancer, such
(identified as the active agent in the A key development was the as breast cancer in humans, did not
irritant croton oil). Because tumor- correlation by Vogelstein et al. (1988) primarily involve alkylating agents.
igenesis in animals is amenable to of morphological change during the Thus, in human cancer RAS activa-
histological examination at all stag- development of colon cancer in hu- tion is a relevant genetic change in
es, morphological criteria can be mans with particular genetic change. tumour tissue, without reference to
used to characterize the process. The concept was applicable to all exogenous agents (Bos et al., 1987).
With the production of malignant tu- tumour types. Thus, in a diagram il- Although the concept of multi-
mours as the end-point, two-stage or lustrating multistage carcinogenesis stage carcinogenesis was estab-
multistage carcinogenesis was read- with respect to human lung cancer, lished through the use of exogenous
ily described in various organ sites in Harris (1992) made no reference to agents that target particular organ
animals, including the liver and the any particular exogenous agents as sites in animals, by 1990 multistage
bladder (Slaga et al., 1978). mediating specific stages in tumori- carcinogenesis was primarily iden-
Thus, in relation to hepatocarcino- genesis, and showed the transitions tified with altered structure or ex-
genesis, agents such as phenobarbi- between stages as being mediated pression of genes associated with
tal, dichlorodiphenyltrichloroethane, by alterations in the structure or ex- cell proliferation, specifically as de-
polychlorinated biphenyls, butylated pression of oncogenes and tumour scribed in human tumours. However,
hydroxytoluene, and estradiol ben- suppressor genes. the focus of that research has not
zoate were identified as promoters Oncogenes and tumour suppres- involved the specification of genetic
(Dohi et al., 1996). The relevant sor genes mediate altered prolifera- change over time in a manner that
experimental observations, in addi- tive activity in a positive and nega- might account for the emergence of a
tion to indicating the possible risk to tive sense, respectively. Classically, metastatic cell population from with-
humans presented by the relevant increased proliferative activity due in normal tissue. Rather, the relevant
chemicals, also led to the contempo- to oncogene expression accounted research has involved the identifica-
rary understanding of the nature of for the transformation of NIH 3T3 tion of disordered signal transduction
malignancy itself. That understand- cells by DNA isolated from tumours pathways, with a view to developing
ing was based on the identification of and not by DNA from normal tissue targeted therapies. The archetype
particular abnormal cell populations, (Shih et al., 1981). Oncogene ac- of such research is that establish-
specifically including chemically in- tivation (e.g. mutation of Ras) has ing the transforming role of the ty-
duced hyperplastic nodules in rat liv- shown that although binding of many rosine kinase BCR-ABL in chronic
er (Farber, 1973). chemical carcinogens to diverse myeloid leukaemia, and its inhibition

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 95
– to the great benefit of patients – by emerge early, whereas others – sus- • enabling replicative immortality;
the low-molecular-weight inhibitor tained angiogenesis, and tissue inva- and
STI-571 (imatinib) (Bilanges and sion and metastasis – are seen later. • activating invasion and metastasis.
Stokoe, 2007; Rosa et al., 2008). Although hallmarks such as sus- It is notable that, in almost every
tained angiogenesis and metasta- instance, the hallmark is not the
Molecular changes sis involve morphological change, name of a phenotype but refers to
Among a series of reviews marking all of the hallmarks were identified a dynamic process. Consistent with
the publication of the 100th volume with reference to changes in gene this perception, the authors wrote,
of the journal Cell, Hanahan and expression and not by reference to, “The hallmarks of cancer comprise
Weinberg (2000) delineated the very or necessarily in correlation with, six biological capabilities acquired
wide (even then) spectrum of stud- a change in morphology. Diversity during the multistep development
ies addressing the genetics of can- between tumour types and within a of human tumours. The hallmarks
cer by reference to phenotype. Six given tumour type was noted, and no constitute an organizing principle for
characteristics of how cancer cells reference was made to any particu- rationalizing the complexities of neo-
behave could be identified in rela- lar type of neoplasm for illustrative plastic disease.”
tion to particular genes or classes of purposes. In addition, a decade of progress
genes. The phenotypic characteris- In such a description of the mani- had enabled the specification of two
tics were: uncontrolled proliferative festation of essential alterations that “emerging hallmarks”:
activity (Hall, 1984), tumour growth
collectively characterize malignant • deregulating cellular energetics;
attributable to familial risk (Hussain
growth, there is no requirement to and
and Harris, 1998), survival of can-
identify exogenous agents as act- • evading immune destruction.
cer cells (Vaux et al., 1988), immor-
ing on normal or premalignant cells The enabling characteristic iden-
talization of cancer cells (Sedivy,
to cause the change. The focus is tified in 2000 as “genomic instabili-
1998), growth of blood vessels in
tumours (angiogenesis) (Cavallaro on the nature of tumours and how ty” was described in 2011 as “geno­
and Christofori, 2000), and metastat- they may be distinguished from rele- mic instability and mutation”, and a
ic growth (Webb and Vande Woude, vant normal tissue. Finally, Hanahan second enabling characteristic was
2000). Accordingly, the hallmarks and Weinberg (2000) identified an identified as “tumour-promoting
of cancer were initially identified as en­abling characteristic: genomic inflammation”. Superficially, such
follows: instability, which is equated with in- reference to mutation and to pro-
• self-sufficiency in growth signals; creased mutability evident during motion might be seen as implying, if
• insensitivity to anti-growth signals; the process of tumour progression not specifying, the roles that DNA-
(Loeb, 1994). damaging and proliferation-induc-
• evasion of apoptosis;
ing agents have in carcinogenesis.
• sustained angiogenesis; A decade on: “the next However, this is not the case.
• limitless replicative potential; and generation”
In this context, “mutation” refers
• tissue invasion and metastasis. to an acceleration of the accumu-
In 2011, Hanahan and Weinberg
The 2000 “hallmarks” review was lation of mutations, due to, among
provided a new assessment of the
concerned primarily with the charac- other things, defects in the DNA
hallmarks (Hanahan and Weinberg,
terization of the genes and associat- maintenance machinery (Kinzler
2011). They commented, “The past
ed signal transduction pathways that and Vogelstein, 1997). As a result,
decade has witnessed remarkable mutation occurs more readily, ir-
mediate these respective activities
in malignant cells and tumours. In progress towards understanding the respective of whether it is mediat-
that paper, hypothetical patterns of mechanistic underpinnings of each ed by exogenous or endogenous
multistage carcinogenesis were illus- hallmark.” One indication of progress agents. Accordingly, DNA adducts,
trated by a linear arrangement of the is that the original hallmarks were strand breakage, and related phe-
pictograms for the hallmarks, without rebadged as follows: nomena are not to be identified with
reference to any morphological crite- • sustaining proliferative signalling; this enabling characteristic and do
ria. From that diagram, it can be in- • evading growth suppressors; not account for, or are not proper-
ferred that some hallmarks – such as • resisting cell death; ly identified with, a particular hall-
self-sufficiency in growth signals – • inducing angiogenesis; mark. Mutation, in the context of

96
carcinogenesis, identifies a mecha- “genotoxic” indicated, among other include bacterial, mammalian, and
nism whereby a chemical carcinogen things, that the covalent binding of other cells, with weight being given
may cause the emergence of any of a carcinogen adduct to DNA, when to the extent to which the test system
the hallmarks, and almost certainly evident, might account for carcino- has been “validated”, as summa-
of several of them, or perhaps of all genesis. Thus, Weisburger and rized by sensitivity and specificity in
of them. The enabling characteristic Williams (1981) categorized carcin- relation to known carcinogens and
“genomic instability and mutation” ogens primarily on the basis of ge- non-carcinogens. In vivo indicators
renders such outcomes more likely notoxicity. Research over the sub- of genotoxicity include, among oth-
(Wang et al., 2012), rather than refer- sequent 30 years did not alter that ers, (i) metabolism of a chemical to
ring to the mechanism through which approach (Hsu and Stedeford, 2010). produce reactive, typically electro-
the change occurs. The multiplicity of agents and the philic, intermediates, which are the
The identification of “tumour-pro- relatively limited understanding of source of adducts bound to DNA and
moting inflammation” as the second their respective mechanisms of ac- other macromolecules, and (ii) ev-
enabling characteristic recognizes tion have precluded the adoption of a idence of subsequent DNA repair
that inflammation causes the emer- scheme for categorizing carcinogens
and/or mutation.
gence of several of the hallmarks, beyond the consideration of geno-

CHAPTER 11
This description of indicators of
including sustaining proliferative sig- toxicity. Arguably, until the present

PART 2
genotoxicity also summarizes the
nalling and inducing angiogenesis. In IARC Scientific Publication, the most
their discussion of this enabling char- relevant mechanism of chemical
authoritative assessment on how
acteristic, Hanahan and Weinberg carcinogenesis as currently under-
carcinogens act was the 35-page
(2011) were concerned primarily with stood (Cohen and Arnold, 2011).
consensus report in the publica-
cellular infiltration by cells of both Thus, carcinogen metabolism and
tion Mechanisms of Carcinogenesis
the innate and the adaptive arms of DNA repair processes have been
in Risk Identification (Vainio et al.,
the immune response. They made used to identify candidate genes
1992); this was the agreed position
scant, if any, reference to exogenous for lung cancer susceptibility stud-
of a Working Group of more than 40
agents provoking an inflammatory ies (Yokota et al., 2010). Compared
scientists in 1991. The consensus re-
response. with the relatively modest number of
port did not centre on a scheme for
From a broad perspective, refer- genes that account for the absorp-
classifying carcinogens according to
ence to the multistep development tion, metabolism, and elimination of a
their mechanism of action.
of human tumours provides a way carcinogen, together with the repair
Across decades, commentaries
to consider the particular impact of of corresponding DNA adducts, the
on chemical carcinogens (Van
carcinogens and other exogenous hallmarks (Hanahan and Weinberg,
Duuren, 1980; Pitot, 1990; Xue and
agents that may contribute to can- 2011) enable the specification of
Warshawsky, 2006; Cohen and
cer development. However, in iden- tens – if not hundreds – of genes
Arnold, 2011) have not been based
tifying the hallmarks, Hanahan and whose expression contributes to the
on any generally agreed categori-
Weinberg did not pursue this matter.
zation according to mechanism of malignant phenotype.
action. Rather, the common theme At the single-gene level, mutation
Identifying mechanisms of
carcinogenesis has been the enumeration of bio- of TP53, specified with reference to
logical parameters that may deter- particular transitions and transver-
As mentioned above, chemical car- mine whether tumours develop in sions, is attributable to miscoding,
cinogens have been categorized response to carcinogens in general. which in turn is a consequence of
primarily with reference to whether DNA adduct formation from rele-
Genotoxicity: progress and
they exhibit genotoxicity. This mech- vant carcinogens, including those in
problems
anistic distinction began with many tobacco smoke (Soussi, 2011). The
then-known carcinogens being iden- Multiple indicators of genotoxicity data provide evidence of particular
tified as mutagens in vitro by use of have been recognized and catego- exposures, but it remains unclear
particular bacterial strains and after rized as involving data generated how tumorigenesis is enhanced
metabolic activation (the Ames test) either in vitro or in vivo (Montesano by such mutation, beyond the
(McCann et al., 1975). The term et al., 1976). In vitro test systems consideration that a functional p53

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 97
Table 11.2. Chemicals cited by Ashby (1992) and Eastmond (2012) as examples of compounds with equivocal
genotoxicity

Chemicals identified by Ashby (1992) Chemicals identified by Eastmond (2012)

3-Amino-4-ethoxyacetanilide Bromate

3-Amino-9-ethylcarbazole.HCl Captan

Chlorinated paraffins Carbon tetrachloride

CI Acid Orange 3 Chloroprene

CI Basic Red 9.HCl Chromium(III)

Cinnamyl anthranilate Chromium(VI)

1,2-Dibromo-3-chloropropane 1,3-Dichloro-2-propanol

di-Menthol 1,4-Dioxane

Methyldopa sesquihydrate Ethylene glycol monobutyl ether

5-Nitroacenaphthene Hydroquinone

4-Nitro-o-phenylenediamine 2-Nitrotoluene

Piperonyl butoxide Trichloroacetic acid

Piperonyl sulfoxide 1,2,3-Trichloropropane

1,2-Propylene

Sulfallate

protein induces apoptosis, cell-cy- 10 times that in lung tumours from not evident from genomic analysis
cle arrest, and senescence, and never-smokers (Govindan et al., (Muzny et al., 2012). In short, the role
that these processes are compro- 2012). of mutation as contributing to cancer
mised after TP53 mutation (Bieging However, analysis of lung cancer development may be elucidated with-
and Attardi, 2012). The hallmarks genomics does not require immedi- out reference to any genotoxic agent,
offer a broadened perspective as ate reference to smokers and nev- even when the role of such an agent
to signalling pathways that may be er-smokers to present relevant data has been otherwise established.
affected by mutation of TP53 or any (Liu et al., 2012; Peifer et al., 2012). Distinguishing genotoxic
tumour suppressor gene. Moreover, the recognition of tobac- from non-genotoxic
In the first such determination co-induced genomic injury does not carcinogens
made, genotoxic injury by tobac- necessarily extend to other sites; for
Even though molecular process-
co smoke in one individual case of example, on the basis of individual
es associated with genotoxicity are
lung cancer accounted for 22 910 genomic analysis, it is not possible
being defined in steadily greater
somatic base substitutions, of which to differentiate between cases of
detail, it is not always possible to
134 were in coding sequences pancreatic cancer in smokers and in immediately discriminate between
(Pleasance et al., 2010). The role never-smokers (Wei et al., 2012). individual chemicals on the basis
of tobacco smoke as a determinant More generally, although mutation of whether particular substances
of the genomic landscape of lung of TP53 is highly relevant to colorec- should be categorized as genotox-
cancer has been confirmed, with an tal cancer, the impact of exogenous ic. Difficulties are evident when rel-
average mutation frequency in lung influences or causal factors on the evant chemicals are considered on
tumours from smokers of more than development of this tumour type is a case-by-case basis. More than

98
Table 11.3. Examples of categories of non-genotoxic carcinogens as variously proposed over more than three
decadesa

Weisburger and Weisburger (1989) Marquardt (1999) Hernández et al. Benigni et al. (2013)
Williams (1981) (2009)

Solid-state Halogenated Cytotoxic Endocrine modifiers Peroxisome proliferators


carcinogens compounds carcinogens

Hormones Immunosuppressants Tumour promoters Receptor-mediated Gap-junction inhibitors

Immunosuppressants Hormones Hormones Non-receptor- DNA-methylating agents


mediated

Co-carcinogens Solid-state materials Immunosuppressants Promoters Agonists/antagonists


of the aryl hydrocarbon
receptor

Promoters Certain Peroxisome Tissue-specific toxicity Oxidative stress inducers

CHAPTER 11
hypolipidaemic proliferators and inflammation
carcinogens inducers

PART 2
Phthalate ester Solid bodies or Cytotoxic agents and Hormonal imbalance
plasticizers particles immunosuppressants inducers

Gap-junction inhibitors
a Typically, the listings have been provided by the respective authors for illustrative purposes, without necessarily specifying an intent
to be comprehensive.

20 years ago, Ashby (1992) reported • the agent’s metabolism and Non-genotoxicity: multiple
on “practical examples of instances toxicokinetics; mechanisms and pathways
in which the term genotoxic is both • structural similarities to recognized
Regardless of any difficulty with par-
needed and capable of having differ- mutagenic carcinogens;
ent meanings”. Two decades later, ticular agents as discussed in the
• the origin of or mechanisms under-
Eastmond (2012) provided insight by previous section, the conceptual ba-
lying the observed effects; and
summarizing data for another set of sis of genotoxicity is unequivocally
• in vivo data, particularly in the
chemicals, different from those dis- focused on a particular pathway to
target organ.
cussed by Ashby (Table 11.2). malignant transformation. No such
Eastmond (2012) illustrated each
Hence, there are some chemicals single focus is available for non-
of these points with two or more
that are not readily categorized in re- genotoxic carcinogens, as illustrat-
examples.
lation to genotoxicity because, for ex- ed by the designation “epigenetic”,
Specifying genotoxicity is com- which, although previously applied
ample, they produce positive results
plex, as becomes evident when to these agents (Weisburger and
when assessed by use of in vitro
all available mechanistic data are Williams, 1981; Benigni et al., 2013),
genotoxicity tests but after their ad-
identified, as occurs, for example, can no longer be unequivocally used
ministration to intact animals, they do
in IARC Monographs evaluations. In in this context.
not cause structural DNA damage or
other manifestations of genotoxicity. some instances, the totality of avail- Epigenetic processes are relevant
As described by Eastmond (2012), able mechanistic data may indicate to both genotoxic and non-genotoxic
apparently contradictory findings that the categorization of a carcino- agents (Pogribny et al., 2008), and
can be reconciled when, for differ- gen as genotoxic is equivocal. There epigenetic change may be deter-
ent individual chemicals, account is does not appear to be a context in mined by mutation (You and Jones,
taken of: which awareness of the hallmarks 2012). From a different perspective,
• the chemical properties of the would provide an improved basis for when discussing non-genotoxic car-
agent, its metabolites, and/or its identifying genotoxic carcinogens cinogens, Meza et al. (2010) iden-
degradation products; specifically. tified tobacco smoke and radon in

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 99
this context. Despite such ambigu- of cell differentiation and cycling, equally recognized (Schetter et al.,
ity, 45 non-genotoxic carcinogens hormonal and nutritional homeosta- 2010). Cell proliferation in this con-
were recognized in 2009 among sis, coordination of cellular stress text does not pertain to proliferation
371 agents classified by IARC in responses (including inflammation after toxic injury by genotoxic agents.
Group 1, Group 2A (probably carci- and apo­ptosis), immune responses, Proliferative activity induced by ge-
nogenic to humans), and Group 2B and ageing. Therefore, it is difficult nomic injury may be considered in
(possibly carcinogenic to humans) to identify AhR-mediated processes relation to the pluripotent stem cells
(Hernández et al., 2009). with a specific hallmark. (Cohen and Arnold, 2011), further in-
Grouping agents on the basis of a The adoption of a mechanistic ap- dicating how a characteristic – such
default criterion – i.e. that the agent proach to categorize non-genotoxic as the hallmark “sustaining prolifer-
is not genotoxic – implies uncertain- carcinogens leads to incongruities ative signalling” – cannot readily be
ty. The scope of uncertainty can be if definitive and exclusive specifica- assigned or restricted to a particular
seen from differences between re- tions are sought. Thus, TCDD may category of carcinogens.
ports indicating categories of agents be readily identified as a promoter
that are reasonably considered (Ray and Swanson, 2009) while also Public health decision-
to be non-genotoxic carcinogens; being recognized as a complete car- making: the definitive
Table 11.3 shows selected examples cinogen on the basis of bioassay and consideration
from 1981 to 2013. epidemiological data (Baan et al.,
Parameters used to identify 2009). Similarly, although PAHs can This IARC Scientific Publication is
non-genotoxic carcinogens include be identified with the genotoxicity of, based on evaluations made in Volume
either the nature of the agent or for example, tobacco smoke, Puga 100 of the IARC Monographs. Two
some indicator of a putative mecha- et al. (2009) noted that exposure to broad issues are addressed: (i) the
nism of action. The terminology is far toxic PAHs raises several toxic and extent to which the occurrence and
from definitive. Thus, while the term carcinogenic responses in experi- anatomical site of agent-attributable
“promoter” may be used to identify a mental animals and humans, medi- cancer in humans may be correlat-
non-carcinogen that contributes to ated for the most part by AhR. Such ed with the occurrence and, where
tumour development, tumour promo- apparent paradoxes indicate that relevant, organ site of tumours in
tion may be identified with the action although mechanistic categorization animals treated with the same agent;
of many non-genotoxic carcinogens of many genotoxic carcinogens is and (ii) whether known mechanisms
(Schulte-Hermann et al., 1999). definitive and exclusive, the same of action of the carcinogenic agents
in question, considered together
The role of receptors has long process applied to non-genotoxic
with current knowledge of cancer
been recognized as key to the car- agents may lead to outcomes de-
etiology, reveal options for catego-
cinogenicity of many non-genotoxic termined by context. The relevant
rizing carcinogens, so as to better
agents (Lucier, 1992) and under- agents cannot be identified with a
indicate the risk posed to humans by
pins current commentaries (Klaunig, single path to malignancy.
exposure.
2010). Relevant receptors include The role of cell proliferation in re-
These two considerations are inti-
the aryl hydrocarbon receptor (AhR), lation to non-genotoxic agents also
mately related. Thus, the occurrence
the peroxisome proliferator-activated depends on the context (Preston-
or absence of tumours in rodents
receptor (PPAR), and various hor- Martin et al., 1990; Marquardt,
treated with particular agents may
mone receptors. 1999). With respect to chemicals, the
be wholly dependent on biological
Arguably, AhR is recognized original focus was on mitogens, in- mechanisms operating, or not oper-
mainly as mediating the carcino- cluding peroxisome-proliferating car- ating, in particular species. Until now,
genicity of 2,3,7,8-tetrachlorodiben- cinogens (Butterworth et al., 1992). mechanistic assessment of carcino-
zo-para-dioxin (TCDD). However, This approach now identifies inflam- gens has not established a compre-
as specified by Matsumura et al. mation as contributing to cancer de- hensive basis for determining wheth-
(2009), apart from mediating toxic velopment, and auto-inflammatory er particular agents are capable of
effects of some pollutants, AhR is disease and the impact of various causing cancer in humans. This situ-
involved in development, regulation cancer-causing infectious agents are ation confirms that evaluations of the

100
IARC Monographs are appropriate available data are ordered according correlates with the enabling charac-
for hazard identification, as distinct to these end-points, it is evident that teristic “genomic instability and mu-
from any simple categorization of for many agents, simple categoriza- tation”. The end-points “alterations in
relevant agents. The fact that agents tion according to a single mechanism telomere length” and “immortaliza-
may be classified into Groups does is not possible or appropriate. tion” address the hallmark “enabling
not alter the need to make evalua- An important consideration is the replicative immortality”.
tions on a case-by-case basis. discrepancy between the extents It would appear that the hallmark
The determination of whether a “evading growth suppressors” cor-
to which end-points have been as-
chemical induces cancer through
sessed. DNA damage and gene responds to end-points identified by
a genotoxic mechanism frequently
mutations have been studied most cell-cycle effects taken together with
plays an important role in evaluating
extensively, and agents for which a subset within the end-point “gene
the risks associated with low expo-
there is unequivocal evidence of ge- mutations”: the subset of mutation of
sures (Eastmond, 2012). For low
notoxicity across in vitro and in vivo tumour suppressor genes as distin-
levels of exposure to non-genotoxic
systems have rarely been studied in guished from mutation of oncogenes
carcinogens, there is expected to
relation to, for example, epigenetic or other genes. The default position
be a dose–response threshold for

CHAPTER 11
the carcinogenic effects; this does alterations. Epigenetic alterations would then be to identify “sustaining

PART 2
not apply to genotoxic carcinogens have been described for estrogenic proliferative signalling” – arguably
(Klaunig, 2010). Low-dose models of hormones (Imamura, 2011), arse- the premier hallmark – with the re-
liver cancer induction in fish by geno­ nic (Jensen et al., 2008), and nickel maining end-points. However, ref-
toxic carcinogens indicate further (Costa et al., 2005), although each erence to those end-points leads to
levels of complexity (Williams, 2012), of these agents had also been char- the recognition that end-points such
and ongoing controversy about acterized as causing DNA damage. as “epigenetic alterations” are the
non-monotonic responses means Evidence of immunosuppression means through which many, if not all,
that such issues remain pertinent may have been considered as a sin- of the hallmarks may emerge.
(Fagin, 2012). Mechanisms that un- gular mechanism of carcinogenesis, Finally, “deregulating cellular en-
derpin, for example, dose–response but while azathioprine can be char- ergetics” remains as the hallmark
curves may become amenable to ge- acterized as immunosuppressive, not addressed through the char-
nomic and related analyses. acteristics identified, because this
this agent also causes DNA damage.
Having been adopted as de- parameter has not been recognized
Systematic appraisal
scribed, the 10 key characteristics in systematic efforts to character-
of mechanisms of
warrant review with reference to the ize mechanisms of carcinogenesis.
carcinogenesis
hallmarks as cataloguing a broad Overall, no particular insight ap-
Information about mechanisms of biological basis for malignancy pears to be gained by attempting to
carcinogenesis for the Group 1 (Hanahan and Weinberg, 2011). One relate the 10 key characteristics with
agents in the IARC Monographs hallmark, “activating invasion and specific hallmarks.
is summarized in this Scientific metastasis”, is not recognized as a
Tobacco smoke, cancer of the
Publication with initial reference to mechanistic end-point because few,
lung, and the hallmarks
24 mechanistic end-points, which if any, agents are identified primarily
were then merged into 10 key with metastatic growth, given that no Generalizing across tumour types,
characteristics (see Chapter 10, by such hazard needs to be established genomic and comparable analyses
Smith). These end-points – which over and above carcinogenicity. are concerned little, if at all, with ex-
include DNA damage, changes in Some hallmarks are singularly iden- ogenous agents that mediate malig-
gene expression, receptor-mediated tified as mechanistic end-points or nant transformation. Paradoxically,
effects, and inhibition of gap junc- enabling characteristics, i.e. those the first tumour genome document-
tional intercellular communication – corresponding to chronic inflam- ed was described with a total focus
have been adopted on the basis of mation, immune effects, cell death, on mutations attributable to tobac-
their wide use to investigate mech- and angiogenic effects. Arguably, co smoke (Pleasance et al., 2010).
anisms of carcinogenesis. Once the the end-point “DNA repair alteration” Although genomic analysis revealed

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 101
Fig. 11.1. Hallmarks of lung adenocarcinoma. Left: The prevalence of mutation or somatic copy number alterations
of genes mapping to cancer hallmarks defined by Hanahan and Weinberg (2011) based on tumour specimens
from a cohort of 183 patients of whom more than 85% had a history of smoking. Top right: Genes comprising
the mutated genes in the hallmark “sustaining proliferative signalling” are shown. Bottom right: A proposed new
hallmark of “epigenetic or RNA deregulation” is shown, depicted as above. Genes shown in grey are candidate
lung adenocarcinoma genes identified in the study of Imielinski et al. (2012) that may additionally contribute to the
hallmark. Reprinted from Imielinski et al. (2012), copyright 2012, with permission from Elsevier.

102
an average mutation frequency in Possible inferences from oncogene activation was achieved
lung tumours from smokers of more hallmark-based studies by genetic manipulation or after ex-
than 10 times that in lung tumours posure to an alkylating N-nitroso
from never-smokers (Govindan et al., Any malignancy is expected to ex- compound (Westcott et al., 2015).
2012), the genomic pattern of squa- hibit the hallmarks, whether it arises Hence, genomic analysis may reveal
mous cell lung cancer, established spontaneously or upon exposure to a distinct patterns of tumour-associ-
from 178 patients of whom 96% had carcinogen. Insight into mechanisms ated changes that are dependent
of carcinogenesis is gained by the on etiology and relevant to the full
a history of smoking, was presented
demonstration of biological change, scope of tumour-associated signal
with no overt reference to tobacco
which may be aligned with a hallmark transduction as identified by the
use (Hammerman et al., 2012).
(He et al., 2014). The public health hallmarks.
The genomic profile of lung ade-
implications of such a discovery may Apart from any mechanistic cate-
nocarcinoma, involving a cohort of
apply to agents not recognized as gorization of carcinogens in relation
patients of whom more than 85%
carcinogenic but shown to be pro- to particular hallmarks, the hallmarks
had a history of smoking, was pre-
moters and/or inducers of inflamma- do provide a basis for innovation.
sented with reference to the hall- tion or angiogenesis. Nicotine is an Genes identified from the perspec-
marks, documenting the prevalence

CHAPTER 11
example of such an agent (Cardinale tive of each hallmark provide a ba-

PART 2
of the enabling characteristic “geno- et al., 2012; Schaal and Chellappan,
sis on which to analyse both known
mic instability and mutation” in 25 ad- 2014). In addition to its contribution
carcinogens and agents of unknown
enoma genes adopted as indicators to a better understanding of tobacco
status in that regard. An indication
(Imielinski et al., 2012). The findings smoke carcinogenesis, this informa-
of agents worthy of attention may
were not presented with reference tion about the properties of nicotine
well be achieved by adding hall-
to smoking status but indicated is relevant to appropriate regulation
mark-related targets in the context of
markedly different fractions of mu- of electronic cigarettes (also known
high-throughput screening assays,
tation (Fig. 11.1), including 42% with as electronic nicotine delivery sys-
as described by Kavlock and col-
respect to “genomic instability and tems) (Dutra and Glantz, 2014).
leagues (Kleinstreuer et al., 2013).
mutation”. This result indicates the Nicotine may contribute to cancer
The outcome may be the recognition
requirement to distinguish between development, for example by stimu-
of new classes of toxins that contrib-
gene mutation being relevant to eti- lating angiogenesis, in a manner not
ute to increased risk of cancer.
ology, whether or not it is caused by likely to result in the compound being
an exogenous agent, and frequency designated a carcinogen.
Summary
of mutation being an indicator of ge- During the past 50 years, the
nomic instability and thus a charac- understanding and use of the term Cancer was once described with ref-
“carcinogenesis” has changed from erence to causative agents, and mul-
teristic of malignancy. Also of note,
that involving a necessary reference tistage development of tumours was
only 6% of tumours had alterations
to one or more exogenous carcin- characterized through the impact of
assigned to all six original hallmarks.
ogens to that involving intracellu- particular chemicals. Subsequently,
Mutation of genes that mediate
lar processes leading to malignant multistage development of cancer
particular hallmarks and are at-
transformation, with no necessary was identified with morphological
tributable to, among other agents,
or implied reference to exogenous change being correlated with altered
N-nitroso derivatives of nicotine and
agents. This understanding has genetic makeup. The more recent
related compounds, and PAHs, is to description of eight hallmarks of ma-
recently included the description of
be expected. However, beyond lung random mutations arising from DNA lignancy is based not on morpholo-
cancer, there are only few references replication in normal non-cancerous gy or on the impact of carcinogens
to genomic analyses that enable indi- stem cells as accounting for sporadic but on changes in gene expression,
vidual tumours attributable to smok- disease (Tomasetti and Vogelstein, sometimes mediated by mutation,
ing to be distinguished from others. 2015). However, another recent and on selection for growth.
Thus, genomic analysis did not re- development is the identification In parallel to this evolution of our
veal likely tobacco causation for par- of different mutational landscapes understanding of cancer, no gener-
ticular pancreatic cancers (Biankin between classes of K-ras-driven ally recognized mechanism-based
et al., 2012). tumours, depending on whether scheme for classifying carcinogens

Part 2 • Chapter 11. Mechanisms of carcinogenesis: from initiation and promotion to the hallmarks 103
has evolved beyond categorization variously resulting in emergence signal transduction pathways as-
of chemical carcinogens accord- of the hallmarks, with the relevant sociated with particular hallmarks
ing to genotoxicity. When appropri- processes being facilitated by ge- may provide new understanding of
ately studied, both genotoxic and nomic instability and inflammation. agent-related carcinogenesis.
non-genotoxic agents may medi- Enhancing –omics-based screening
ate genetic and epigenetic change, procedures to specifically include

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106
part 2.
mechanisms of carcinogenesis

chapter 12.

The role of genotoxicity


in carcinogenesis
David M. DeMarini

CHAPTER 12
PART 2
The process of mutagenesis be called DNA-damaging agents. of a molecule between a pair of
Instead, it is the cell that produces bases. Again, DNA damage is itself
The process of agent-induced mu- the mutation – either through faulty not a mutation and generally does
tagenesis consists of three parts: the DNA repair of the mutagen-induced not alter the linear sequence of nu-
induction of DNA damage, the sen- or spontaneous DNA damage, or cleotides. A mutation is defined as a
sing of the DNA damage by the cell by replicating past the unrepaired change in the sequence or number
(the DNA damage response), and DNA damage, thereby introducing a of nucleotides in the DNA.
the processing of the DNA damage replication error (Shaughnessy and When DNA damage occurs, the
by the cell, which may or may not DeMarini, 2009). cell detects it by means of the DNA
result in a mutation. A key under- A description of the process of damage response system and de-
lying concept is that mutagenesis is mutagenesis begins with the induc- termines how it will be processed;
a cellular process, frequently invol- tion of DNA damage by an endoge- the DNA damage response includes
ving DNA replication. Another key nous or exogenous event. Examples DNA repair and apoptosis path-
concept is that there is a distinct dif- of DNA damage are DNA adducts ways, which are described in detail
ference between DNA damage and (i.e. a molecule bound covalently to by Ciccia and Elledge (2010). The
mutation. Thus, mutagens, despite DNA) and single- or double-strand DNA damage response can mediate
what their name suggests, generally breaks (i.e. breakage of the phos- the repair of the damage, attempt to
do not produce mutations; instead, phodiester backbone). Other types repair the damage but instead pro-
mutagens produce DNA damage, of DNA damage are oxidized or frag- cess it into a mutation, or direct the
and they might more appropriately mented bases and the intercalation cell to undergo apoptosis. Another

Part 2 • Chapter 12. The role of genotoxicity in carcinogenesis 107


possibility is that the damage is not induces mutations in a mutation integrated viral genome (Todaro and
repaired at all, and when the cell rep- assay permits it to be classified as Huebner, 1972), and alteration of im-
licates, the DNA polymerase correct- mutagenic. munological factors by carcinogens,
ly bypasses the damage, resulting in permitting the formation and growth
a normal DNA sequence. A brief history of the nexus of tumours (Baldwin, 1973). As time
between mutagens and has shown, all of the above-men-
The cell can process DNA dam-
carcinogens
age into three general classes or tioned mechanisms can play a role
types of mutation: gene mutation in the carcinogenic process, espe-
As reviewed by Claxton et al. (2010),
(mutations that occur within a gene), cially in the light of the accumulating
there was little direct evidence for
chromosomal mutation (mutations evidence for the important role of
the role of mutagenesis in carcino-
involving more than one gene, typ- epigenetic mechanisms (Baylin and
genesis until the early 1970s, and
ically called chromosomal aberra- Jones, 2011).
before that time only a few carcino-
tions), and genomic mutation (mu- How did the paradigm shift occur
gens had been shown to be muta-
tations involving the whole genome that showed a connection between
gens (Burdette, 1955). Indeed, it is
– generally aneuploidy, which is mutagenesis and carcinogenesis?
surprising to recall that at the time it
the gain or loss of a whole chromo- The first screening studies to test
was somewhat bold to propose that
some). The standard definition of a there was any direct connection be- the hypothesis that some carcino-
tween the two processes (Miller and gens might also be mutagens were
gene refers to a segment of DNA that
Miller, 1971; Knudson, 1973). Many performed by Demerec et al. (1951)
codes for an mRNA that codes for a
studies in the 1950s and 1960s in Escherichia coli and then by
protein. The recent Encyclopedia of
showed binding of carcinogens to nu- Szybalski (1958), who tested more
DNA Elements (ENCODE) project
cleic acids (Wiest and Heidelberger, than 400 compounds in E. coli.
indicates that at least 80% of the
1953; Brookes and Lawley, 1964). Although additional testing proceed-
human genome is transcriptionally
However, before 1972 there was no ed throughout the 1960s in a varie-
active, but only a small proportion
direct proof that the electrophilicity ty of systems in bacteria, fungi, and
of the expressed regions code for
of some chemical carcinogens had mammalian cells, few carcinogens
protein (Maurano et al., 2012).
a necessary role in the potential mu- other than the direct-acting alkylating
In the context of the process of
tagenic activity of such compounds, agents were found to be mutagens,
mutagenesis described above, the
or even that DNA, as opposed to leading to the conclusion that carcin-
term “mutagen” refers to an agent
protein, was the ultimate target of ogens were generally not mutagenic.
that can induce DNA damage that
carcinogens (Miller, 1970). However, this view began to shift
the cell processes into a mutation.
Although sound theoretical rea- when Malling (1966) combined a
The more general term “genotox- chemical hydroxylating mixture with
sons had been proposed to support
in” refers to an agent that induces the carcinogens diethylnitrosamine
the notion that carcinogens might act
DNA damage that may or may not and dimethylnitrosamine, which
through a mutagenic mechanism,
be processed by the cell into a mu- were not mutagenic in vitro, and
a clear demonstration of this con-
tation. Some assays for genotoxici- showed that the resulting metabo-
nection did not yet exist (Miller and
ty, for example, measure only DNA lites were mutagenic in the fungus
Miller, 1971). Thus, binding to DNA of
damage, such as 32P-postlabelling metabolites of carcinogens had been Neurospora crassa. Malling (1971)
and the comet assay, whereas oth- identified, but there were no data to then prepared an enzymatic activa-
er assays measure mutation, such show that these DNA adducts were tion system composed of the super-
as the Salmonella typhimurium re- processed into mutations or that mu- natant from mouse liver homogenate
verse mutation test, the Hprt gene tations themselves played a role in centrifuged at 30 000g (microsomes)
mutation assay in Chinese hamster carcinogenesis. Consequently, mu- plus cofactors, and showed that di-
ovary cells, and transgenic mouse tagenesis was viewed at that time as methylnitrosamine was mutagenic
mutation assays. Thus, finding that an equally plausible mechanism for in S. typhimurium in a liquid suspen-
an agent induces DNA damage carcinogenesis, along with epigenet- sion assay in the presence of this ac-
would permit it to be called genotox- ic changes (Miller, 1970; Miller and tivation mixture. Additional evidence
ic, and showing that the agent also Miller, 1971), altered expression of an that carcinogens could be mutagens

108
after mammalian metabolism was Indeed, a comprehensive analysis present at high frequencies in tu-
provided by Legator and Malling showed that more than 90% of the mours, DNA sequencing methods
(1971) with the host-mediated assay. IARC Group 1 chemical carcinogens were introduced in 1977 (Pettersson
Ames et al. (1972) introduced the are genotoxic (Waters et al., 1999). et al., 2009), which provided the tech-
use of the plate incorporation assay The current genetic toxicity test nical means to directly determine the
in Salmonella and demonstrated that battery is based on this relationship presence and types of mutations in
DNA-reactive metabolites of known between mutagenesis and carcino- any gene or chromosome.
carcinogens were direct-acting mu- genesis. Consequently, mutagenic- DNA sequencing of mutations
tagens. The connection between ity assays continue to be used as induced in selected genes by a lim-
mutagenesis and carcinogenesis a potential screen for carcinogens, ited number of mutagenic carcino-
was extended when Ames et al. and the results are used for regula- gens in microbes in the 1980s and in
(1973) combined a rat liver homoge- tory purposes throughout the world mammalian cells and tumours in the
nate centrifuged at 9000g (S9 frac- (Eastmond et al., 2009). For exam- 1990s began to show that any par-
tion) plus cofactors prepared as de- ple, a positive result in the Salmonella ticular mutagen produced an array
scribed by Garner et al. (1972) with mutagenicity assay indicates a 70% of mutations and that these varied
Salmonella and a variety of rodent among the genes and cells exam-

CHAPTER 12
probability that the test chemical is

PART 2
carcinogens then considered to be a rodent carcinogen (Zeiger, 1998). ined. A variety of mutagens produce
non-mutagenic in the plate incorpo- When a randomly selected set of similar mutation spectra, and the
ration assay and showed that these 100 organic compounds was tested predominant base substitution that
carcinogens were, in fact, muta- in the Salmonella mutagenicity as- an agent induces in one system
genic. Additional refinements of the say, about 20% of them were posi- is generally the same one that the
Salmonella tester strains and the tive (Zeiger and Margolin, 2000). agent produces predominantly in all
conduct of multiple testing studies, Thus, out of an estimated 80 000 other systems across the phyloge-
involving not only Salmonella but such compounds in commercial use, netic scale, from bacteria to humans
also other test systems (Tennant 16 000 (20%) may be positive for (DeMarini, 1998, 2000). Thus, in
et al., 1987), resulted in the current mutagenicity in the Salmonella mu- terms of the predominant base sub-
recognition that many carcinogens, tagenicity assay, and 11 200 (70%) stitution produced by agents, there is
by themselves or after metabolic of those may be potential rodent concordance across species in that
activation, are mutagens, and that carcinogens. the DNA damage induced by a par-
mutagenesis is a critical feature of ticular agent is processed similarly
carcinogenesis. Mutations in tumours by a wide range of species.
Despite the recognized impor- With regard to mutations in tu-
tance of mutagenicity as a part of Soon after the discovery of the mours, generally elucidated without
cancer induction and progression, by correct number of human chromo- reference to any exogenous caus-
the 1990s it appeared that many ro- somes (46) by Tjio and Levan in ative agent, the technology in use
dent and human carcinogens were, 1956 (Gartler, 2006; Harper, 2006), from the 1980s until the early 2000s
in fact, not clearly mutagenic or ge- cytogenetic studies began to show permitted the determination of mu-
notoxic. Some operate through re- that tumours (specifically leukaemic tations in only a few cancer-related
ceptor binding, which can result in an cells) had higher frequencies of chro- genes, such as TP53 and KRAS.
alteration in gene expression, often mosomal aberrations than did nor- The first gene mutation in a human
leading to increased cell replication. mal cells (Nowell and Hungerford, tumour was determined in 1982
However, an analysis of a set of so- 1960). A decade later, the develop- (Reddy et al., 1982), and by the end
called non-genotoxic carcinogens ment of quinacrine fluorescence and of the 20th century, there was clear
found that most of them were, in fact, Giemsa staining enabled the first evidence that some tumours had
genotoxic (inducing DNA damage discovery that a specific chromoso- mutations in certain oncogenes and
and/or mutation) when tested ade- mal aberration was associated with tumour suppressor genes that could
quately for both gene, chromosomal, a specific type of leukaemia (Rowley, be associated with the types of mu-
or genomic (aneuploidy) damage 1973). As evidence accumulated tations produced by the carcinogen
and mutation (Jackson et al., 1993). that chromosomal aberrations were associated with the induction of the

Part 2 • Chapter 12. The role of genotoxicity in carcinogenesis 109


tumour – both in rodents and in hu- specific to particular tumour types be required for some haematopoietic
mans (Dogliotti et al., 1998; Hainaut and subtypes (Hoang et al., 2013; tumours, whereas at least five or
and Wiman, 2009). Examples in- Alexandrov and Stratton, 2014). six are required for solid tumours
As discussed elsewhere in (Stratton, 2011). In addition, all tu-
clude CC  →  TT mutations in the
this Scientific Publication (see mours have many genes with altered
TP53 gene in skin tumours associat-
Chapter 11, by Stewart, and gene expression (Baylin and Jones,
ed with exposure to sunlight, G → T
Chapter 19, by Caldwell et al.), can- 2011).
mutations in codon 259 of the TP53 cer is a genetic disease that pro- A recent discovery is “shattered”
gene in liver tumours associated with ceeds by a type of Darwinian evolu- chromosomes in tumours, a phe-
exposure to aflatoxin B1 (Ceccaroli tion (Hanahan and Weinberg, 2011). nomenon termed chromothripsis,
et al., 2015), A → T mutations in the In this way, changes in gene function which results in massive chromo-
TP53 gene in tumours of the upper (by mutation) and in gene expression somal rearrangements in 1–3% of
urinary tract associated with expo- (by epigenetic mechanisms) that re- human tumours (Stephens et al.,
sult in a cell having a specific growth 2011). Studies indicate that these
sure to aristolochic acid (Grollman,
advantage may be selected for in shattered, highly rearranged chro-
2013), and the different locations of
certain tissue microenvironments mosomes may appear exclusively
G  →  T mutations in the TP53 and
(Hanahan and Weinberg, 2011; in micronuclei (Crasta et al., 2012;
KRAS genes in lung tumours as- Solomon et al., 2011; Whitfield and Maher and Wilson, 2012), providing
sociated with exposure to cigarette Soucek, 2012). a new insight into the potential role
smoke (IARC, 2012) or to emissions Stratton (2011) estimated that of micronuclei in tumours (Hatch and
from smoky coal (DeMarini et al., most human tumours contain 1000 Hetzer, 2015).
2001). There are about 20 carcino- to 10 000 base substitution muta- As evidence had accumulated
gens that are known to produce ei- tions; tissues exposed more directly that mutation and mutagenesis are
to the environment, such as the lung essential features of carcinogenesis,
ther unique or distinctive mutation
and the skin, have tumours with more the notion that tumours might be mon-
spectra in tumours linked epidemi-
than 100 000 mutations. However, oclonal became popular, because
ologically to specific exposures in only about 400 genes (~2% of the of the monoclonality of haemato-
humans (Ceccaroli et al., 2015). coding genome) appeared to be poietic malignancies and because
With the advent of next-genera- involved directly in tumorigenesis; this notion appeared to support the
tion DNA sequencing, exome and the rest were likely to be passenger prevalent initiation–promotion model
genome sequencing of tumours mutations, i.e. mutations not relat- of carcinogenesis. However, as mo-
was first reported by Wood et al. ed to the carcinogenic process and lecular analyses of tumours became
possibly resulting from the genomic more sophisticated, it soon became
(2007) and Parsons et al. (2008).
instability of the tumour (Bozic et al., clear that tumours are not monoclo-
These initial studies revealed that tu-
2010). nal and that they are, in fact, highly
mours had large numbers of mutat-
Genome or exome sequencing heterogeneous (Parsons, 2008).
ed genes; however, it appeared that has identified 20 distinct mutational The most exquisite evidence for
only a few (six to eight) genes were signatures among human tumours this has been provided by Gerlinger
involved directly in the carcinogenic and confirmed that tumours of the et al. (2012), who showed that ap-
process. As few as three driver gene most “protected” organs (e.g. the proximately two thirds of all somat-
mutations are required for the devel- brain) have only a few mutations, ic mutations were not present in all
opment of lung cancer or colorec- whereas those in organs exposed regions of a set of kidney tumours
more directly to the environment analysed by a combination of exon
tal cancer (Tomasetti et al., 2015).
(e.g. the lung and the skin) have sequencing, chromosomal aberra-
Large-scale sequence analysis of
thousands of mutations (Alexandrov tion analysis, and mRNA expression
the genomes of thousands of human
et al., 2013). The vast majority of mu- analysis. Recently, Martincorena
tumours has identified new genes tations in tumours are base substitu- et al. (2015) demonstrated that this
that are important for cancer and tions (Vogelstein et al., 2013). Only heterogeneity is established early
new mutational signatures that are one or two mutated genes appear to on, by showing that physiologically

110
normal human skin contains a epigenetic changes per se are not disease and (ii) an agent that causes
patchwork of thousands of evolving mutations because the sequence of cancer induces alterations in gene
clones, with more than one quarter nucleotides has not been changed, function (by mutation) and/or gene
of such cells having cancer-causing as evidenced above, mutation may expression (by epigenetic changes),
mutations. be the basis for some epigenetic either by direct interaction with DNA
Although there is now also over- events. or chromatin or by indirect mecha-
whelming evidence for the essential nisms, such as through generation
role of epigenetic changes in the car- Models of agent-induced of reactive oxygen species, inflam-
cinogenic process (Grønbaek et al., carcinogenesis mation, and/or receptor-mediated
2007; Baylin and Jones, 2011) and interactions. These considerations
Data generated in recent years have
for the fact that many carcinogens suggest that carcinogens must be ge-
led to a reconsideration of the dichot-
can induce such changes (Ceccaroli notoxic in the broadest sense of the
et al., 2015; Nicolaidou and Koufaris, omy between so-called non-geno- term, i.e. they damage DNA or alter
2015), as discussed below there is toxic versus genotoxic carcinogens its expression either directly or indi-
emerging information that mutation (Waters et al., 1999) and indicate rectly, leading to a change in function
itself might underlie some, if not that some epigenetic events may or expression of genes. Such chang-

CHAPTER 12
have a mutational basis (You and

PART 2
most, of these epigenetic changes. es in the appropriate genes with pro-
There are three primary epigenet- Jones, 2012). In addition, chronic motion through cell replication and
ic mechanisms by which cells regu- inflammation, which is associated selective pressure can then lead to
late gene expression: methylation of with increased cancer risk (Colotta a tumour. For colorectal tumours
DNA (Hsiao et al., 2009), modifica- et al., 2009), causes DNA damage this concept has been characterized
tions of histones (Ellis et al., 2009), (etheno-base lesions and other exo- as the “Big Bang” model for tumour
and binding of microRNAs and other cyclic DNA adducts) that appears to growth, in which tumours start early
non-coding RNAs to the genome or be the basis for the increased risk, on producing mixed subclones that
to other RNAs (Garzon et al., 2009). as demonstrated by the fact that re- are not subject to stringent selection,
However, studies have shown that pair of the damage by base excision thus explaining the heterogeneity of
mutations in genes involved in these repair enzymes (alkyl glycosylases) tumours (Sottoriva et al., 2015).
three processes may be the basis for reduces the risk of cancer (Calvo This greater appreciation for how
many of the epigenetic events me- et al., 2012). Indeed, an analysis of a chemical, physical, and biological
diated by these mechanisms (You dozen human studies found strong- agents may induce cancer leads to
and Jones, 2012). For example, mu- ly increased risks of cancer among a model for agent-induced carcino-
tations in specific chromatin-modi- individuals with high levels of DNA genesis that integrates portions of
fying genes appear to occur in spe- adducts relative to those with low lev- the classic initiation–promotion mod-
cific cancers, such as in JARID1C els, and the cancer risks were even el with elements of the hallmarks of
in renal cancer, in SMARCA4/BRG1 higher for the group with high adduct cancer. Such a model would envi-
in lung cancer, and in ARID1A in levels when other risk factors, such sion a carcinogenic agent establish-
ovarian cancer (Jones et al., 2010). as infection and inflammation, were ing the process by either genetic or
Also, mutations in the DNA meth- taken into account (Poirier, 2012). As epigenetic mechanisms that cause
yltransferase genes DNMT1 and noted in Chapter 19, by Caldwell et changes in gene function and ex-
DNMT3A are found in colorectal al., host susceptibility factors mod- pression, resulting in the plethora of
cancer or acute myeloid leukaemia, ulate all of these events and are a characteristics of cancer cells, i.e.
the histone lysine methyltransferas- critical element in the overall cancer the hallmarks of cancer: mutations
es or demethylases HK4, H3K9, and risk. in key oncogenes, altered gene ex-
H3IK27 are mutated in kidney can- Within the context of both the ini- pression, changes in cell signalling,
cer and colon cancer, and the his- tiation–promotion model of carcino- altered cell growth, evasion of ap-
tone acetyltransferases H3K18 and genesis and the “hallmarks” of can- optosis, sustained angiogenesis,
H3K27 are mutated in acute lymph- cer (Hanahan and Weinberg, 2011), increased genomic instability, and
oblastic leukaemia (Peltomäki, 2012; these data have led to the view that eventual metastasis. Much of this
Ryan and Bernstein, 2012). Although (i) cancer is essentially a genetic can be modulated by various sus-

Part 2 • Chapter 12. The role of genotoxicity in carcinogenesis 111


ceptibility factors, including genetic theory of cancer, such as tissue changes in cell signalling that result
or epigenetic factors, as well as by organization field theory (Baker, in the hallmarks of cancer, with the
a large number of environmental and 2015) and tissue programming theo- entire process being modified by a
lifestyle factors (see Chapter 19, by ry (Burgio and Migliore, 2015). variety of susceptibility factors.
Caldwell et al.). However, a geno- The ability of carcinogenic agents
toxic carcinogen may not necessar- Summary to induce mutation and/or alter gene
ily cause cancer via a genotoxicity expression, with either ability being
mechanism alone or predominantly, Work since the 1970s demonstrat-
sufficient to initiate the process of
and further mechanistic studies are ed that many carcinogens are either
tumour formation (Grønbaek et al.,
needed to delineate the carcinogenic directly or indirectly genotoxic or
2007; Halazonetis et al., 2008), is
mechanisms of any particular agent. mutagenic and/or alter gene expres-
now an established feature of
This generalized model no longer sion. Analyses of tumours, first by
agent-induced carcinogenesis. This
makes a distinction between ini- cytogenetic methods in the 1970s,
deeper understanding of the rela-
tiation and promotion, which was an then by single-gene analysis in the
tionship between genotoxicity and
operational model derived largely 1990s, and most recently by exome
carcinogenicity is the culmination of
from mouse skin-painting studies. or whole-genome sequencing, have
research that provided the first evi-
Similarly, it does not divide carcin- demonstrated clearly that mutagen-
dence for such a relationship only
ogens into genotoxic and non-ge- esis is a central feature of carcino-
about 40 years ago.
notoxic categories. Instead, an in- genesis. Thus, it is not surprising
tegrated model of agent-induced that more than 90% of the known
Acknowledgements
carcinogenesis as described above human chemical carcinogens (IARC
emphasizes the ability of the car- Group 1) are positive in convention- The author thanks J.A. Ross and
cinogen (chemical, physical, or bi- al short-term tests for genotoxicity C.E. Wood for their helpful com-
ological) to alter gene structure (by (Waters et al., 1999). ments on this manuscript.
mutation) and/or gene expression Cancer is now recognized as an
(by genetic or epigenetic changes), essentially genetic disease, with car- Disclaimer
leading to functional changes in the cinogens causing genetic damage
genome that manifest themselves and/or changes in gene expression This article was reviewed by the
through changes in cell signalling, either directly or indirectly. This rec- National Health and Environmental
altered cell growth, and genomic ognition should prompt a reconsid- Effects Research Laboratory,
instability, resulting in the hallmarks eration of the distinction between United States Environmental
of cancer, with susceptibility factors genotoxic and non-genotoxic car- Protection Agency and approved for
modifying various aspects of these cinogens. A generalized model of publication. Approval does not signi-
processes and outcomes. The im- agent-induced carcinogenesis would fy that the contents reflect the views
portance of epigenetic changes, no longer make a distinction between of the agency, nor does mention of
cell signalling, and tissue–cell inter- initiation and promotion but would in- trade names or commercial products
actions have suggested alternative stead emphasize the initial effects of constitute endorsement or recom-
models to the somatic mutation the agent that then lead to a series of mendation for use.

112
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CHAPTER 12
PART 2

Part 2 • Chapter 12. The role of genotoxicity in carcinogenesis 115


part 2.
mechanisms of carcinogenesis

chapter 13.

Alterations in cell proliferation,


cell death, or nutrient supply
Jane C. Caldwell

CHAPTER 13
PART 2
Introduction these agents – that can be used to 1986; Jayat and Ratinaud, 1993;
identify and organize mechanistic in- Stacey and Hitomi, 2008; Irish and
Mechanistic data have been in- formation related to cancer induction Doxie, 2014). Although for many of
cluded in Volume 100 of the IARC (see Chapter 10, by Smith; see also the human carcinogens Volume 100
Monographs, and they vary with the Smith et al., 2016). One of these 10 of the IARC Monographs contains
agent studied. These data are espe- mechanistic categories of data is a more descriptive data under this cat-
cially dependent on the type of study composite that includes information egory, primarily as changes in cell
and the contemporary understand- on the ability of a carcinogen to alter
proliferation, these changes are in-
ing of the state of the science at the cell proliferation, cell death, or nutri-
herently related to alterations in cell
time of publication of the study. ent supply.
signalling and/or cell-cycle control.
As an outcome of the two-part Alteration of cell proliferation is
Many challenges are associated
IARC Workshop on Tumour Site identified through assays that detect
with the use of data described for
Concordance and Mechanisms replicative DNA synthesis, 5-bro-
of Carcinogenesis, a mechanistic mo-2′-deoxyuridine (BrdU) labelling, alterations in cell proliferation, cell
database was assembled for the proliferating cell nuclear antigen death, or nutrient supply to examine
IARC Group 1 carcinogens (see (PCNA) labelling, and hyperplasia mechanistic and tumour site concor-
Chapter 22, by Krewski et al.). These or the occurrence of multinucleated dance between humans and experi-
agents were examined with regard to cells by light microscopy, and through mental animals. Several key mecha-
10 key characteristics – one or more analysis of some of these end-points nistic characteristics can result in or
of which are commonly exhibited by by use of flow cytometry (Gray et al., arise from changes in cell signalling

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 117
(e.g. inflammation, genotoxicity, and This chapter focuses on issues as- ellers, growth factors, growth factor
epigenetic alterations) and can have sociated with the understanding and receptors, signal transducers, and
both genetic and epigenetic origins. interpretation of available data for apoptosis regulators (Narayanan
There are other levels of interde- this key mechanistic characteristic. et al., 2015). In response to mito-
pendence between the key mecha- gens, cell proliferation is triggered by
nistic characteristics. Inflammation, Genetic drivers of cell increased translocation into the nu-
excessive oxidative stress, and ge- proliferation and apoptosis: cleus of ERK 1 and 2 (ERK1/2), the
complex relationships and
nomic instability are related (see last proteins in the MAPK/ERK cas-
pleiotropic roles of cell
Chapter 17, by Kane). Mutagenesis cade. Activating mutations upstream
signalling molecules
may also underlie some epigenetic or within the ERK1/2 cascade are
events that change cell signalling. Cell signalling is a process whereby present in several human cancers,
For example, mutations in genes proteins or other chemical messen- but ERK1/2 activation also occurs in
involved in the methylation of DNA, gers activate receptors at the cell cancers without mutation of compo-
modification of histones, and bind- surface and then transmit signals nents of the cascade (Plotnikov et al.,
ing of microRNAs to the genome or inside the cell via membrane-to-nu- 2011).
to other RNAs may initiate epige- cleus pathways. In healthy adults, Ras, a small upstream guano-
netic changes (see Chapter 12, by cell proliferation, cell differentiation, sine-5′-triphosphate (GTP)-binding
DeMarini, and Chapter 20, by Rice and cell death determine the size protein in several signalling path-
and Herceg). of the proliferating cell population ways, has two isoforms, H-Ras and
Cell signalling pathways that reg- in soft tissues, for example surface K-Ras, with different potencies to
ulate cell proliferation are not inde- epithelia, mucosal lining cells of ex- activate the MAPK/ERK pathway;
pendent of those associated with cretory ducts, columnar epithelia the KRAS gene is more frequent-
other key mechanistic characteris- lining the gastrointestinal tract and ly mutated in human cancer, which
tics of IARC Group 1 carcinogens. uterus, transitional epithelium of the can result in constitutive activation
Many of the genes associated with urinary tract, and bone marrow and (McCubrey et al., 2007). H-Ras
cell proliferation are also linked with haematopoietic cells. These pro- has been implicated as contributing
apoptosis, inflammation, and several liferating cells replace dead cells to the cancer phenotype, through
pleiotropic responses. Dysregulation throughout life. Pathological effects evasion of anti-growth signalling,
in the mitogen-activated protein (e.g. injury resulting from hepato- angiogenesis, genetic instability,
kinase (MAPK) pathway affects cellular necrosis or partial hepatec- tissue invasion and metastasis, tu-
most, if not all, processes involved tomy) and physiological conditions mour-promoting inflammation, and
in cancer (Dhillon et al., 2007). The (e.g. estrogen-induced effects on the changes in the tumour microenviron-
extracellular signal-regulated kinase endometrium during the menstrual ment (Engström et al., 2015). K-Ras
(ERK) pathway of the MAPK family cycle) can involve stimulation of cell promotes metabolic reprogramming,
is most commonly associated with proliferation (Engström et al., 2015). activation of proliferative signalling
regulation of cell proliferation (Reuter The relationships between cell pathways, glycolysis, reduction of
et al., 2010). signalling molecules and pathways oxidative metabolism in the tricar-
The ability to use this character- that control cell proliferation and pro- boxylic acid cycle, and channelling of
istic to evaluate both mechanistic grammed cell death (apoptosis) are glucose intermediates into anabolic
and tumour site concordance is in- complex. Numerous enzymes and pathways, such as the hexosamine
fluenced by recent developments in cell signalling pathways are modulat- biosynthesis pathway.
cancer research, by the overarching ed during apoptosis, and dysfunction The tumour suppressor protein
issue of how carcinogens may ex- of cell death pathways is associated p63, which is activated by DNA dam-
press certain characteristics, and with initiation and progression of tum- age, cellular stress, and oncogenic
by the question as to the biological origenesis; the products of proto-on- signal transduction, has pleiotropic
basis of the differences between cogenes (genes that encode proteins anti-proliferative and metabolic ef-
species, strains, target organs, and that stimulate cell proliferation, inhibit fects that include metabolic cell-cy-
target cells in cell signalling and apoptosis, or do both) include tran- cle arrest (Robey et al., 2015).
cell-cycle control. scription factors, chromatin remod- Numerous pathways have been

118
identified to be involved in disruption trinucleotides that identify amino protein-coding genes, which are like-
of resistance to cell death, and p53 acids, and the regulatory code that ly to mediate species differences (Lin
has been described as implicated determines how DNA sequences di- et al., 2014).
in cell-cycle arrest, apoptosis, reg- rect gene expression are highly con- Human-to-mouse transgenic ex­ -
ulation of metabolism, DNA repair, served between species. However, peri­ments demonstrate recapitu­-
and every pathway linked to these species differ in the composition and lation of human gene regulation in
processes (Narayanan et al., 2015). the length of the DNA sequences mice, even in the case of human
Disruption of the MAPK cascades, that use this language in the regu- genes that lack murine orthologues.
which are central signalling pathways latory regions of their genes (Nitta However, for distinct biological path-
that regulate a wide variety of cellular et al., 2015). ways, the expression profiles of
processes, is associated with induc- The Mouse ENCODE (Encyclo- many mouse genes diverged from
tion and progression of various dis-
pedia of DNA Elements) Consortium those of human orthologues (Yue
eases, including not only cancer but
reported that the degree of conser- et al., 2014). A core set of candidate
also diabetes, autoimmune disease,
vation is high: the mouse genome is regulatory sequences were con-
and developmental abnormalities
similar to the human genome in size, served and display similar activity
(Plotnikov et al., 2011).

CHAPTER 13
structure, and sequence composi- profiles in humans and mice: expres-
The role of gene activation in

PART 2
tion, and more than 80% of mouse sion patterns for genes that encode
carcinogenesis is also complex and
genes have human orthologues. proteins in the nuclear and intracel-
evolving. Activation of protein kinase
The chromatin landscape in a cell lular organelle compartments, and
C, which acts as a catalyst for sever-
lineage is relatively stable in both genes involved in RNA processing,
al cellular functions that are related
humans and mice, transcription fac- nucleic acid metabolism, chromatin
to cancer (e.g. cell survival, prolif-
eration, apoptosis, and migration), tor networks are substantially more organization, and other intracellular
has been thought to enable tumour conserved, and both the human and processes. However, less interspe-
development. However, protein ki- mouse genomes are pervasively cies concordance was observed for
nase C isozymes have recently been transcribed (Vierstra et al., 2014; Yue genes involved with the extracellular
reported to be suppressed in human et al., 2014). The pattern of chromatin matrix, cellular adhesion, signalling
cancers, possibly through loss of states (defined by histone modifica- receptors, and immune responses
function that suppresses other on- tions) and the large-scale chromatin (Yue et al., 2014).
cogenic signals (Antal et al., 2015). domains are highly similar between Within orthologous mouse and hu-
The gene for an RNA-binding protein mice and humans, but there is a di- man cell types, there is conservation
that is highly active in blood cancers vergence in the regulatory landscape across species of the global fraction
(i.e. RNA-binding protein Musashi that confers plasticity both between of regulatory DNA sequences that
homolog 2) is not directly mutated cell types and between species (Yue encode recognition sites for each
in tumours, but its activation affects et al., 2014). transcription factor (Vierstra et al.,
the ability of RNA to be translated Organ-specific genes are more 2014). However, between humans
into proteins (Wang et al., 2015), and highly expressed than housekeep- and mice there is variation in regu-
consequently its role in cancer has ing genes (i.e. those present in all latory regions that govern individual
not been identified through mutation tissues), and the highest organ-spe- gene systems and the occupancy
or gene expression patterns. cific gene expression is observed in pattern of transcription factors, with
the testes, brain, liver, muscle (car- extensive cis-regulatory “rewiring”,
Genetic variability in cell
diac and/or skeletal), and kidney (Lin mediated by elements that recognize
signalling between species,
strains, and target organs et al., 2014). Comparisons of gene transcription factors. Although they
expression between human and mu- have a common language in regula-
Genetic variability between species rine tissues showed similarities in tion, active elements in one species
has been described in terms of their gene expression profiles at the tissue may be reassigned to a different
genomic content and the regulation and organ level. However, there were tissue in another species (Vierstra
and expression of their genes. Both greater similarities within each spe- et al., 2014). Thus, differences in the
the genetic code, which specifies cies for non-coding and conserved regulation of gene expression and

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 119
cell signalling between species and mutations, one chemically induced i.e. it was lower in areas of active
tissues may affect mechanistic and and the other genetically engineered chromatin and transcription (Polak
tumour site concordance. transgenic, produced tumours with et al., 2015).
different gene expression patterns To create a comprehensive cat-
Variability in mutation targets alogue of genes responsible for the
(Westcott et al., 2015) and showed
and cell signalling across initiation and progression of cancer,
differences not only in tumour sus-
tissues and in tumours
ceptibility but also in model-de- 27 types of cancer were studied
pendent signalling pathways. (See through sequencing of matched tu-
Effects from activation of the MAPK/
mour and normal tissue samples as
ERK pathway, such as cell growth, Chapter 19, by Caldwell et al., for
part of the Cancer Genome Atlas and
prevention of apoptosis, and cell-cy- a discussion of host susceptibility
the International Cancer Genome
cle arrest, depend on the cell line- factors that influence tumour site
Consortium (Lawrence et al., 2013).
age; for example, activation of this concordance.)
However, as sample sizes increase,
pathway is associated with prolifera- As noted above, human tumours
the number of putative significant
tion and drug resistance in haemato- carry mutations in genes that en-
genes also increases and the risk of
poietic cells, but the activation is sup- code components of cell signalling
false positives resulting from tissue
pressed in some prostate cancer cell pathways associated with cell prolif-
lines (McCubrey et al., 2007). heterogeneity between cancer types
eration and cell death. High mutation in mutation type, distribution, and
The difficulty in predicting
frequencies have been associated frequency is highly variable. Across
cell-specific effects on cell signal-
with tumours induced by particular the 27 types of cancer, the median
ling is also illustrated by differences
carcinogens or mixtures of carcin- frequency of non-synonymous mu-
in certain biological responses be-
ogens, for example melanoma in- tations varied over more than three
tween histological subtypes of lung
duced by exposure to ultraviolet light orders of magnitude; half of the var-
cancer, i.e. adenocarcinoma versus
squamous cell carcinoma, in human and lung cancer induced by exposure iation in mutation frequency was as-
patients and in chemically induced to tobacco smoke (Lawrence et al., sociated with tissue type of origin.
lung cancer in mouse models. In 2013). Within cancer types, patient-specific
A/J mice treated with urethane to However, cancer is not a disease mutation frequencies also spanned
induce adenocarcinoma, or with of uniform origin, progression, or cell three orders of magnitude. This mu-
N-nitrosotris-(2-chloroethyl)urea to biology. Different types of cancer tational heterogeneity was strongly
induce squamous cell carcinoma, show variation in overall mutation correlated with DNA replication tim-
inhibition of vascular endothelial rate, predominant mutation type, ing and with transcriptional activity,
growth factor has the opposite ef- and distribution of mutations along i.e. it was higher in late-replicating
fect in these two tumour subtypes DNA regions and lower in highly
the genome. Epigenetic patterns
(increased apoptosis vs increased expressed genes. Higher mutation
of chromatin accessibility, histone
proliferation) (Larrayoz et al., 2014). frequencies occurring in late-rep-
modification, gene expression, and
In another example, gene ex- licating genes may be responsible
DNA replication timing are also cell
pression profiling was poor at dis- for potentially false-positive putative
lineage-specific (Polak et al., 2015).
tinguishing histological subtype and cancer genes, such as the olfacto-
A study of 173 cancer genomes from
cell type of origin for human breast ry receptor genes and some genes
eight different types of cancer, rep-
cancer, but a mouse model could cited in association with lung cancer
resenting a wide range of tissues of
demonstrate the correct genetic le- (Lawrence et al., 2013). Thus, the
origin, carcinogenic mechanisms, tissue of origin greatly affects mu-
sion and cell type to model human
disease, by confirming that the ori- and mutational signatures, showed tation patterns and is linked to DNA
gin of BRCA1 mutation-associated that chromatin features of the cell replication timing and tissue-specific
breast cancer is a luminal estrogen type of origin, and not of matched transcriptional activity.
receptor (ER)-negative mammary cancer cell lines, were the best pre- Genomic sequencing of estab-
epithelial progenitor (Molyneux and dictors of local frequency of somatic lished tumours to study their caus-
Smalley, 2011). However, different mutations. Mutation density was as- es has its limitations, because such
mouse models with the same K-ras sociated with epigenomic features, an approach is unable to study the

120
evolution of the clones, the accumu- 2013). Two distinct expression ar- It is difficult to identify associa-
lation of mutations in normal somatic rays of breast cancer cells, with al- tions that directly support a primary
cells, the variability among individ- most no genes – and thus no pro- metabolic link between environmen-
uals in driver mutation profiles, and tein changes – in common, can be tal exposures and cancer, for several
the variability among cancer genes equally useful for predicting clinical reasons: metabolic control does not
in clonal dynamics. In a study of 74 behaviour, and analyses with gene occur in a single step in a metabol-
cancer genes in sun-exposed skin, expression arrays may not provide a ic pathway; controlling factors differ
i.e. a polyclonal quilt of mutations between intact cells and in vitro cell-
true understanding of cancer biology
in key cancer genes consistent free systems; observed changes in
(Weinberg, 2014).
with damage from ultraviolet light, individual pathway elements do not
Sequencing of entire tumour ge-
multiple cancer genes were found always lead to changes in metabol-
nomes has yet to demonstrate de-
to be under strong positive selec- ic flux; and cancer cell phenotypes
finitively the number of somatic mu-
tion even in physiologically normal are neither fixed nor cancer-specific.
tations required to create a human
skin (Martincorena et al., 2015). The review also noted the function-
Positively selected mutations were tumour. A few conceptual insights
al interdependence of dysregulated
found in 18–32% of normal skin into cell and tissue behaviour have

CHAPTER 13
metabolism and other hallmarks of
resulted from elaborate maps of

PART 2
cells. The size of clonal expansions cancer, considering that, for exam-
varied across genes, and gene size interacting signalling components
ple, proliferating cancer cells have
was not necessarily correlated with and computer models of signalling
shared regulatory factors associ-
the potential of the somatic mutation (Weinberg, 2010). The paradigm
ated with the fundamental anabol-
to induce malignant transformation. of somatic evolution and multistep
ic and catabolic demands of the
Consistent with findings in tumours, tumorigenesis does not provide a
hallmark “sustaining proliferative
the mutation rate also varied along logical reason why oncogenesis
signalling” (Robey et al., 2015).
the genome, with higher rates found recapitulates ontogenesis (Huang Increased body mass index has
in less frequently expressed genes et al., 2009). been associated with increased risk
and in repressed chromatin. These
of cancer: obesogens – chemicals
findings were inconsistent with the Alterations in nutrient supply
that disrupt normal development
assumption that driver mutations oc-
and the balance of lipid metabol-
cur infrequently in long-lived cell line- Although dysregulated metabolism
ism – are able to cause permanent
ages and that those arising in cancer is one of the most common and
changes in metabolism that may ren-
are too small to be detected clinically recognizable features of cancer and
der the subject more susceptible to
(Martincorena et al., 2015). is associated with other phenotyp-
If a gene signature – a group of cancer later in life (see Chapter 19,
ic indicators, the results of a recent
genes with a characteristic com- by Caldwell et al.). Inflammation is
literature review attempting to link
bined expression pattern – is associ- associated with metabolic changes
cancer development and dysregulat-
ated with a prognosis, it is assumed and has been linked with several
ed metabolism suggested that there
to be likely to encode a biological chronic diseases, including cancer.
are major gaps in the understanding
signature driving carcinogenesis. Extracellular pro-inflammatory met-
of exposure-related carcinogenesis abolic signals are adenine nucleo-
However, this assumption has been
and metabolic reprogramming, for tides, succinate, oxidized nicotin-
questioned in view of findings that
example with respect to the specif- amide adenine dinucleotide (NAD+),
random changes in gene sets are
ic causal and temporal relationships and urate (McGettrick and O’Neill,
associated with prognosis and
that prognostic signatures in ER- between exposures, dysregulated 2013; Tannahill et al., 2013). The
negative breast cancer – associated metabolism, and the development gut microbiome has an important
with hypoxia and angiogenesis – of cancer and the associated phe- role in carbohydrate absorption and
are more similar to those in ovarian notypic hallmarks of cancer (Robey metabolism in humans and plays a
cancer than to those in ER-positive et al., 2015). This review did not con- significant part in inflammatory re-
breast cancer, which are driven by sider lifestyle-related exposures and sponses as well (see Chapter 19, by
proliferation pathways (Beck et al., IARC Group 1 carcinogens. Caldwell et al.).

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 121
The transgenerational character modes of action, and the third de- out the need for either continued
of metabolic disturbances and ef- scribes the ability of a transformed exposure or sustained proliferation
fects on cell signalling is demonstrat- cell to escape normal cell-cycle con- during exposure (see Chapter 19, by
ed by studies of multigenerational trol and to continue replication. The Caldwell et al.). It has been noted for
undernutrition in rats (i.e. for 50 gen- interpretation of mechanistic data some time that enhanced cell divi-
erations). The undernourished rats for cell proliferation and cell death sion does not always predict carcino-
were predisposed to insulin resis- is dependent on the development genesis (Melnick et al., 1993). After
tance, had altered levels of several of appropriate animal models (see exposure to a carcinogen, the de-
metabolic regulators (e.g. circulating Chapter 19, by Caldwell et al.), and velopment of cancer in experimental
insulin, homocysteine, endotoxin, although cell proliferation has been animal models is influenced by the
leptin, adiponectin, vitamin B12, and used in descriptions of hypothesized circumstances under which expo-
folate), and had a higher susceptibili- modes of action (Wood et al., 2015), sure occurs (e.g. sustained vs tran-
ty to streptozotocin-induced diabetes it should be viewed in the context of sient) and by the presence or ab-
compared with properly fed control the newer understandings of cancer sence of inflammatory mediators or
rats. These changes were not re- mechanisms. DNA damage.
versed by feeding rats a normal diet For DNA damage to lead to a
in the two subsequent generations Liver cancer
mutation, DNA replication and cell
(Hardikar et al., 2015). division are typically required (see The complex interactions between
Studies on altered cell signalling Chapter 12, by DeMarini). As noted proliferation, mutation, and inflam-
have traditionally been performed in the United States Environmental matory cell signalling have been
on putative target cells of cancer, Protection Agency guidance assess- studied extensively for liver cancer.
but the contribution of the gut mi- ing the risk of cancer from early-life In humans, hepatocellular carcinoma
crobiome (i.e. the microbiota living exposures (EPA, 2005), more fre- (HCC) is markedly heterogeneous,
on and in humans) has only recently quent cell division during develop- both histomorphologically (Yeh et al.,
been investigated as a factor in can- ment can result in enhanced fixation 2007) and genetically, with a wide
cer susceptibility (see Chapter 19, of mutations because of the reduced diversity in gene expression patterns
by Caldwell et al.). The microbiome time available for repair of DNA le- (Chen et al., 2002). Histopathological
plays a role in the control of nutri- sions, and clonal expansion of a variability is also associated with ge-
ent supply (e.g. the gut microbiome mutated cell produces a larger pop- ographical region: slow-growing, dif-
is highly enriched in carbohydrate ulation of mutant cells. For adult or- ferentiated HCC nodules surrounded
metabolism genes, compared with ganisms, sustained cell proliferation by a fibrous capsule are common
the human genome overall; Bultman, has also been postulated to increase in this type of cancer in Japanese
2014), in metabolic pathways, and in risk of cancer, based on the same ra- patients, whereas a febrile form of
host susceptibility to metabolic dis-
tionale, and it has been proposed as HCC, characterized by leukocytosis,
ease (Suez et al., 2014).
a factor in increased cancer suscep- fever, and necrosis within a poorly
tibility. Sustained cell proliferation differentiated tumour, is common in
Cell proliferation as a
component or cause of is a feature of several hypothesized this cancer type among black peo-
cancer modes of action for cancer develop- ple in South Africa (Feitelson et al.,
ment, for example the induction of 2002).
There are at least three scenarios kidney cancer via alpha2u-globulin HCC signature genes vary con-
related to cancer and mechanisms of accumulation (EPA, 1991). siderably and depend on etiologi-
cancer induction in which alterations Although alterations in cellu- cal and accompanying pathological
in cellular replication and/or cell-cy- lar replication or cell-cycle control conditions, such as viral infection,
cle control have been described. The are important features of carcino- cirrhosis, inflammation, and fibrosis.
first invokes the predisposition of genesis, cell proliferation in and of The study of tumour formation in the
replicating cells with unrepaired DNA itself is not able to induce cancer. liver is also affected by continuous
damage to develop into cancer cells. Several carcinogenic substances changes in the transcriptome that
The second identifies sustained can cause cancer in humans after accompany hepatectomy and age
replication as a key event in various perinatal or prenatal exposure with- (Colak et al., 2010). A comparison of

122
conserved genes between rats and simple paradigm to explain the differ- cell signalling and is consistent with
humans (human orthologues) with ences in strain sensitivity, for exam- inflammation contributing to cancer
respect to early expression profiles ple between C3H/HeJ and C57BL/6J development. After exposure of ro-
of HCC signature genes showed mice, which show a difference of dents to trichloroethylene, hepato-
some conservation between species up to 40-fold in multiplicity of liv- cyte proliferation is confined to only
for components of the MAPK/ERK, er tumours (Hanigan et al., 1988; a small population of cells without
phosphoinositide 3-kinase (P13K)/ Maronpot, 2009). regenerative hyperplasia, sustained
Akt, and transforming growth factor The activation of oncogenic hepatocellular proliferation, and
beta (TGF-β) pathways (Colak et al., pathways appears to be more het- hepatocellular necrosis. Any tran-
2010). erogeneous in human HCC than in sient DNA synthesis, peroxisome
Development of liver cancer af- other types of cancer (El-Serag and proliferation, or cytotoxicity is not
ter exposure to carcinogens is more Rudolph, 2007). The high degree of correlated with trichloroethylene-in-
common in rodents, especially in heterogeneity in the ways in which duced liver carcinogenicity (EPA,
the mouse, than in humans. There cell signalling is disturbed before 2011). Thus, induction of liver cancer
are obvious differences between hepatocellular neoplasia may make by trichloroethylene is not a result of
rodents, non-human primates, and induction of liver cancer a useful sustained cell proliferation.

CHAPTER 13
PART 2
humans in background susceptibil- marker for changes that can lead to Exposure to one of the most
ity to hepatocarcinogenesis and, cancer elsewhere, depending on cel- studied carcinogens, 2,3,7,8-tetra-
as noted above, in the regulation of lular context and target (Vogelstein chlorodibenzo-para-dioxin (TCDD),
gene expression and cell signalling. and Kinzler, 2004). induces liver cancer in rodents, but
With respect to the ability to respond Thus, pathway concordance be- short-term effects do not include
to a mitogenic stimulus such as par- tween species may not always result induction of hepatocellular prolifer-
tial hepatectomy, the liver responds in site concordance for expression ation. Rather than simply inducing
differently and much more slowly in of cancer. The analysis of liver tu- cell proliferation, TCDD is thought
non-human primates and in humans mour site concordance is complicat- to cause cancer by altering the cel-
compared with rodents (Gaglio et al., ed by the heterogeneity of disease lular ability to proliferate, migrate,
2002). in humans, as well as by rodent undergo apoptosis, senesce, and
Global gene expression patterns susceptibility. terminally differentiate in a multistep
in HCCs from seven different mouse For induction of cancer in the liv- process focused on the accumula-
models were compared with those er in rodents, the nature of cell pro- tion of mutations and heritable epige-
in human HCCs from groups with liferation also determines the risk of netic changes (Safe, 2001; Marlowe
poorer survival and better survival. cancer (Caldwell et al., 2008). When and Puga, 2005; Ray and Swanson,
Expression patterns in HCCs from both necrosis and inflammation are 2009). In addition, the upregulation
Myc-Tgfa transgenic mice and in di- present, the resultant hepatocellular of drug-metabolizing enzymes by
ethylnitrosamine-induced HCCs in proliferation is fundamentally differ- TCDD may enhance the formation of
mice were most similar to those in ent from the transient proliferation highly reactive intermediates during
human HCCs from the group with caused by peroxisome proliferators metabolic activation and/or trans-
poorer survival, whereas the pat- or other primary mitogens. After formation of several key hormones
terns in HCCs from Myc, E2f1, and treatment with a mutagenic agent, (e.g. enzyme induction as a source
Myc-E2f1 transgenic mice were most transient proliferation induced by pri- of reactive oxygen species forma-
similar to those in human HCCs from mary mitogens has not been shown tion, which is linked to decoupling of
the group with better survival (Lee to lead to cancer induction, whereas the cytochrome P450 catalytic cy-
et al., 2004). partial hepatectomy or necrogenic cle) and result in DNA damage and
Many factors, such as diet, hor- treatments with carbon tetrachlo- mutations (IARC, 2012c).
mones, oncogene activation, methyl- ride have (Ledda-Columbano et al., Although liver data provide an
ation, imprinting, and cell prolifera- 1993; Gelderblom et al., 2001). example of the role of inflammatory
tion or apoptosis, are modulators of The mechanism by which necro- signals under some circumstances,
spontaneous and induced murine sis may enable cancer development inflammation in itself may not induce
hepatocarcinogenesis. There is no involves concurrent inflammatory cancer without other concurrent

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 123
cofactors. For many of the agents ducts in portal spaces, Mdr2 knock- Several key mechanistic charac-
discussed in Volume 100C of the out mice rarely develop tumours of teristics of IARC Group 1 carcino-
IARC Monographs (IARC, 2012a), the bile duct (Nickoloff et al., 2005). gens induce traits of cancer cells de-
inflammation is a key characteristic The relationship between chron- scribed as the hallmarks of cancer,
of their effects (see Chapter 17, by ic inflammation and cancer is com- including effects on cell proliferation,
Kane). One of the most recognized plex: inflammation may have roles cell death, and nutritional status (see
examples of how inflammation con- in initial genetic mutations or epi- Chapter 11, by Stewart). The descrip-
tributes to neoplastic development is genetic changes that not only drive tion of the hallmarks attempts to bring
the induction of mucosa-associated cell transformation but also provide together a fundamental understand-
lymphoid tissue (MALT) lymphoma a microenvironment that enables ing of how cancer cells manifest a
and gastric adenocarcinoma asso- progression and metastasis and distinct phenotype. More recently, a
ciated with exposure to Helicobacter prevents immune responses against series of reviews in Carcinogenesis
pylori. The MALT proliferations of the tumour. Chronic inflammation reported the findings from an inter-
B-cell lymphoid follicles are the pre- favours accumulation of DNA dam- national team of cancer biologists
cursor of a low-grade lymphoma of age and chromosomal damage and toxicologists who participated
B cells. A large proportion (98%) of (see Chapter 12, by DeMarini, and in the Halifax Project (Harris, 2015).
patients with gastric MALT lympho- Chapter 17, by Kane). They reviewed the literature on each
ma are also infected with H. pylori; of the hallmarks of cancer to exam-
The hallmarks of cancer ine the carcinogenic potential of ex-
however, only a small percentage of
H. pylori-positive individuals devel- posure to low doses and mixtures
In their updated paper, Hanahan
op MALT lymphoma (Bassig et al., of chemicals. Relevant reviews for
and Weinberg (2011) noted that the
alterations in cell proliferation, cell
2012). Little is known about the pos- most fundamental trait of cancer
death, or nutrient supply included
sible role of environmental cofactors cells involves their ability to sustain
the potential of chemical mixtures to
in the predisposition to H. pylori-in- chronic proliferation. As part of the
enable sustained proliferative signal-
duced gastric lymphomagenesis. “hallmarks” of cancer, alterations in
ling (Engström et al., 2015), to confer
Other factors are certainly involved, cellular replication and/or cell-cy-
resistance to cell death (Narayanan
including susceptibility (IARC, cle control figure prominently in the
et al., 2015), and to induce metabol-
2012b). discussions of cell proliferation, in- ic reprogramming and dysregulated
flammation, and changes in cell sig-
Inflammation metabolism (Robey et al., 2015). A
nalling that are part of cancer cell related review that encompasses
Certain types of inflammatory pro- physiology. The authors noted that many aspects of cell signalling re-
cesses in skin, and possibly in other the precise identities and sources ported on environmental immune
tissues, may serve a tumour suppres- of the proliferative signals in general disruptors, inflammation, and risk
sor function. Some clinical conditions remain poorly understood, but that of cancer (Thompson et al., 2015).
show that inflammation is a critical mitogenic signalling in cancer cells The overlap in the descriptions of
component of tumour progression, is characterized and known in some- pathway disruption and functions of
for example reflux esophagitis be- what more detail. The mechanism by cell signalling molecules between
fore oesophageal cancer, or inflam- which necrosis enables cancer in- these papers is striking and is con-
matory bowel disease that precedes duction was also described in terms sistent with the discussion presented
colorectal cancer. However, a condi- of the release of pro-inflammatory above with regard to the complex re-
tion such as psoriasis is known as a signals by necrotic cells and the in- lationship and pleiotropic roles of cell
chronic cutaneous inflammatory dis- fluence of cytokines on proliferation signalling molecules involved in cell
ease that is seldom, if ever, accom- and invasiveness of cancer cells. proliferation and apoptosis.
panied by cancer. Similarly, despite Thus, tumour-promoting inflamma- The paradigms of cytotoxicity,
extensive inflammation, activation of tion was considered by Hanahan and cell proliferation, and initiation–pro-
nuclear factor kappa-light-chain-en- Weinberg (2011) to be an enabling motion as mechanisms of carcino-
hancer of activated B cells (NF-κB), characteristic for acquisition of core genesis have been superseded by a
and abundant proliferation of bile hallmark capabilities. more nuanced understanding of the

124
process of carcinogenesis (Hanahan The biological basis for how cell delayed cancer induction after expo-
and Weinberg, 2011). As stated signalling and cell-cycle control dif- sure to toxicants and the bystander
above, cell proliferation, inflamma- fer between species, organs, and effect of radiation on tumour devel-
tion, or cytotoxicity alone do not lead tumour cells, as well as the variabil- opment. With regard to cell signal-
to cancer, and they are interrelated ity in mutation targets, are also dis- ling, spontaneous or environmentally
through changes in cell signalling. cussed here. Determining the caus- induced epigenetic alterations are
The hallmarks of cancer describe es of cancer through examination of increasingly recognized as early mo-
gene expression profiles in tumours lecular events in cancer formation,
well the characteristics that are
is difficult, especially in terms of in- and these alterations may potential-
manifested after the development of
creased and sustained cell prolif- ly be more adverse than nucleotide
cancer. However, by the time of di-
eration, which is a characteristic of mutations, because their effects on
agnosis, tumour cells already carry
cancer itself. Alteration in nutrient regional chromatin structure can
large numbers of mutations and are
supply is a common and recogniza- spread out, thereby affecting multiple
also very heterogeneous in gene
ble feature of cancer, and is also not genetic loci (Weidman et al., 2007).
product profiles; the multiple cell di- independent of activities associated The key characteristics of the
visions and the consequent damage with increased cell proliferation or IARC Group 1 carcinogens have

CHAPTER 13
processing obscure the initial lesion,

PART 2
the hallmarks of cancer. some overlap with the hallmarks
rendering it difficult, if not impossi- Some of the information col- of cancer and perhaps can provide
ble, to make a distinction between lected in Volume 100 of the IARC insight into the “environment” that
causal and consequential events in Monographs was presented in the creates the neoplastic cell. The elu-
carcinogenesis. context of the older hypotheses for cidation and understanding of sus-
mechanisms of cancer induction. ceptibility factors may help determine
Conclusions The understanding of cancer mech- what parts of that environment are
anisms and the descriptive data already present in individuals, spe-
Among the 10 mechanistic charac- associated with them continues to cies, or target tissues where cancer
teristics more commonly observed evolve (see Chapter 11, by Stewart). develops as a result of exposure to
for IARC Group 1 carcinogens is a The discussion of mechanistic data environmental carcinogenic agents.
composite that includes information for ionizing radiation in Volume 100D This may also help in evaluating
on alteration of cell proliferation, of the IARC Monographs (IARC, mechanistic and tumour site concor-
cell death, and nutrient supply. This 2012d) and Chapter 12, by DeMarini, dance between species. However,
chapter examines many of the chal- provide more current discussions the mechanistic data provided on
lenges associated with the use of about the understanding of cancer agents identified as carcinogenic
this type of information to determine and the key mechanistic character- to humans need to be examined in
mechanistic and tumour site con- istics of IARC Group 1 carcinogens. the context of more recent informa-
cordance between humans and ex- With the present state of knowledge, tion on carcinogenesis. Cancer is a
carcinogenesis cannot be confident- heterogeneous disease, even with-
perimental animals, and discusses
ly attributed to an underlying purely in the same target site. Because,
how this mechanistic characteristic
genetic or purely epigenetic pro- among other considerations, some
shows interdependence with others,
cess. Mechanistically, it is probably a epigenetic events may have a mu-
such as genotoxicity and inflamma-
mixture of the two. tational basis, the dichotomy once
tion. Many of the indicators of chang-
Epigenetic alterations may identified between genotoxic and
es in cell proliferation or cell death
precede DNA sequence mutations, non-genotoxic carcinogens should
are non-specific for the induction with subsequent mutations occurring be reconsidered.
of cancer, and although they result not in a random fashion but in re- Enhanced cell proliferation and re-
primarily from effects on the MAPK/ sponse to specific types of epigenet- duced cell death are key mechanistic
ERK cell signalling pathway, they are ic changes induced by the environ- characteristics of IARC Group 1 car-
influenced by a large array of cell ment (Karpinets and Foy, 2005). This cinogens, are a hallmark of cancer,
signalling molecules with pleiotropic selection for enhanced growth has and are necessary for DNA damage
effects. been suggested to explain both the to be processed into a mutation.

Part 2 • Chapter 13. Alterations in cell proliferation, cell death, or nutrient supply 125
However, such changes alone are Acknowledgements Approval does not signify that the
not sufficient to induce cancer. A key contents reflect the views of the
The author thanks D.M. DeMarini,
mechanistic question emerges as to agency, nor does mention of trade
M.V. Evans, and S. Vulimiri for their
what events or cellular environment names or commercial products con-
helpful comments on this manuscript.
may precede or cause such chang- stitute endorsement or recommen-
es and may stimulate the formation Disclaimer dation for use.
and selection of DNA sequence mu-
tations and epigenetic changes that This article was reviewed and
induce a cell and its descendants to approved for publication by the
acquire the hallmarks of cancer, in- National Center for Environmental
cluding increased cell proliferation Assessment, United States En-
and evasion of apoptosis. vironmental Protection Agency.

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part 2.
mechanisms of carcinogenesis

chapter 14.

Receptor-mediated
mechanisms
Maarten C. Bosland

CHAPTER 14
PART 2
Introduction have genotoxic effects; polycyclic biphenyls (PCBs) can be metabo-
aromatic hydrocarbons (PAHs), such lized to reactive, DNA-damaging in-
Carcinogenic agents can cause as benzo[a]pyrene, are examples of termediates that may contribute to
cancer by inducing DNA damage this group. Yet other agents have can- their receptor-mediated carcinogen-
through various genotoxic mecha- cer-enhancing or tumour-promoting icity (Ludewig and Robertson, 2012;
nisms that result in mutations. Many effects through pathways involving Lauby-Secretan et al., 2013; IARC,
of the agents classified as carcino- receptors but also cause formation of 2016).
genic to humans (Group 1) by the reactive oxygen species or enhance The purpose of this chapter is
IARC Monographs are not directly or the bioactivation of pro-carcinogens to provide an overview of recep-
indirectly genotoxic but cause cancer to ultimate carcinogens, both of tor-mediated mechanisms thought
via mechanisms that, by themselves, which can potentially damage DNA to be involved in carcinogenesis,
do not generate or involve DNA dam- and result in mutations; 2,3,7,8-tetra- followed by a discussion reflecting
age. One non-genotoxic mechanism chlorodibenzo-para-dioxin (TCDD) is on the complexity of these mech-
involves receptor mediation (Pitot, an example of this group. anisms and how such mechanistic
1995). Thus, the distinction between information can be used for rational
Hormonally active agents that are genotoxic carcinogens and carcino- hazard identification of carcinogenic
associated with carcinogenic effects gens that act through receptor-me- agents. Receptor-mediated carcino-
typically act as ligands for nuclear diated mechanisms is often not genesis was often discussed in the
receptors and, in some cases, for black and white. Insight continues 1990s as a major mechanism of
receptors located at the cell surface. to evolve about the mechanisms by cancer induction by non-genotoxic
There are also agents that are con- which some carcinogens act through carcinogens (Pitot, 1995). However,
sidered to be carcinogenic through receptors. For example, there is now carcinogenesis research has since
receptor mediation but in addition evidence that certain polychlorinated moved beyond these receptors to the

Part 2 • Chapter 14. Receptor-mediated mechanisms 129


downstream mechanisms involved in onset and a long duration, where- the half-life of the agent in vivo and
their action or has often focused on as the effects of the second group on its bioaccumulation. Notorious in
just one agent’s effects. Indeed, a re- of receptors are typically rapid and this regard are compounds such as
cent PubMed search for articles with transient. Both classes of receptors TCDD and PCBs, which are stored
“receptor-mediated carcinogenesis” can be involved in mechanisms of in fat tissue and have a long half-life
in the title yielded only eight hits, four carcinogenesis. (6–10 years); consequently, even
of which are reviews, all published Although some exogenous li- a short-term exposure can result
between 1992 and 1999, and one gands act as agonists by compet- in a sustained internal dose (IARC,
journal issue devoted to this topic ing for binding with an endogenous 1997).
(volume 333 of Mutation Research, ligand, others may bind but lack the Importantly, many agents that act
in 1995). Despite a very large num- intrinsic activity to activate the re- by receptor mediation display non-
ber of relevant primary publications, ceptor they bind to and thus have linear dose–response relationships
there have been no major general an antagonist effect. There are also for end-points such as cell prolifer-
reviews on this subject since the receptors for which no endogenous ation, similar to the dose–response
ligand has been identified with cer- of steroid hormones (Reddel and
mid-1990s. Other reviews identified
tainty, such as the aryl hydrocarbon Sutherland, 1987; Pitot, 1995; Sewall
by the keywords “receptor-mediated”
receptor (AhR); these receptors are and Lucier, 1995; Gaido et al., 1997;
and “carcinogenesis” or “toxicity” are
conceivably activated only by exoge- Toyoshiba et al., 2004; Walker et al.,
devoted to specific carcinogens, tu-
nous agents, because they are often 2005); androgenic and estrogenic
mour types, or target tissues, such
involved in detoxification process- steroid hormones typically stimu-
as the liver (Andersen et al., 2014).
late in vitro proliferation of recep-
Yet, several of the IARC Group 1 es that have evolved as protective
tor-positive cells at low, physiological
carcinogens act entirely or in part mechanisms. Exogenous agents
concentrations but are inhibitory at
via receptor-mediated mechanisms, may also indirectly affect recep-
supraphysiological and pharmaco-
including benzo[a]pyrene, TCDD, tor-mediated mechanisms by mod-
logical concentrations (Lee et al.,
3,3′,4,4′,5-pentachlorobiphenyl (PCB ulating the amount of endogenous
1995; Almstrup et al., 2002; Simons,
126), 2,3,4,7,8-pentachlorodibenzo- ligand available for receptor bind-
2008). This phenomenon has impor-
furan (PeCDF), and the hormonal ing and activation, through effects
tant implications for quantitative risk
agents that are used in combined on the biosynthesis, bioavailability,
assessment. However, this is outside
oral contraceptives and in treatment bioactivation, and degradation of
the scope of this chapter, which, like
for menopausal symptoms (IARC, the bioactive ligand. In addition, ex-
the IARC Monographs, focuses on
2012a, b). ogenous agents may influence the
hazard identification.
abundance of receptors by modify-
Oral exposure can result in a
General comments ing receptor biosynthesis and deg-
first-pass effect if elimination of the
radation. Finally, exogenous agents
agent by the liver is efficient, thereby
Biological mechanisms involving re- may indirectly affect receptor-medi-
reducing the dose to organs beyond
ceptor activation fall into two broad ated mechanisms by having effects the liver. When hepatic elimination is
categories: (i) those that involve in- on or acting as co-activators and slow, the systemic dose of a carcin-
tracellular receptors that translocate co-repressors of nuclear receptors. ogen may be high and may lead to
into the nucleus and act as transcrip- The effects of agents that act via
cancer induction. This was demon-
tion factors regulating gene expres- receptor mediation depend in many
strated in mouse strains that dif-
sion (genomic action), and (ii) those cases on the dose, duration and
fered in inducibility of CYP1A1/2 en-
that involve cell surface receptors route of exposure, and timing of ex-
zymes – members of the cytochrome
posure during the life of an organism.
and some intracellular receptors that P450 (CYP450) family of enzymes –
For example, the effects in animals of
activate signal transduction path- by the AhR mechanism when treated
exposure to diethylstilbestrol in ute-
ways, resulting in biological respons- ro or neonatally are known to differ with benzo[a]pyrene (Nebert et al.,
es, including gene transcription from those after exposure in adult life 1979).
(non-genomic action). The effects on (IARC, 2012b) (see also Chapter 20, The downstream biological ef-
gene transcription of the first group by Rice and Herceg). The duration of fect of receptor binding is really
of receptors typically have a slow internal exposure depends in part on what determines the potential for

130
carcinogenicity of an agent that Birnbaum et al., 1990; Fernandez- carcinogenic processes (Pitot, 1995;
acts via a direct receptor-mediated Salguero et al., 1995). Although Shawver et al., 1995).
mechanism. Effects that are gener- some of these studies evaluated 12-O-tetradecanoylphorbol-13-ac­
ally considered to be associated with the involvement of AhR in effects of etate (TPA) may act as a tumour
carcinogenic activity of such agents TCDD that are mechanistically as- promoter through activation of mem-
include, but are not limited to, the sociated with carcinogenesis, this brane-located protein kinase C re-
following: enhancement of carcino- approach has not been applied to ceptors, activating downstream sig-
gen bioactivation; reduction of DNA determine whether AhR is necessary nalling pathways that contribute to
repair; induction of oxidative stress; for TCDD-induced tumour formation. the enhancing effects of TPA on for-
stimulation of cell proliferation, an- Studies with mice that overexpress or mation of skin tumours (Niedel et al.,
giogenesis, and invasive or migratory lack estrogen receptor alpha (ER-α) 1983; Nishizuka, 1984). However,
cell properties (including epithelial– have demonstrated that this receptor conclusive evidence for this mech-
mesenchymal transition); inhibition of is involved in many of the develop- anism is still lacking (Gschwendt
cellular apoptosis, senescence, and/ mental and carcinogenic effects of et al., 1995; Marks and Gschwendt,
or differentiation; evasion of immune neonatal treatment with diethylstil- 1995). Moreover, TPA is a complete
surveillance; and epigenetic effects bestrol in female animals (see below) carcinogen for the skin in mice when

CHAPTER 14
(Hanahan and Weinberg, 2011) (see (Couse et al., 1997, 2001; Couse and given at sufficiently high doses with

PART 2
Chapter 10, by Smith). Korach, 2004). a sufficiently long period of obser-
Finding any one such effect, or vation (Iversen and Iversen, 1979).
even a combination of several of Non-genomic receptor- The involvement of protein kinase C
these effects, as a result of exposure mediated mechanisms and in TPA-induced tumour promotion is
to an agent that binds to cellular re- carcinogenesis highly complex and is only partially
ceptors is not by itself sufficient evi- understood (Griner and Kazanietz,
Non-genomic regulation of gene 2007), and more recent research is
dence that the agent is a carcinogen,
expression and cellular function in- focused on downstream signalling
but such findings do generate suspi-
volves signal transduction pathways effects (Hsu et al., 2000; Lu et al.,
cion that the agent might cause can-
that are activated by a wide range 2007). TPA has also been shown to
cer. If such receptor binding is asso-
of receptor molecules located at the act through other receptor mecha-
ciated with the mechanism by which
plasma membrane. These receptor nisms (Marks and Gschwendt, 1995)
a recognized human carcinogen op-
modules are activated by an equal- and, when applied to the skin in
erates, the evidence for carcinogeni-
ly large number of ligands, ranging mice, to induce inflammation, which
city in humans may be sufficient. An
from locally produced autocrine and in itself can stimulate cell prolifera-
example of this is the recent upgrad-
paracrine growth factors to circulat- tion and generate reactive oxygen
ing by IARC of PCB 126 and PeCDF
ing cytokines and hormones. The species (Aldaz et al., 1985; Wei and
to Group 1 carcinogens, based on major groups of these receptors in- Frenkel, 1993).
the similarity of their binding to AhR clude tyrosine kinase receptors such Receptors for several steroid
(IARC, 2012a, 2016). as the epidermal growth factor (EGF) hormones are not only intracellular
Proof that a receptor-mediated receptor, serine/threonine kinase re- nuclear receptors (see below) but
mechanism is involved in carcino- ceptors such as the protein kinase C may also be present at the plasma
genesis in mammals would require receptor, G protein-coupled recep- membrane, where their activation
evidence that blocking the activation tors, and receptors for cytokines, by agonists causes rapid non-ge-
of a specific receptor, or genetic re- chemokines, and other ligands. nomic signal transduction-medi-
moval of the receptor or reduction They all catalyse phosphorylation ated effects. These non-classical
of its expression in mouse models, of downstream signalling molecules receptor-mediated effects have
interferes with tumour induction by upon ligand binding, some via activa- been shown to occur for the andro-
the carcinogen in question. The latter tion of G protein signalling, ultimately gen receptor (AR) (Bennett et al.,
approach was used to demonstrate resulting in regulation of expression 2010; Lang et al., 2013), the gluco-
that several of the toxic effects of of specific genes or sets of genes corticoid receptor (Matthews et al.,
TCDD are indeed mediated by AhR that control cellular function and 2008; Samarasinghe et al., 2012),
in vivo (Poland and Glover, 1980; behaviour and potentially influence the progesterone receptor (PR)

Part 2 • Chapter 14. Receptor-mediated mechanisms 131


(Wheeler et al., 2000; Peluso, 2011), action of these agents. In addition, ligands have been identified, but they
and the estrogen receptor (ER) indirect non-genomic activity of car- do bind to exogenous ligands. These
(Acconcia and Kumar, 2006; Song cinogenic agents could conceivably receptors are thought to mediate
and Santen, 2006). affect receptor abundance or stim- protection against harmful xenobiot-
Activation of rapid membrane-ini- ulate signal transduction pathways ics, predominantly by inducing ex-
tiated estrogen signalling by estro- that lead to pro-carcinogenic effects. pression of specific drug-metaboliz-
genic agonists causes non-geno- ing CYP450 enzymes (Boutros et al.,
mic signal transduction-mediated Nuclear receptor- 2009). Examples of the latter catego-
effects (Acconcia and Kumar, 2006; mediated mechanisms and ry are the pregnane X receptor and
carcinogenesis
Song and Santen, 2006). The bio- the constitutive androstane receptor
logical significance and contribution (Nebert and Dalton, 2006; Tompkins
Nuclear receptor-mediated mecha-
of these effects to carcinogenesis and Wallace, 2007; Elcombe et al.,
nisms involve intracellular receptors,
are uncertain at present. One of 2014), both of which frequently en-
most of which belong to the so-called
the two rapidly acting non-genomic gage in cross-talk with various other
nuclear receptor superfamily (Evans,
membrane-located forms of PR, PR nuclear receptors and transcription
1988; Mangelsdorf et al., 1995).
membrane component 1 (PGRMC1), factors (Lim and Huang, 2008).
There are a large number and a wide
is expressed in human ovarian cells Several other nuclear receptors
variety of nuclear receptors that act
and tumours (Wendler et al., 2012). with endogenous ligands are in-
via genomic mechanisms involving
PGRMC1 expression is associated volved in physiological functions, but
direct binding to specific DNA se-
with cell proliferation in ovarian can- quences (response elements) or in- they are also activated by xenobiot-
cers and may have anti-apoptotic direct binding to AP1 or Sp1 sites in ics. An example of this category is the
effects (Wheeler et al., 2000; Peluso the promoter regions of the specific group of peroxisome proliferator-ac-
et al., 2008). PGRMC1 also inter- genes they regulate (Kushner et al., tivated receptors (PPARs), which are
acts with and changes the function 2000; Aranda and Pascual, 2001; involved in lipid metabolism but are
of some CYP450 enzymes and may Safe and Kim, 2008). Once the nu- also activated by xenobiotics with
affect carcinogen metabolism (Rohe clear receptor is bound to a response peroxisome proliferating activity; the
et al., 2009; Szczesna-Skorupa and element, various co-regulator mole- PPARα subtype has been implicat-
Kemper, 2011). There are no pub- cules are recruited that are critical ed in the hepatocarcinogenicity in
lished studies to date indicating a for regulation of the initiation of gene rats of some of these xenobiotics
contribution of these non-genomic transcription in a cell type-specif- (Green, 1995; Cattley, 2004; Corton
ER- and PR-mediated effects to ic manner (Edwards, 2000; Lonard et al., 2014), which are probably not
cancers associated with exposure to and O’Malley, 2012). Many mem- human hepatocarcinogens (IARC,
estrogens and progestins. bers of the nuclear receptor super- 1994, 1996). Many nuclear receptors
None of the IARC Group 1 carcin- family are involved in physiological need to homodimerize before they
ogens are known to be carcinogenic functions that mediate the effects of can bind to response elements, and
by acting via non-genomic recep- steroid hormones and other endog- several others heterodimerize with
tor mechanisms, but this cannot be enous ligands (Evans, 1988; Tsai the retinoid X receptor before binding
ruled out, for several reasons. Both and O’Malley, 1994; Whitfield et al., (Dawson and Xia, 2012).
estrogens and progestins can act 1999; Jacobsen and Horwitz, 2012). AhR, which binds to TCDD and
via transmembrane receptors, and ER, PR, and AR are examples of ligands that are structurally similar
TCDD can affect signalling pathways receptors that bind to and are acti- to TCDD, does not belong to the
via mechanisms that do not involve vated by steroid hormones, whereas nuclear receptor superfamily and
AhR (Tijet et al., 2006; Biswas et al., the retinoic acid receptors and the is in a class by itself. There are no
2008; Boutros et al., 2009; Kim et al., retinoid X receptors bind to vita- established physiological endoge-
2009; Denison et al., 2011). Thus, it min A metabolites (Rochette-Egly nous ligands for AhR, even though
is conceivable that carcinogenicity and Germain, 2009; Duong and this receptor has physiological
associated with exposure to estro- Rochette-Egly, 2011; Dawson and functions, because mice that lack
gens, progestins, or dioxins may Xia, 2012). There are also nuclear AhR expression show developmen-
involve mediation by non-genomic receptors for which no endogenous tal abnormalities (Gonzalez and

132
Fernandez-Salguero, 1998; Carlson A second form of ER was discovered 2011) and often have opposite activi-
and Perdew, 2002). However, some in 1996 and was named ER-β to dis- ty in breast, prostate, and colon can-
endogenous compounds that bind to tinguish it from the receptor previous- cer cells (Bardin et al., 2004; Roger
and activate AhR have been identi- ly cloned, ER-α (Kuiper et al., 1996). et al., 2008; Chen and Iverson, 2012;
fied, such as certain tryptophan de- Genomic activity of ER, also Nelson et al., 2014).
rivatives, but their physiological role termed nuclear-initiated steroid sig- There are several isoforms of
has not yet been firmly established nalling, is achieved through two ER-α and ER-β that result from alter-
(Denison and Nagy, 2003; Henry main mechanisms. The first involves native mRNA splicing (Moore et al.,
et al., 2006). The tryptophan metab- the direct binding of ER to its target 1998; Flouriot et al., 2000; Wang
olite kynurenine has been identified gene. Estrogen binding to its recep- et al., 2005). Of these variants, ER-
as an endogenous ligand of human tor in the cytosol triggers a series of α36 and ER-α46 are predominantly
AhR. After receptor binding, down- events, starting with loss of chaper- localized to the plasma membrane
stream effects are the suppression one molecules such as heat shock and cytoplasm, mediating mem-
of anticancer immune mechanisms protein 90 (Hsp90), and followed by brane-initiated rapid non-genomic
and the promotion of cancer cell migration of the receptor from the cy- signalling (Flouriot et al., 2000; Wang
survival and motility in human brain tosol into the nucleus. Dimerization et al., 2005). Of note, the so-called

CHAPTER 14
tumours (Opitz et al., 2011), but the

PART 2
of the receptor then induces a con- estrogen-related receptors show a
role of kynurenine in carcinogenesis formational change that allows sub- high degree of sequence homology
has not been established. To exert sequent binding of the receptor dimer with the ERs, but they are orphan re-
its effects, AhR heterodimerizes with to specific sequences of DNA known ceptors for which no ligand has been
a unique molecule, the AhR nuclear as estrogen response elements. The identified (Deblois and Giguère,
translocator (ARNT). DNA–receptor complex recruits oth- 2011, 2013). Although activation of
AhR, ER, and PR mediate ef- er proteins such as co-activators, the α-isoform of estrogen-related
fects of several agents that are which are responsible for the ini- receptors resulted in suppression
classified as IARC Group 1 human tiation of transcription of downstream of growth of xenografted human
carcinogens (IARC, 2012a, b) and DNA into mRNA, resulting in trans- breast cancer cells in nude mice
are discussed in more detail below. lation to proteins to produce chang- (Chisamore et al., 2009), there is no
Although AR has been implicated es in cellular function (Dickson and evidence that these nuclear recep-
in the causation of prostate cancer Stancel, 2000; Aranda and Pascual, tors are involved in estrogen-induced
in humans, this has not been firmly 2001). The second mechanism of carcinogenesis.
established, and there is only limited transcriptional regulation does not Two isoforms of PR have
evidence of the carcinogenicity of involve estrogen response elements been identified, PR-A and PR-B,
androgenic steroids in humans; they but is based on interaction of ER which are encoded by the same
are classified by IARC as probably with the transcription factors Fos and gene but controlled by different
carcinogenic to humans (Group 2A), Jun to bind to AP1 sites, or with Sp1 estrogen-regulated promoter re-
because there is sufficient evidence to bind to Sp1 sites (Nilsson et al., gions and different translation ini-
of their carcinogenicity in experimen- 2001). tiation events (Conneely et al., 1989;
tal animals (IARC, 1979, 1987). In The two basic forms of ER, ER-α Kastner et al., 1990; Kraus et al.,
rats, testosterone is a weak complete and ER-β, show some sequence 1993). These two receptor forms
carcinogen and a strong tumour homology and are widely distribut- play different roles in various tissues
promoter in the prostate (Bosland, ed in tissues of the body, including and cell types and may have oppo-
2014). target tissues of estrogen carcino- site molecular effects (Jacobsen
genicity, but they differ greatly in cell and Horwitz, 2012). Although PRs
Estrogen receptor (ER) and
type-specific abundance. In addition, are in many respects similar to ERs
progesterone receptor (PR)
although their basic molecular mech- in the way they initiate transcrip-
ER was discovered by Jensen anism of action is similar, they differ tion, dimerization may not always
(Jensen et al., 1968; Jensen and in specificity and binding affinity for be necessary, and PR monomers
DeSombre, 1973), and its gene was ligands (Matthews and Gustafsson, can bind to progesterone response
cloned in 1986 (Greene et al., 1986). 2003; Thomas and Gustafsson, element half-sites (Jacobsen et al.,

Part 2 • Chapter 14. Receptor-mediated mechanisms 133


2009; Jacobsen and Horwitz, 2012). that observed with estrogen alone breast of women treated with estro-
The gene expression and other bi- (Beral, 2003). The stimulating ef- gen–progestin menopausal therapy
ological responses mediated by re- fect of adding progestin treatment (Hofseth et al., 1999), and similar
ceptor isoforms are often progestin- to estrogen exposure in the induc- effects have been observed in mice
and PR subtype-specific (Graham tion of breast cancer has also been (Raafat et al., 2001; Haslam et al.,
and Clarke, 2002; Jacobsen and demonstrated in a rat model (Blank 2002). However, there are partially
Horwitz, 2012; Moore et al., 2012). et al., 2008). Furthermore, the pure conflicting data in humans (Harvey
The expression of both isoforms is anti-estrogen ICI 182780 inhibited et al., 2008), and progesterone has
induced by estrogens, but progestins growth stimulation of transplanted been found to inhibit estrogen-stim-
downregulate PRs (Jacobsen and ER-positive mouse mammary tu- ulated proliferation of breast cancer
Horwitz, 2012). mours by medroxyprogesterone in cells in vitro, depending on dose and
intact mice (Giulianelli et al., 2012). timing (Groshong et al., 1997; Lippert
Breast cancer induced by However, precisely how these two et al., 2000).
hormonal therapies hormones and their respective re- There are convincing data indi-
ceptors interact in increasing breast cating that the hormonal changes
Combined estrogen–progestin treat­
cancer risk is far from understood. during the normal menstrual cycle
ments, as therapy for menopausal
Both ER-α and ER-β can mediate are associated with cyclic changes
symptoms or as oral contracep-
the growth stimulatory and, at higher in the rate of proliferation of epitheli-
tives, are carcinogenic to the female
doses, inhibitory effects of estrogen, al cells in the breast, which reaches
breast, but only the menopausal
and the ratio of abundance of the two a maximum during the luteal phase,
therapy is carcinogenic to the endo-
ER isoforms and the cellular context when circulating levels of both estra-
metrium, whereas only the contra-
in which they operate appear to be diol and progesterone are high
ceptive treatment is carcinogenic to
critical determinants of the eventual (Anderson et al., 1989; Pike et al.,
the uterine cervix and liver (IARC,
effect (Sotoca et al., 2008; Soldati 1993). The cell proliferation rate of
2012b). The mechanisms by which et al., 2010). In addition, these ERs the breast epithelium is increased
these hormonal regimens cause may cross-talk with signalling path- during both the follicular phase and
these cancers in women are not ways and interact with PR (Giulianelli the late luteal phase (Anderson et al.,
clear. et al., 2012; Cotrim et al., 2013). 1989). These findings suggest that
The outcome of the estrogen- Initially, ER- and PR-mediated cyclicity in circulating hormone levels
alone arm of the Women’s Health induction of cell proliferation had may be a driving force in stimulating
Initiative randomized clinical trial was been postulated to be responsible cell proliferation in the normal breast,
remarkable in that breast cancer in- for the carcinogenic effects of es- resulting in a higher risk of breast
cidence was reduced (Anderson trogens and progestins (Anderson cancer with a higher number of years
et al., 2012), whereas breast cancer et al., 1989; King, 1991; Yager et al., during which a woman menstruates
incidence was increased in women 1991; Pike et al., 1993; Feigelson (Henderson et al., 1982). If this no-
treated with combined estrogen– and Henderson, 1996). However, tion were correct, one would expect
progestin (Chlebowski et al., 2010). this may be too simple a proposition, that continuous exposure to estrogen
These findings clearly indicate that because these hormonal factors also plus progestin would not increase
the addition of progestins (in the have a wide range of other biological breast cancer risk, but the Women’s
form of continuous medroxyproges- effects. Although cell proliferation Health Initiative randomized clinical
terone) to treatment with estrogens is no doubt a necessary compo- trial with continuous exposure to
(conjugated equine estrogens) is a nent of the carcinogenic process, both agents demonstrated that this
determining factor in breast carcino- is it probably not sufficient, and ad- is clearly not the case (Chlebowski
genesis induced by these hormones. ditional factors, some of which may et al., 2010). Furthermore, similar
In the prospective Million Women also be receptor-mediated, are likely effects in increasing breast can-
Study, estrogen-only treatment in- to be required to induce malignant cer risk were found for both se-
creased risk of breast cancer, and cell transformation (Dickson and quential and continuous combined
combined estrogen–progestin treat- Stancel, 2000) (see also below). estrogen–progestin treatment in the
ment was associated with a greater Enhanced cell proliferation has been prospective Million Women Study
increase in breast cancer risk than found in some substructures of the (Beral, 2003).

134
The breast cells that proliferate inantly expressed in luminal cells, the interplay of these molecularly
are not the cells that strongly express whereas PR expression is enriched diverse mechanisms is likely to be
ER or PR, suggesting that paracrine in the basal cell compartment, which highly complex and is still poorly
mechanisms, possibly involving may contain progenitors of luminal understood (Yager and Davidson,
breast stromal cells, play a major cells and possibly cancer progenitor 2006). In addition, the understanding
role. Breast density (i.e. the relative cells (Hilton et al., 2012). This obser- of how these receptors function is
proportion of the stromal component vation may have a bearing on the no- still evolving in many ways. For ex-
of the breast) has been found to be tion that there are four basic breast ample, it has become apparent that
associated with the rate of cell pro- cancer types (and possibly subtypes microRNAs regulate ER and PR ex-
liferation (Clarke et al., 1997; Russo of these) (Sorlie et al., 2001; Guiu pression and in turn are regulated by
et al., 1999; Harvey et al., 2008). et al., 2012; Elsawaf et al., 2013). these receptors as well (Adams et al.,
Although it is currently not known 2007; Maillot et al., 2009; Pandey
Thus, in vitro studies with benign
which of these four breast cancer and Picard, 2010; Cochrane et al.,
or malignant breast epithelial cells
types are associated with estrogen– 2012), but it is not known whether or
exposed to the sex hormones con-
progestin treatment, breast can- how these mechanisms are involved
tained in estrogen–progestin treat-
cer risk associated with hormonal in breast carcinogenesis.

CHAPTER 14
ments have limited significance,
menopausal therapy varied among Cancer of the endometrium and

PART 2
because they do not involve a stro-
different histological types of inva- ovary
mal component, although once they
sive and in situ breast cancer in the
are malignantly transformed, breast The reduction of risk of cancer of
Million Women Study (Reeves et al.,
cancer cells may become inde- the endometrium and ovary by use
2006). Of interest in this respect is
pendent of paracrine mediation and of combined oral contraceptives is
the observation that there may be
regulate their growth by autocrine well established, whereas menopau-
cross-talk between PR subtypes
mechanisms (Obr and Edwards, sal estrogen therapy unopposed by
and human epidermal growth factor
2012). receptor (HER)/ErbB receptors in progestins causes cancer of the en-
Also, cell culture-based studies HER-positive breast cancers (Lindet dometrium (IARC, 2012b). In the nor-
have yielded a somewhat confusing et al., 2012). mal premenopausal endometrium,
overall picture of how estrogen and Genotoxicity of estrogen metab- cell proliferation rates are high dur-
progestin treatments interact in af- olites potentially plays a role in the ing the follicular phase, when estra-
fecting cell proliferation. Progestins carcinogenicity of estrogen and es- diol levels peak, but they decline as
can inhibit estrogen-stimulated pro- trogen–progestin treatments (Yager progesterone levels rise in the luteal
liferation of breast cells (Seeger and Liehr, 1996; Cavalieri et al., phase (Whitehead et al., 1981; Key
et al., 2003a, b), but they also in- 2000; Yager and Davidson, 2006). In and Pike, 1988).
crease the ratio of apoptosis to prolif- addition, some estrogen metabolites It is well established that the
eration (Krämer et al., 2005; Seeger (the 4- and 16α-hydroxylated but longer a woman menstruates, the
et al., 2005). Dose and type of pro- not the 2-hydroxylated metabolites) higher the risk of cancer of the en-
gestin appear to be critical in deter- can also have cell proliferative ef- dometrium (Key and Pike, 1988;
mining the eventual effect (Seeger fects through poorly defined mecha- Karageorgi et al., 2010). This rela-
et al., 2003b, 2005). Despite their nisms that may involve ER mediation tionship is similar for cancers that
limitations, these in vitro studies have (Seeger et al., 2006). differ in the degree of genomic insta-
shown that ER- and PR-mediated Overall, there is little doubt that bility (Amankwah et al., 2013) and for
apoptosis must be considered as an ER- and PR-mediated mechanisms, both the common endometrioid form
important effect of combined estro- including stimulation of breast cell of endometrial cancer (type I) and
gen–progestin treatment, and they proliferation, are involved in the the less common type II endometri-
have provided evidence that growth carcinogenicity of estrogen–pro- al cancers (including serous, clear
factors can be determinants of these gestin treatments (Sutherland et al., cell, and mixed Müllerian tumours)
effects (Krämer et al., 2006). 1998; Anderson and Clarke, 2004). (Setiawan et al., 2013).
More recent observations in nor- However, other mechanisms are Estrogen therapy during men-
mal human breast tissue have pro- clearly also operational and are opause also increases endometri-
vided evidence that ER-α is predom- probably critically important as well; al cell proliferation, and this effect

Part 2 • Chapter 14. Receptor-mediated mechanisms 135


is counteracted by progesterone female and male offspring of women oral contraceptives cause malignan-
(Whitehead et al., 1981), whereas exposed to this synthetic estrogen cies of the liver, uterine cervix, and
progestin-only contraceptives sup- during pregnancy (Titus-Ernstoff breast, but the precise mechanisms
press cell proliferation in the en- et al., 2008; Kalfa et al., 2011). by which these tumours are caused
dometrium (Landgren et al., 1990; Whether these epigenetic effects are and how exactly these hormonal
Moyer and Felix, 1998). It is not ER-mediated is unknown (Newbold treatments are involved mechanis-
clear whether changes in cell prolif- et al., 2006). It is also unclear wheth- tically is far from clear. Most recent
eration are the only explanation for er the immunosuppressive effects of research has focused on molecular
the induction of endometrial cancer diethylstilbestrol are ER-mediated alterations in cancers at these sites
by estrogens and for the preventive (Brown et al., 2006). Overall, the and the mechanisms by which they
effects of combined oral contracep- contribution of ER-mediated mecha- can develop, but how hormonal fac-
tives on endometrial and ovarian nisms in the carcinogenic effects of tors intersect with these process-
cancer and the preventive effects in utero exposure to diethylstilbestrol es remains unknown (Merritt and
of progestins on endometrial cancer is not clear. It seems likely that any Cramer, 2010).
induced by estrogens. Even less is ER-mediated effect of prenatal expo- Even more nebulous is how com-
known about hormonal effects on the sure to diethylstilbestrol facilitates its bined oral contraceptives provide
ovary that may be involved in ovarian genotoxic effects by stimulating cell lasting protection against cancer
carcinogenesis. proliferation in target cells, or other- of the endometrium and ovary, and
wise by increasing the induction of how precisely progestins protect
Diethylstilbestrol the DNA alterations that underlie the the endometrium against estro-
carcinogenicity of diethylstilbestrol gens used in menopausal therapy.
The human transplacental carcin- (IARC, 2012b). Downregulation of PR may in part
ogen diethylstilbestrol is not only Studies with mice that overex- explain these protective effects, be-
genotoxic but is also probably the press or lack ER-α suggest that cause it may result in reduction of
strongest known estrogen. Its ge- endogenous estrogen in the adult cell proliferation, but other mecha-
notoxic effects in human and rodent animal acts as a tumour promoter nisms are also likely to be involved.
tissues and cells are unlikely to be via ER mediation, after induction of Similarly, just evoking stimulation of
receptor-mediated. Non-genotoxic permanent genotoxic and epigenetic cell proliferation as the mechanism
effects of diethylstilbestrol mediat- effects by exposure to diethylstilbes- by which unopposed estrogen caus-
ed by ERs are hormonal in nature trol early in life (Couse et al., 1997, es endometrial cancer is probably
and involve estrogenic stimulation 2001; Couse and Korach, 2004). an oversimplification as well.
of cell proliferation, which has a bi- This notion would be consistent with Involvement of ER in the mechanism
phasic dose–response relationship the observation that malignancies by which diethylstilbestrol causes
in breast cancer cells in vitro (Reddel induced by diethylstilbestrol in wom- cancer in women after prenatal ex-
and Sutherland, 1987). en exposed in utero do not appear posure may be limited to the effects
Diethylstilbestrol induces epige- until after menarche (Couse and of endogenous estrogens acting as
netic changes in DNA methylation Korach, 2004). The role of ER in the tumour promoters.
in target tissues in rodents (Li et al., increased risk of breast cancer ob-
Aryl hydrocarbon receptor
2003; Newbold et al., 2007; Tang served in women exposed to diethyl-
(AhR)
et al., 2008). These changes may be stilbestrol during pregnancy is not
heritable (Anway and Skinner, 2006) clear, but a combination of genotoxic AhR is a member of the PER-ARNT-
and conceivably underlie the mech- effects and stimulation of cell prolif- SIM family of basic helix–loop–helix
anism responsible for the develop- eration is likely (IARC, 2012b). transcription factors (Burbach et al.,
mental and carcinogenic effects ob- In conclusion, cell proliferation 1992). This receptor is induced by
served in the second, and possibly appears to be involved in how hor- and binds to a very large number
the third, generation of rodents after mones used in menopausal thera- of exogenous ligands, including
in utero exposure to diethylstilbestrol py cause cancer of the breast and PAHs such as benzo[a]pyrene,
(Newbold et al., 1998, 2000); these endometrium and possibly cancer planar PCBs, polychlorinated
effects have also been reported in of the ovary, and how combined dibenzofurans, and its most potent

136
ligand, TCDD (Denison et al., 2002; interact directly with phosphorylat- relevant that there are interspecies
Tsuchiya et al., 2003; Flaveny et al., ed retinoblastoma protein, leading and rat and mouse strain-specific
2009). to cell-cycle arrest, and with ER-α differences in AhR binding affinity for
Endogenous ligands for AhR and ER-β in various ways, resulting TCDD, as well as in functional AhR
have not been identified with cer- in repression of ER-mediated signal- polymorphisms, AhR abundance,
tainty, but it is likely that these exist ling and anti-estrogenic effects (Safe and cofactors required for AhR ac-
and that AhR has a physiological role et al., 1998; Safe and Wormke, 2003; tivity, which may all be critical to the
(Gonzalez and Fernandez-Salguero, Dietrich and Kaina, 2010; Denison carcinogenic process (Barrett, 1995;
1998; Carlson and Perdew, 2002; et al., 2011). Interestingly, ARNT by De Souza et al., 2009; Flaveny et al.,
Bock and Köhle, 2009). Certain en- itself can activate ER-α, and par- 2009).
dogenously formed tryptophan deriv- ticularly ER-β (Rüegg et al., 2008). The binding affinity of human AhR
atives that bind to and activate AhR AhR may activate cyclin A via JunD for dioxin is much weaker than that
have been identified, but their phys- and ATF2, thereby inhibiting contact found for AhR in some rodent spe-
iological role has not yet been firm- inhibition in vitro, which would be a cies (Ema et al., 1994; Ramadoss
ly established (Denison and Nagy, pro-carcinogenic effect (Weiss et al., and Perdew, 2004). Rodents have
2003; Henry et al., 2006; Perdew 2008; Dietrich and Kaina, 2010). a high degree of AhR sequence ho-

CHAPTER 14
PART 2
et al., 2015). Factors that can acti- The great diversity of genes reg- mology (Korkalainen et al., 2001), but
vate AhR in cultured liver cells have ulated by AhR and the many mech- there is less homology with human
been found in the serum of knockout anisms involved are remarkable and AhR (Ema et al., 1994; Flaveny et al.,
mice that lack AhR (McMillan and illustrate the considerable complexity 2008). There are also differences
Bradfield, 2007), but whether these in how AhR functions (Denison et al., between humans and rodents in the
are the elusive endogenous ligands 2011). This complexity is exacerbated response of hepatic gene expres-
is uncertain. because (i) ligand-dependent differ- sion to TCDD, but little difference
This cytosolic receptor, once ences have been observed in co-ac- in inducibility of CYP450 enzymes
bound to a ligand, translocates to tivator recruitment and in binding (Mandal, 2005).
the nucleus, loses various chaper- specificity of AhR towards sequence Mice that carry AhR variants
one molecules, such as Hsp90, and variants in xenobiotic response ele- with different dioxin-binding affini-
heterodimerizes with ARNT. The ments, (ii) cell type-specific co-acti- ties vary in their response towards
AhR–ARNT complex then binds to vators and co-repressors can mod- TCDD toxicity and PAH-induced
xenobiotic response elements, also ulate DNA binding specificity of the carcinogenesis (Garte and Sogawa,
termed dioxin response elements, AhR–ARNT complex, (iii) AhR and 1999; De Souza et al., 2009; Flaveny
in the promoter regions of the genes the AhR–ARNT complex may them- et al., 2010). Thus, there are differ-
it regulates, including those encod- selves act as co-activators for other ences between humans and rodents
ing CYP1A1, CYP1A2, CYP1B1, transcription factors, and (iv) exten- in AhR binding affinity that may
glutathione-S-transferase M, and sive cross-talk exists with important account for interspecies variation
nicotinamide adenine dinucleotide signalling pathways (Beischlag et al., in AhR-mediated carcinogenicity.
phosphate NAD(P)H:quinone ox- 2008; Denison et al., 2011). Toxicogenomic comparisons of hu-
idoreductase, all of which are in- It is not surprising that activation man, rat, and mouse cells or tissues
volved in metabolism of xenobiotics of AhR results in distinct species- exposed to TCDD indicate consider-
(Beischlag et al., 2008; Denison and tissue-specific changes in gene able interspecies differences in the
et al., 2011). Involvement of AhR in expression profiles and that these response to dioxin (Boverhof et al.,
immune regulation, the cell cycle, may be subject to temporal changes 2006; Black et al., 2012). Of note,
the function of mucosal barriers, and can be dependent on, as well there are AhR polymorphisms in hu-
and development has recently been as independent of, TCDD and diox- mans, some of which could conceiv-
identified (Hubbard et al., 2015). in-like compounds such as PCBs ably be associated with differences
In addition, AhR can interact with and dibenzofurans (Vezina et al., in toxic effects and cancer risk in re-
molecules other than ARNT and can 2004; Tijet et al., 2006; Kopec et al., sponse to exposure to TCDD (Garte
engage extensively in cross-talk with 2008, 2013; N’Jai et al., 2008; Dere and Sogawa, 1999; Hung et al.,
various signalling pathways. It can et al., 2011a, b). In this regard, it is 2013; Luo et al., 2013).

Part 2 • Chapter 14. Receptor-mediated mechanisms 137


AhR-mediated carcinogenesis ly to be required for the carcinogeni- Many of these effects and mech-
city of 2,3,7,8-TCDD, its precise role anisms may be ligand-specific, as
TCDD and dioxin-like PCBs are in this process remains unclear.” Ten indicated by comparative toxicoge-
considered to be AhR-mediated nomic studies of the liver of mice in
years later, Walker (2007) concluded
carcinogens. PCBs as a group have vivo and of primary rat hepatocytes
that “despite … decades of research,
recently been designated by IARC in vitro after short-term exposure
we still do not have a clear mecha-
as Group 1 carcinogens (Lauby- to dioxin-like compounds (Kopec
nism of action leading from ligand
Secretan et al., 2013; IARC, 2016). et al., 2008, 2010; N’Jai et al., 2008;
binding of TCDD or a related ligand
TCDD is a multisite human carcin- Rowlands et al., 2011). In toxicoge-
to the AhR and the ultimate develop-
ogen (Warner et al., 2011; IARC, nomic studies, longer-term in vivo
ment of toxicities [including carcino-
2012a, 2016). There is sufficient ev- exposure (13 or 52 weeks) of rats to
genesis]”. Research from the past
idence from epidemiological studies the AhR ligands TCDD, PeCDF, and
several years does not change this
that exposure to PCBs causes mela- PCB 126, as well as the non-AhR
conclusion (Budinsky et al., 2014).
noma (Loomis et al., 1997; Gallagher ligand 2,2′,4,4′,5,5′-hexachlorobiphe-
TCDD, PCB 126, and PeCDF
et al., 2011; IARC, 2012a, 2016). For nyl (PCB 153), revealed consider-
are complete carcinogens as well
non-Hodgkin lymphoma, there are able ligand specificity (Vezina et al.,
as tumour promoters in rodents,
mixed data, suggesting that risk may 2004; Ovando et al., 2010).
and some other PCBs and poly-
be elevated in men but not in women Nevertheless, these gene expres-
chlorinated dibenzofurans have
(Bertrand et al., 2010; Laden et al., sion profiling studies did show partial
tumour-promoting activity as well
2010; Maifredi et al., 2011; Bräuner overlap of genes regulated by TCDD,
(Hébert et al., 1990; Anderson et al.,
et al., 2012; Kramer et al., 2012; PCB 126, and PeCDF (Vezina et al.,
1991; Waern et al., 1991; Hemming
IARC, 2016). The most recent up- 2004; Kopec et al., 2008; Ovando
et al., 1995; IARC, 1997). Induction
date of the follow-up study on people et al., 2010). PeCDF and PCB 126
of xenobiotic-metabolizing CYP450
affected by the poisoning incident in appeared to share more gene re-
enzymes has been recognized as
1968 in Japan, where rice oil was sponses than either compound
one of the major effects of activa-
contaminated with PCBs and poly- shared with TCDD (Vezina et al.,
chlorinated dibenzofurans, indicat- tion of AhR relevant to its contrib-
2004). These studies used equi-
ed small but statistically significant ution to the carcinogenic effects of
potent doses on the basis of so-
associations with risk of all cancers planar PAHs, TCDD, and dioxin-like
called toxic equivalency factors, and
and risk of cancer of the liver and compounds (IARC, 1997, 2012a).
showed AhR-mediated gene expres-
lung (Onozuka et al., 2009). The epi- However, many other major effects
sion responses that were shared
demiology for PeCDF is very sparse of AhR activation can conceivably
among TCDD, PCB 126, and PeCDF
(IARC, 2012a). also contribute to the carcinogeni- or 2,3,7,8-tetrachlorodibenzofuran.
TCDD has an extremely wide city of these AhR ligands, including However, the non-coplanar PCB
range of biological and toxic ef- induction of cell proliferation, oxida- 153, which does not activate AhR but
fects, many, but not all, of which tive stress, cross-talk with signalling may antagonize it (Suh et al., 2003),
are mediated by AhR (Poland and pathways involved in carcinogenic also shared some gene responses
Glover, 1980; Birnbaum et al., 1990; mechanisms, induction of cell pro- with TCDD and PCB 126 (Kopec
Fernandez-Salguero et al., 1995). liferation, and alteration of cell–cell et al., 2010; Ovando et al., 2010).
AhR is critical to the carcinogenicity interactions (Schwarz and Appel, Exposure to TCDD, and possibly
of TCDD and at least some diox- 2005; Dere et al., 2006; Dietrich and to 2,2′,3,3′,4,4′-hexachlorobiphenyl
in-like compounds, such as certain Kaina, 2010). Sustained activation of (PCB 128) and PeCDF, affects vari-
PCBs and polychlorinated dibenzo- AhR has been postulated to be nec- ous metabolic genes and cell regula-
furans, as summarized in Volume 69 essary for its carcinogenic activity in tory pathways known to be involved
(IARC, 1997), Volume 100F (IARC, the liver in rodents (Budinsky et al., in cancer, such as genes of the G
2012a), and Volume 107 (IARC, 2014). Carcinogenicity of PCBs is protein-coupled receptor protein
2016) of the IARC Monographs. In likely to involve not only AhR activa- signalling pathway and genes of the
Volume 69, it was stated that “even tion but also other such mechanisms polyamine and glutathione pathways
though Ah receptor activation is like- (IARC, 2016). (Jennen et al., 2011). Exposures to

138
combinations of PCBs have not been Stemmann et al., 2009; Puga et al., et al., 1997; Oliver and Roberts,
studied but would be of interest, giv- 2009) and the transforming growth 2002). TCDD has been found to in-
en that, for example, PCB 153 signif- factor beta (TGF-β) (Gomez-Duran duce hepatocellular replicative DNA
icantly increased the carcinogenic et al., 2009) and nuclear factor kap- synthesis in vivo in some studies
potency of PCB 126 (NTP, 2006c). pa-light-chain-enhancer of activated (Lucier et al., 1991) but not in oth-
Overall, the mechanism of action of B cells (NF-κB) pathways (Tian et al., ers (Fox et al., 1993; Bauman et al.,
TCDD and dioxin-like compounds 1999; Vogel and Matsumura, 2009). 1995), and it inhibits growth of pri-
appears to be highly complex and to There is also evidence for cross-talk mary hepatocytes in vitro (Hushka
be dependent on ligand, dose, du- with the constitutive androstane re- and Greenlee, 1995). Lack of AhR in
ration of exposure, and sex, and it ceptor and one of its target genes in knockout mice resulted in increased
is only partly understood (Silkworth mice, Cyp2b10 (Patel et al., 2007), hepatocellular apoptosis (Zaher
et al., 2008; Budinsky et al., 2014). and with cyclin G2 (Ahmed et al., et al., 1998), but TCDD treatment
Oxidative stress and oxidative 2012). of rats that express AhR also re-
DNA damage have long been recog- The extensive cross-talk with sulted in increased apoptosis in the
nized as important factors in carcino- ER signalling mentioned above ex- liver during hepatocarcinogenesis
genesis (Klaunig et al., 2011) (see plains the anti-estrogenic properties (Stinchcombe et al., 1995). In human

CHAPTER 14
PART 2
Chapter 15, by Bucher). Oxidative of TCDD (Safe and Wormke, 2003; skin cells in vitro, TCDD caused in-
stress has been demonstrated to Denison et al., 2011). There is evi- hibition of cellular senescence, pre-
occur after in vivo and in vitro expo- dence that this AhR–ER cross-talk sumably via AhR, which may explain
sure to AhR ligands including TCDD, can occur at different levels of AhR in part the tumour-promoting activi-
PCB 126, and PeCDF (Yoshida involvement in the regulation or ty of TCDD in initiated skin in mice
and Ogawa, 2000; Hennig et al., dysregulation of gene expression, (Ray and Swanson, 2009). However,
2002). Upregulation of CYP1A1 and including non-classical modes with whether this mechanism occurs in
CYP1B1 by activated AhR increases AhR–ARNT or just AhR acting as vivo and in the liver is not known.
the chance of production of reactive co-repressor (Ohtake et al., 2003; PCBs can also stimulate hepatic cell
oxygen species upon uncoupling Labrecque et al., 2012). proliferation, but this is not consis-
of the catalytic cycle of these en- Mediated by AhR, TCDD down- tently found and may or may not be
zymes, as has been shown for ex- regulates the EGF receptor and in- AhR-dependent.
posure to dioxin-like coplanar PCBs hibits binding of EGF to its receptor The coplanar PCB 126 and the
(Schlezinger et al., 2006; Green et al., in several tissue types (Madhukar non-coplanar PCB 153 both in-
2008). In addition, in experiments et al., 1984; Kitamura et al., 2006; duced proliferation, but only af-
with mice, AhR activation has been Haarmann-Stemmann et al., 2009). ter a tumour-initiating dose of
associated with mitochondrial pro- This reduces EGF signalling, which diethylnitrosamine, and both had
duction of reactive oxygen species may be an anti-carcinogenic rath- liver tumour-promoting activity
in a process that does not appear er than a pro-carcinogenic effect. (Rignall et al., 2013). However, in
to involve CYP450 enzymes (Senft Conversely, EGF receptor signalling other studies, PCB 153 stimulated
et al., 2002). appears to inhibit AhR action at the hepatocellular proliferation in rats
Many signalling pathways are DNA level (Sutter et al., 2009), indi- but not when preceded by treatment
mechanistically involved in carcino- cating significant cross-talk with EGF with diethylnitrosamine, whereas
genesis, including those involved pathways. the coplanar 3,3′,4,4′-tetrachlorobi-
in regulation of cell proliferation, Induction of cell proliferation has phenyl (PCB 77) inhibited or stim-
apoptosis, senescence, and angio- long been considered to be a causal ulated hepatocellular proliferation
genesis (Hanahan and Weinberg, factor in carcinogenesis in humans (Tharappel et al., 2002; Lu et al.,
2011) (see Chapter 10, by Smith). and experimental animals (Preston- 2003, 2004). PCB 126, but not PCB
There is considerable evidence of Martin et al., 1990; Schwarz et al., 153, stimulated proliferation of cul-
extensive cross-talk of AhR with 1995), with receptor mediation and tured liver cells (Vondrácek et al.,
several of these and other path- an imbalance between proliferation 2005). In studies with exposures of
ways, including various nuclear and apoptosis in favour of cell growth up to 2 years, PCB 153 did not stim-
transcription factors (Haarmann- as major mechanisms (Roberts ulate hepatic cell proliferation and

Part 2 • Chapter 14. Receptor-mediated mechanisms 139


had equivocal carcinogenic activity, null mice (Fritz et al., 2007). These be involved in the carcinogenic pro-
whereas PCB 126 did stimulate pro- findings suggest that AhR can func- cess. Nonetheless, it is clear that
liferation after exposure for 1 year tion as a tumour suppressor, which AhR is critical to the carcinogen-
and was carcinogenic (NTP, 2006a, is supported by the observation of icity of TCDD and some or many
b). In similar studies, PeCDF and the increased cell proliferation and re- dioxin-like compounds as well as
coplanar 2,3′,4,4′,5-pentachlorobi- duced apoptosis in liver tumours some important PAHs, and it is ev-
phenyl (PCB 118) also stimulated he- induced by diethylnitrosamine in ident that this receptor can be in-
patic cell proliferation and had carci- AhR-null mice (Fan et al., 2010). volved in cancer-initiating as well as
nogenic activity (NTP, 2006c, 2010). There is some evidence from tumour-promoting mechanisms.
Overall, the involvement of AhR- studies with human tumour cells
mediated changes in cell prolifera- in culture to support the idea that Summary
tion and apoptosis in TCDD carcino- AhR has tumour-suppressive po-
genesis is not clear and may depend tential (Zudaire et al., 2008). This How do we evaluate – on the basis
on context (in vivo or in vitro), tumour notion may appear to be in conflict of mechanistic data – the carcino-
induction protocol (initiation–promo- with the reduced or absent tumour genicity to humans of chemicals
tion or continuous treatment), tissue, development in mice that lack AhR that have not been tested in chronic
dose and duration of TCDD treat- in response to treatment with ben- animal bioassays or adequate epi-
ment, and species (Budinsky et al., zo[a]pyrene and dibenzo[a,l]pyrene demiological studies? Are all chemi-
2014). This may also be the case for (Shimizu et al., 2000; Nakatsuru cals that bind to and activate AhR, or
dioxin-like PCBs and dibenzofurans. et al., 2004). However, mice that all agents that activate both ER and
AhR appears also to be critical lack the Arnt gene specifically in PR, human carcinogens?
to the carcinogenicity of PAHs that the skin did not develop skin cancer A case in point is the designation
are preferentially metabolized by when treated with benzo[a]pyrene of PCB 126 and PeCDF as carcino-
CYP1A1 and CYP1A2, such as ben- plus TPA, whereas they did so when genic to humans (Group 1) in Volume
zo[a]pyrene and dibenzo[a,l]pyrene treated with a carcinogen that does 100F of the IARC Monographs (IARC,
(Shimizu et al., 2000; Nakatsuru not require AhR mediation, such as 2012a). This classification was based
et al., 2004), but not 7,12-dimethyl­ N-methyl-N-nitrosourea, followed by on the ability of these chemicals
benz[a]anthracene (DMBA) (Ide TPA (Shi et al., 2009). This finding to activate AhR, induce CYP1A1,
et al., 2004). This is presumably be- strongly suggests that CYP1A1 in- CYP1A2, and other drug-metaboliz-
cause DMBA is metabolically activat- duction by the AhR–ARNT complex ing enzymes, stimulate cell prolifer-
ed by CYP1B1, which does not need in response to benzo[a]pyrene treat- ation, induce oxidative stress, and
induction by AhR but is constitutively ment is required for benzo[a]pyrene have carcinogenic and tumour-pro-
expressed in rodent target tissues, to be carcinogenic. moting activities in ways similar to
and is required for the carcinogen- In conclusion, AhR is activated those observed with TCDD. A critical
icity of DMBA (Buters et al., 1999; by many chemical agents; some of point was the ability of these agents
Ide et al., 2004). Diethylnitrosamine, these are carcinogens, and others to produce a spectrum of neoplasms
which is not an AhR ligand and are not. Most or all carcinogenic that is similar to the tumours found
is metabolically activated not by compounds that activate AhR are after treatment with TCDD in the
CYP1A1 but by CYP2E1, induced likely to also act via other mecha- same rodent species. This designa-
liver tumours in mice, and this effect nisms to cause cancer. Thus, AhR tion would probably not have been
was markedly enhanced in mice that activation is probably best consid- justified in the absence of such ani-
lack AhR (Fan et al., 2010). ered as an important mechanistic mal bioassay data.
TRAMP (transgenic adenocarci- effect of carcinogens rather than as There may be compounds that
noma mouse prostate) mice, which the sole or predominant carcinogen- activate AhR and induce CYP1A1
have disrupted retinoblastoma and ic mechanism of such compounds. and CYP1A2 but are not carcinogen-
p53 function in the prostate, result- In view of the complexity of AhR- ic. β-Naphthoflavone is an example
ing in tumour development in this mediated mechanistic events, it is of such a compound that activates
organ, displayed increased tumour likely that there are many ways in AhR and induces CYP1A1 but is
formation when crossed with AhR- which this important receptor can not a carcinogen (Denison et al.,

140
2011), even though it is a liver tu- liferation or reduction of apoptosis; compounds, such as those used in
mour promoter after treatment with in that concept, receptor-mediated combined oral contraceptives and
diethylnitrosamine (Hayashi et al., carcinogens are tumour promoters, hormonal menopausal therapy, the
2012). Omeprazole also activates co-carcinogens, enhancers of sus- dose–response is typically biphas-
AhR and induces CYP1A1 but is not ceptibility, or indirect inducers of ge- ic (inverted U-shaped), and the ef-
carcinogenic (Kleeberg et al., 1999). notoxicity, for example via induction fect threshold is observed at very
However, many other agents that of oxidative stress. For example, in- low doses (Reddel and Sutherland,
activate AhR and induce CYP1A1 creased cell proliferation has been 1987; Groshong et al., 1997).
and CYP1A2 are carcinogenic. shown to neoplastically transform rat A further complicating issue is the
Furthermore, induction of oxidative hepatocytes only after treatment with evidence for a wide range of modi-
stress by TCDD and dioxin-like com- a genotoxic agent (Lee et al., 1989). fying factors that can affect the re-
pounds is non-specific and is – like NIH 3T3 cells will undergo complete ceptor-mediated action of suspected
various other pleiotropic effects of neoplastic transformation under fa- carcinogenic agents; not only is the
these agents – perhaps not required vourable conditions after sustained dose critical, but species, sex, age,
for AhR-mediated carcinogenesis. proliferation, but these cells are al- tissue or cell context, duration and
Such effects can only be considered ready immortalized (Yao et al., 1990; timing of exposure, and route of ex-

CHAPTER 14
Chow and Rubin, 2000). posure are all crucially important

PART 2
additional evidence suggesting that
a compound that activates AhR and Thus, it appears that stimula- modifying factors, as pointed out
induces CYP1A1 and CYP1A2 is car- tion of cell proliferation by itself is above. Often neglected are co-ex-
cinogenic to humans or experimental not sufficient to induce cancer, but posures that may be important in de-
animals. that it can be a powerful enhancer termining the ultimate effect; for ex-
Stimulation of cell proliferation of carcinogenesis by facilitating ini- ample, exposure to cigarette smoke
is often invoked as an effect that tiating events, for example through and its constituents may influence
indicates carcinogenic potential of fixation of pro-mutagenic DNA le- the outcome of exposure to TCDD
sions, affecting DNA repair, inducing or other dioxin-like compounds
chemicals and as a cause of human
error-prone DNA synthesis (Mimura (Kitamura and Kasai, 2007).
cancer (Preston-Martin et al., 1990;
and Fujii-Kuriyama, 2003), and/or The diversity of ER-, PR-, and
Oliver and Roberts, 2002). But one
by acting as a driving force during AhR-mediated effects observed
can ask whether receptor-mediated
tumour promotion and progression. across species, sexes, tissues, and
effects resulting in enhancement of
These notions obviously have impli- cell types and across exposure con-
cell proliferation and/or reduction of
cations for the use of cell prolifera-
apoptosis are sufficient for carcino- ditions (dose, duration, timing, and
tion as a criterion in hazard identi-
genicity, or whether more is needed. route of exposure) poses consid-
fication of suspected carcinogens
Induction of cell proliferation erable obstacles to rational testing
and in carcinogen risk assessment
by estrogens and progestins or by for carcinogenic and mechanistic
(Melnick et al., 1996).
TCDD and dioxin-like compounds effects that predict human carcino-
Receptor-mediated events are
is not a simple effect but is highly genicity of agents that act via these
typically dose-dependent, and
complex and dependent on a wide receptors. Whole-animal models are
dose–response relationships be-
variety of factors, as was pointed out essential to sort out these effects,
tween ligands and receptor-mediated
above. Only in certain circumstances effects are often nonlinear. This has and animal bioassays that are de-
will these agents stimulate cell pro- led to the concept of a dose thresh- signed to detect relevant carcino-
liferation in target tissues, whereas old for receptor-mediated carcino- genic effects remain indispensable.
in other situations they may inhibit gens that is used in carcinogen risk However, no good animal models
proliferation (Hushka and Greenlee, assessment. However, non-thresh- exist for exposures to some of the
1995). Furthermore, there is only old dose–responses also occur, and important receptor-mediated carcin-
scant direct evidence that increased there are many factors that can af- ogens. It is not clear how to best test
cell proliferation by itself causes tu- fect the shape of dose–responses of for effects of agents that are used or
mours or malignant transformation. non-genotoxic or receptor-mediated intended as oral contraceptives, and
Most likely, genetic damage is re- carcinogens (Melnick et al., 1996). there are no truly relevant animal
quired in combination with cell pro- Moreover, for steroid hormone-like models reflecting conditions found

Part 2 • Chapter 14. Receptor-mediated mechanisms 141


during and after human menopause. Perhaps the most difficult aspect of complexity as to how such carci-
Mice or rats ovariectomized at age of evaluating the carcinogenic po- nogenic agents mechanistically act
1–1.5 years may be useful as a mod- tential of agents that act via recep- via receptors, and insight into these
el for menopause, but this approach tors, particularly AhR, ER, and PR, mechanisms continues to evolve.
has not been used much. More re- is the sheer complexity of how these Receptor binding and activation by
search will be needed to develop and receptors are involved in mediat- chemical agents or the induction of
validate approaches with appropriate ing the effects of chemical agents cell proliferation or oxidative stress
animal models for these important (Pandey and Picard, 2010; Denison are by themselves insufficient as ev-
exposure conditions in humans. et al., 2011; Jennen et al., 2011; Ruiz- idence for carcinogenicity in animals
As indicated above, there are Aracama et al., 2011; Thomas and or humans.
also no validated models to evaluate Gustafsson, 2011; Cochrane et al., To make progress in the identi-
the protective effects of combined 2012; Jacobsen and Horwitz, 2012). fication of receptor-mediated car-
oral contraceptives and hormonal Signature changes in gene expres- cinogens, it will be important (i) to
agents used in menopausal therapy. sion induced in target cells or tissues identify signature changes in gene
Although standard animal models for by suspected carcinogens would be expression in relevant tissues and
cancer of the colon and breast could a most useful tool for carcinogen cells in validated animal models that
be used, there is a paucity of mod- identification if they would suggest are predictive of carcinogenic activi-
els for cancer of the endometrium, or predict the carcinogenicity of the ty of groups of structurally and func-
ovary, and cervix. To investigate ef- agents. However, no consensus al- tionally related chemical agents, and
fects of environmental exposures to terations in gene expression profiles (ii) to further develop and validate
receptor-mediated dioxin-like com- have yet been identified for groups such in vivo models. It also remains
pounds, the Harlan Sprague-Dawley
of agents that are thought to act critical to continue generating data
rat has proven to be suitable and
via similar mechanisms, such as di- that allow evaluation of coherence in
sensitive in a large number of studies
oxin-like compounds or estrogenic mechanisms and concordance in ex-
with these agents. Mice that lack or
substances. In fact, as pointed out posure-associated tumour types and
overexpress AhR have been useful
above, even within the group of di- target sites between animal models
to examine mechanisms underlying
oxin-like compounds there is consid- and humans.
the effects of TCDD, but differences
erable variation in the gene expres- An improved understanding is
related to genetic background and
sion changes they induce (Vezina still needed of the relationships be-
strain hamper the generalization of
et al., 2004; Kopec et al., 2008, tween carcinogenic activity and tim-
the results of such research. Studies
2010; Ovando et al., 2010; Jennen ing, dose, and duration of exposure
with diethylstilbestrol have demon-
et al., 2011). In addition, knowledge to receptor-mediated carcinogens,
strated that for exposure to hormonal
about the mechanisms by which and of the cancer-inhibitory activity
agents in early life, particularly in ute-
these chemicals act continues to of exposure to some of these agents
ro, neonatal treatment of CD-1 mice
and Sprague-Dawley rats is informa- evolve in major ways. For example, for some tissue sites, such as that
tive and relevant for effects observed recent data indicate that some PCBs demonstrated for the reduction of
in humans. However, in vitro models can be genotoxic because they are risk of cancer of the endometrium,
may not be very relevant for studying metabolized to DNA-damaging re- ovary, and colorectum by combined
the effects of most carcinogens that active intermediates, overturning the oral contraceptives and of risk of
act via receptor mediation, because notion that PCBs are purely recep- cancer of the colon by estrogen-only
of the likely importance of paracrine tor-mediated agents (Ludewig and menopausal therapy (IARC, 2012b).
mechanisms involving stromal cells, Robertson, 2012; IARC, 2016).
as shown for ER- and PR-mediated In conclusion, receptor-mediat-
events. However, as a screening ed mechanisms are central to the
tool, approaches based on the use carcinogenicity of important groups
of cell cultures will remain very im- of human carcinogens, but they are
portant for initial investigations and often incompletely or poorly under-
studies that are not feasible in whole stood. Research during the past two
animals. decades has revealed a high degree

142
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chapter 15.

Oxidative stress and


radical-induced signalling
John R. Bucher

CHAPTER 15
PART 2
Throughout evolution, aerobic An imbalance between the normal Pierre et al., 2002). Peroxisomes are
organisms have developed mul- production of oxygen radicals and a source of H2O2, through reactions
tiple defence systems to protect their capture and disposal by pro- involving acyl-CoA oxidase (which
themselves against oxygen radicals tective enzyme systems and antiox- is involved in oxidation of long-chain
(Benzie, 2000). One-, two-, and idants results in oxidative stress, and fatty acids), d-amino acid oxidase,
three-electron reductions of molecu- this condition has been proposed and other oxidases (Schrader and
lar oxygen give rise to, respectively, to be the basis of many deleterious Fahimi, 2006).
superoxide (O2• −), hydrogen peroxide chronic health conditions and dis- When stimulated, inflammatory
(H2O2, a radical precursor), and the eases, including cancer. cells such as neutrophils, eosino-
highly reactive hydroxyl radical (•OH) phils, and macrophages produce ox-
or equivalent transition metal–oxy- Sources of oxygen radicals ygen radicals during the associated
gen complexes (Miller et al., 1990). respiratory burst (the rapid release of
Reactions of oxygen radicals with Mitochondrial oxidative phosphor- reactive oxygen species from cells)
cellular components can deplete an- ylation is a major source of oxy- that involves nicotinamide adenine
tioxidants, can cause direct oxidative gen radicals of endogenous origin. dinucleotide phosphate (NADPH)
damage to lipids, proteins, RNA, and Mitochondrial complex I (reduced oxidase (Babior, 1999). This reac-
DNA, and can result in the forma- nicotinamide adenine dinucleotide tion produces superoxide, which is
tion of a variety of other reactants [NADH]:ubiquinone oxidoreductase) converted by superoxide dismut-
with varying oxidative potentials, in- and complex III (ubiquinol:cy- ase to the more readily diffusible
cluding carbon- or nitrogen-centred tochrome c oxidoreductase) are oxidant H2O2 and is involved in cell
radicals (West and Marnett, 2006). sites of superoxide production, with killing functions. Inflammatory cells
A growing body of literature presents as much as 1–2% of the electron such as macrophages are also ca-
radicals as mediators of various cell flux shunted through one-electron pable of producing nitric oxide (•NO),
signalling processes (Ma, 2010). reduction of molecular oxygen (St- through an inducible form of nitric

Part 2 • Chapter 15. Oxidative stress and radical-induced signalling 153


oxide synthase (Hibbs et al., 1988). product 8-oxo-2′-deoxyguanosine is Oxygen radicals in cancer
• NO is also involved in cell killing but often used as a marker of oxidative
Hanahan and Weinberg (2011), in
can also react with superoxide at DNA damage, although other bas-
their landmark review “Hallmarks
diffusion-limited rates to form per- es are also susceptible to oxidation.
of cancer: the next generation”,
oxynitrite, a potent oxidant with a DNA bases can be modified by lip-
identified sustaining proliferative
longer half-life and diffusion distance id peroxidation reaction products
signalling, reprogramming of en-
than the hydroxyl radical (Beckman, (trans-4-hydroxy-2-nonenal, 4-hy- ergy metabolism, evading growth
1996). droperoxy-2-nonenal, and malondi- suppressors and immune destruc-
Exogenous agents are also impli- aldehyde) to form various pro-mu- tion, resisting cell death, enabling
cated in the generation of reactive tagenic exocyclic adducts (Bartsch replicative immortality, inducing
oxygen. Metals such as cadmium and Nair, 2006). angiogenesis, and activating inva-
and arsenic can participate in reac-
sion and metastasis as signal trans-
tions that generate oxygen radicals Defence mechanisms
duction pathways key to unravelling
(Liu et al., 2008; Kojima et al., 2009).
Cytosolic and mitochondrial forms the cancer phenotype. They also
Miller et al. (1990) presented a list of
of superoxide dismutase catalyse described how genomic instability
endogenous and exogenous agents
the reduction of superoxide to H2O2, and tumour-promoting inflammation
that are capable of reducing oxygen
and when coupled with catalase are principal drivers of these events.
to superoxide or that “autoxidize”,
within peroxisomes or with cytosolic Oxygen radicals clearly contribute to
probably through reactions catalysed
glutathione peroxidase, can further genomic instability, are produced by
by transition metals. Metabolism of
convert these reactive species to inflammation, and – along with oth-
many exogenous agents through cy-
water (Benzie, 2000). Sequestration er radical species – play key roles
tochrome P450-mediated reactions
of transition metals, principally iron in many of the processes identified
can also result in the release of ox-
and copper, in their oxidized forms above as necessary for conversion
ygen radicals (Hrycay and Bandiera,
through deposition in transport or of normal cells into cancer cells.
2015), as can exposure to ionizing
Oxidative damage is considered
radiation. In addition, several life- storage proteins, or as chelates
to be a major factor in the generation
style factors, such as obesity, tobac- that do not support redox reactions,
of mutations, which are estimated to
co smoking, and alcohol consump- also limits radical reactions (Hatcher
occur at a frequency of 10 000 per
tion, as well as chronic inflammatory et al., 2009).
cell per day in humans (Lu et al.,
conditions and viral infections are Dietary and endogenously pro-
2001). More than 100 different oxi-
thought to involve radical-induced duced antioxidants also contribute in
dative DNA lesions (Klaunig et al.,
injury (Mena et al., 2009). the defence against radical damage
2011) and at least 24 base modifica-
by serving as radical scavengers.
Oxidative damage tions (Wilson et al., 2003) have been
Theoretically any oxidizable sub-
identified, along with DNA–protein
The hydroxyl radical or equivalent strate can act as a radical scavenger;
cross-links (Cadet et al., 1997), all
transition metal–oxygen complexes ascorbic acid, tocopherols, uric acid,
of which potentially lead to genomic
(Bucher et al., 1983) are highly re- and sulfhydryl-containing amino ac- instability. RNA has also been shown
active entities, capable of abstract- ids provide considerable scavenging to be susceptible to radical attack (Li
ing electrons from lipids, proteins, capacity (Benzie, 2000). et al., 2006) but may be less chem-
or DNA (Miller et al., 1990), and the Interestingly, high concentrations ically susceptible than DNA (Thorp,
resulting target molecule radical of antioxidants in the presence of 2000). Oxidative damage to DNA
can then combine with molecular transition metals can actually drive can lead to point mutations, dele-
oxygen to participate in subsequent formation of oxygen radicals (Tien tions, insertions, or chromosomal
radical reactions, such as propaga- et al., 1982). The importance of a translocations, which may cause
tion of lipid peroxidation. Radical balance between pro- and antioxid- activation of oncogenes and inacti-
reactions with DNA result in single- ant capacities is also emphasized vation of tumour suppressor genes
and double-strand breaks (Toyokuni by an emerging understanding of and may lead to initiation of carcino-
and Sagripanti, 1996), cross-links, the role of radical species in cellular genesis (reviewed by Bartsch and
and modified bases. The oxidation signal transduction. Nair, 2006; Klaunig et al., 2011).

154
Clearly, high levels of oxygen rad- tions of glutathione peroxidases neonates of certain of these species
icals can be fatal to the cell through along with thyroid peroxidase, to survive such exposures with little
overt necrosis or the induction of and high levels of glutathione per- evidence of injury. The neonates of
apoptosis, but lower levels may also oxidases can interfere with the species resistant to pulmonary injury
contribute to the process of carcino- synthesis of thyroid hormones. are capable of increasing their levels
genesis through stimulation of cellu- Immunostaining for 8-oxo-2′-deoxy- of antioxidant enzymes in response
lar proliferation and alterations in oth- guanosine shows greater intensity in to hyperoxia, in contrast to the adults,
er cellular functions. There appear to thyroid follicular cells near the lumen which are incapable of mounting a
be a myriad of potential mechanisms where H2O2 is generated than in the similar response (Frank et al., 1978).
for these effects, involving induction spleen, liver, or lung, suggesting a Several metals, metalloids, and
of transcription factors for numer- high level of oxidative DNA damage fibres that contain metals or are fre-
ous signalling pathways, particular- in the thyroid (Maier et al., 2006). quently contaminated with metals
ly nuclear factor erythroid 2-related Environmental insults may aug- have been demonstrated to cause
factor 2 (Nrf2), mitogen-activated ment oxidative DNA damage in the cancer of the lung in humans and
protein kinase (MAPK)/AP1, nucle- thyroid. Thyroid uptake of iodine-131 experimental animals (IARC, 2012a).
ar factor kappa-light-chain-enhanc- released during the accident with the These include certain forms of ar-

CHAPTER 15
er of activated B cells (NF-κB), and

PART 2
Chernobyl Nuclear Power Plant in senic, asbestos, beryllium, cadmi-
hypoxia-inducible transcription fac- Ukraine is thought to be responsible um, chromium, and nickel. Although
tor 1 alpha (HIF-1α) (reviewed by for the high rate of papillary carci- carcinogenesis induced by metals
Klaunig et al., 2011). Protein kinase noma observed in exposed children appears to involve many mecha-
C, which is also susceptible to acti- (Bennett et al., 2006). Rats and mice nisms common to the process for
vation by cellular oxidants, is a fam- exposed to iodine-131 develop fol- other carcinogens, oxidative stress
ily of serine/threonine kinases that licular cell tumours (IARC, 2012b). has been implicated as an important
is central to the regulation of many Thiocyanate from cigarette smoke contributing factor for several met-
cellular functions, including prolifer- may, by inhibiting uptake of iodine, als (reviewed by Beyersmann and
ation, cell-cycle control, differentia- cause oxidative damage through Hartwig, 2008).
tion, cytoskeletal organization, cell iodine deficiency. Production of Certain metals may undergo di-
migration, and apoptosis (Wu, 2006). thyroid-stimulating hormone is in- rect redox cycling, as demonstrated
There is also evidence that the ac- creased during periods of iodine by the participation of nickel(II) in a
tivated oncogene v-Ha-Ras may act deficiency, and this hormone stimu- Fenton-like reaction with H2O2, or in
as a sustained proliferative stimulus lates H2O2 production in the thyroid. the metabolism of trivalent to pen-
in transformed fibroblasts through Levels of antioxidant enzymes have tavalent arsenic. Other metals, such
superoxide-mediated signalling been shown to be elevated by iodine as cadmium, may inhibit antioxidant
pathways (Irani et al., 1997).
deficiency (Krohn et al., 2007). enzymes or deplete antioxidants
The lung is also a vulnerable tar- (cadmium and arsenic bind with
Vulnerable tissue sites
and selected carcinogenic get for oxidative damage, by virtue sulfhydryl groups in glutathione) or
exposures of its exposure to air, which contains potentially delocalize iron or copper
21% oxygen, as opposed to the from protected storage sites (e.g. tri-
The synthesis of thyroid hormones much lower oxygen concentrations valent arsenic can release iron from
requires iodination of thyroglobu- in systemic tissues (Carreau et al., ferritin), making them available for
lin in a peroxidase-catalysed re- 2011). A key role of oxygen radicals participation in oxygen radical reac-
action that is dependent on H2O2. in the pulmonary toxicity resulting tions. Still other metals, such as ar-
Krohn et al. (2007) reviewed evi- from prolonged exposure to hyper- senic (reviewed by Shi et al., 2004),
dence suggesting that the thyroid is oxia is demonstrated by the dramatic may activate signalling pathways
particularly sensitive to formation of difference in the sensitivity of adult through increased production of oxy-
malignant nodules induced by oxida- animals of various species, which gen radicals, and potentially promote
tive stress. During active hormone succumb to oxygen toxicity after less radical reactions through a variety
synthesis, H2O2 levels are held in than a week of exposure to 100% of mechanisms. Finally, many stud-
check with increased concentra- oxygen, compared with the ability of ies have shown that asbestos or

Part 2 • Chapter 15. Oxidative stress and radical-induced signalling 155


asbestos-like materials (respirable Summary intimately involved in many well-rec-
elongated mineral particles or fibres) ognized mechanisms of carcino-
are capable of generating oxygen Many substances recognized as genesis, such as inflammation,
carcinogens in both humans and genomic instability, and cell prolifer-
radicals, primarily through reactions
experimental animals are capable ation. Because of the fundamental
catalysed by iron that is present in
of influencing redox processes and involvement of the oxygen radical in
coordination bonding within the min-
redox balance within target cells. many of these processes, substanc-
eral structure, is associated with the Oxygen radicals are capable of in- es that have been shown to promote
surface, or is chelated and released teracting with and influencing many cellular injury induced by oxygen
from the fibre by various intracellular cellular processes believed to be radicals should be considered as pu-
organic acids, such as citrate (Aust involved in the dysregulation of nor- tative human carcinogens until it has
et al., 2011). mal cellular physiology, thus sending been adequately demonstrated oth-
the cells down the pathway to can- erwise (Bucher and Portier, 2004).
cer. Oxygen radical reactions are

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Part 2 • Chapter 15. Oxidative stress and radical-induced signalling 157


part 2.
mechanisms of carcinogenesis

chapter 16.

Immunosuppression
Jerry M. Rice

CHAPTER 16
PART 2
Introduction from exposure to non-viral antigens, pharmaceutical drugs or by ionizing
such as after organ transplantation or ultraviolet radiation is dose-de-
Immunosuppression is a reduction (Ponce et al., 2014). pendent – the intensity and duration
in the capacity of the immune sys- Potentially neoplastic cells that of the effect increases with increasing
tem to respond effectively to foreign arise naturally, or that have been dose or continuing exposure – and is
antigens, including surface antigens transformed by carcinogens acting usually transient: immune function
on tumour cells. Immunosuppression by a mechanism such as genotox- generally recovers after cessation
can result from killing of immune ef- icity or by the various mechanisms of exposure. In contrast, infection
fector cells or from blockage of intra- of action associated with oncogenic with certain pathogens, such as hu-
cellular pathways essential for anti- viruses, may escape immune sur- man immunodeficiency virus type 1
gen recognition or of other elements veillance in immunosuppressed indi- (HIV-1) or malaria parasites, is per-
of the immune response. viduals. As a result, survival of these sistent, and the immune deficiency
Persistent immunosuppression cells and their replication to form that results is progressive unless the
presents a risk of cancer. Individuals tumours is greatly facilitated. infection is effectively treated.
who are latently infected with an on- Certain pharmaceutical drugs, Immunosuppression as a medi-
cogenic virus are at greatly increased ionizing and ultraviolet radiation, cal therapy is used to treat autoim-
risk for developing virus-related or infection with certain viruses mune diseases such as lupus ery-
cancers when they become immu- and parasites can cause immu- thematosus or rheumatoid arthritis.
nosuppressed (Grulich et al., 2007; nosuppression. After exposure to Immunosuppressive drugs, usually
Schulz, 2009; Wieland et al., 2014), X-rays or other types of ionizing in much higher dosage, are used to
and there is excess risk of B-cell radiation, immunosuppression is maintain the functional and anatom-
non-Hodgkin lymphoma (NHL) when most pronounced if the entire body, ical integrity of foreign tissues graft-
immunosuppression is accompanied rather than a limited area, is irra- ed onto another individual, such as
by continuing immune stimulation diated. Immunosuppression by a kidney or heart transplant. A graft

Part 2 • Chapter 16. Immunosuppression 159


from any individual except oneself and solar radiation, and most of the leukaemia by a genotoxic mech-
or an identical twin will provoke an chemical alkylating agents used in anism after its use in anticancer
immune reaction against the graft- anticancer chemotherapy. Radiation chemotherapy (IARC, 2012b).
ed tissues, the intensity of which and chemical alkylating agents are
varies with the degree of antigenic considered to cause cancer primar- Immunosuppressive
difference between graft and host. ily by inducing DNA damage, rather carcinogens
In the absence of adequate immu- than by immunosuppression.
Several Group 1 agents reviewed in
nosuppression, the host will destroy Cyclophosphamide is an anti-
Volume 100 of the IARC Monographs
the graft. Whole organs (e.g. kidney, neoplastic drug and is classified as
act entirely or largely by immuno-
heart, liver, or lung) can be trans- carcinogenic to humans (Group 1).
suppression, often in concert with
planted with maintenance of func- This drug has very marked immuno-
other Group 1 agents, especially
tion that may continue for a normal suppressive properties. In addition to
oncogenic infectious agents. The
lifetime when appropriate levels of its application in anticancer chemo-
Group 1 agents that act by immuno-
immunosuppression are maintained. therapy, cyclophosphamide is used
suppression are HIV-1 and the phar-
However, the risk of primary cancer clinically as an immunosuppressant
maceutical drugs ciclosporin and
in the transplant recipient increases to treat certain autoimmune diseas-
azathioprine.
with increasing intensity and dura- es, such as severe systemic lupus
tion of immunosuppression (Kinlen, erythematosus (Valeri et al., 1994). HIV-1 infection
1996; Yu et al., 2014). The drug, which must be metabo-
An uncommon but potentially lized to act as an alkylating agent, Infection with HIV-1 is the cause of
dangerous side effect of immuno- causes acute myeloid leukaemia and the acquired immune deficiency syn-
suppression to support organ trans- carcinoma of the urinary bladder in drome (AIDS). The severe immune
plants is that suppression of the patients in whom it has been used deficiency that is characteristic of
immune response can allow occult as an antineoplastic agent (IARC, AIDS results from a deficiency in
tumours or metastatic tumour cells 2012b). All available evidence, in- CD4-positive T lymphocytes and
within the transplanted tissues or cluding the organ sites of tumour a severe loss of memory B cells
development and the specific kinds (IARC, 2012a). In addition to severe
organs to survive, grow, and me-
of neoplasms induced, indicates infections, several cancers occur at
tastasize in the transplant recipient.
that cyclophosphamide exerts its high frequency in patients with AIDS.
Occult metastatic melanoma in the
carcinogenic activity via a genotoxic NHL, especially primary brain NHL,
donated organ is especially dan-
mechanism (McCarroll et al., 2008), as well as Kaposi sarcoma and cer-
gerous for the transplant recipient
rather than via immunosuppression. vical carcinoma are AIDS-defining
(Penn, 1996; Loren et al., 2003).
Chlorambucil, like cyclophos- conditions in severely immunosup-
Such transplanted cancers regress
phamide, is a bifunctional alkylating pressed patients.
when immunosuppressive therapy is
agent that also is an antineoplastic There is no evidence that HIV-
withdrawn (Wilson et al., 1968; Loren
drug and is classified as carcinogen- 1 causes NHL or other cancers
et al., 2003).
ic to humans (Group 1). It is used clin- through a direct effect. Unlike what is
Immunosuppression and ically as an immunosuppressant to known about other cancer-associat-
genotoxicity treat childhood nephrotic syndrome ed viruses, there is no evidence that
(Neuhaus et al., 1994), rheumatoid HIV-1 infection by itself leads to cell
The fact that a carcinogen has im- arthritis, and other autoimmune dis- transformation or immortalization.
munosuppressive properties does eases. It has been used to treat poly- The HIV-1 genome is not present
not necessarily mean that this is the cythaemia vera (a malignancy) and in cancer cells, in contrast to what
mechanism by which it causes human is used, often alone, as initial therapy is observed with infectious agents
cancer. DNA-damaging agents are for chronic lymphocytic leukaemia that are directly oncogenic (IARC,
generally also immunosuppressants, and in combination with other drugs 2012a).
especially at high levels of exposure; to treat other cancers. Chlorambucil, Kaposi sarcoma, which is caused
these include external ionizing radi- like other antineoplastic alkylating by Kaposi sarcoma herpesvirus
ation (X-rays and γ-rays), ultraviolet agents, can cause acute myeloid (KSHV), is the most common cancer

160
in patients with HIV-1 infection. Its virus and hepatitis C virus, and are al population (Kasiske et al., 2004).
occurrence is highly correlated with therefore at elevated risk for hepa- Non-melanoma skin cancers other
the severity of suppression of CD4- tocellular carcinoma (Grulich et al., than Kaposi sarcoma also occur at
positive T lymphocytes. The stan- 2007). high frequency in organ transplant
dardized incidence ratio for Kaposi recipients (Forchetti et al., 2014).
Therapeutic
sarcoma in a Swiss cohort was more There is a 65-fold increase in the
immunosuppression
than 500 in patients with a CD4- incidence of squamous cell carci-
positive lymphocyte count of less Therapeutic immunosuppression, noma and a 10-fold increase in the
than 100 cells/mm3 but approximate- generally by various combinations incidence of basal cell carcinoma in
ly 76 in patients with a CD4-positive of drugs such as ciclosporin and organ transplant recipients relative
lymphocyte count of greater than azathioprine, is administered to or- to the general population (Yu et al.,
500 cells/mm3 (Clifford et al., 2005; gan transplant recipients to maintain 2014).
IARC, 2012a). their transplanted organ or organs.
Ciclosporin
NHL, chiefly of the B-cell type, Recipients are at high risk for some
is the second most common ma- of the same cancers that occur in Ciclosporin, a cyclic lipophilic un-
lignancy in patients with AIDS. In a

CHAPTER 16
patients with AIDS. A comparison decapeptide, is a calcineurin inhibi-

PART 2
meta-analysis of six studies, NHL of AIDS-related and transplanta- tor and a potent immunosuppressant
had a standardized incidence ratio tion-associated tumours, from which that is virtually non-myelotoxic but
of 77 in patients with HIV-1 infection this text is excerpted, is presented in is markedly nephrotoxic. It is used
relative to the general population IARC (2012a). in organ and tissue transplanta-
(Grulich et al., 2007), and NHL is Although individuals with AIDS tion to prevent graft rejection after
frequently associated with Epstein– and those with iatrogenic immuno- bone marrow, kidney, liver, pan-
Barr virus (EBV) co-infection. The suppression after organ transplan- creas, heart, lung, and heart–lung
severe depletion of CD4-positive T tation have immunodeficiency in transplantation, and for prophylaxis
lymphocytes induced by HIV-1 leads common, the immunological abnor- and treatment of graft-versus-host
to dysregulated control of B lympho- malities appear to differ consider- disease (IARC, 2012b).
cytes and to the expression of co-in- ably between these two conditions. The immunosuppressive activity
fecting lymphotropic viruses (Engels, However, the spectra of neoplasms of ciclosporin is consistent with an
2007). that occur in patients with AIDS and increased risk of cancer as a result
The third most common malig- in organ transplant recipients large- of impaired immune surveillance,
nancy in HIV-1-positive individuals, ly overlap. An obvious similarity be- particularly for virus-related can-
and also an AIDS-defining condition, tween organ transplant recipients cers such as EBV-related NHL and
is cervical carcinoma associated and patients with AIDS is the in- HPV-related cervical cancer (IARC,
with human papillomavirus (HPV) creased incidence of B-cell NHL as- 2012b). Patients who receive ciclo-
infection. Anogenital intraepitheli- sociated with EBV infection. Specific sporin also are at increased risk for
al neoplasms and carcinomas are differences include more frequent squamous cell tumours of the skin,
also increased in frequency, and so high-grade lymphomas in patients which may be due in part to effects
are skin cancers associated with with AIDS and a more frequent EBV of the drug other than immunosup-
HPV infection (IARC, 2012a). In ad- association and polymorphic lesions pression. Ciclosporin has the ability
dition to NHL and Kaposi sarcoma, in organ transplant recipients. to generate reactive oxygen species,
infection with HIV-1 causes cancer The second important malignan- and this is probably relevant to its
of the cervix, anus, and conjunctiva, cy that is greatly increased in inci- carcinogenicity (IARC, 2012b).
as well as of the vulva, vagina, and dence in both individuals with HIV-1
Azathioprine
penis (IARC, 2012a). The primary infection and transplant recipients is
cause of these squamous epithelial Kaposi sarcoma (Zattra et al., 2014). Azathioprine, a substituted 6-mer-
neoplasms is co-infection with HPV. A study of renal transplant recipi- captopurine, is used in immunosup-
Finally, individuals with HIV-1 infec- ents reported a more than 20-fold pressive treatments to prevent re-
tion have a greatly increased inci- increase in the incidence of Kaposi jection of kidney allografts. The drug
dence of infection with hepatitis B sarcoma compared with the gener- is usually used in conjunction with

Part 2 • Chapter 16. Immunosuppression 161


other immunosuppressive therapy, disorders that generally have a viral immune protection associated with
including local radiation therapy and etiology; and (ii) because it caus- prenatal or chronic exposure to P. fal-
treatment with corticosteroids and es 6-thioguanine to accumulate in ciparum is common in children living
other cytotoxic agents. patients’ DNA, it also contributes to in malaria-endemic regions (Chelimo
One large prospective cohort cancer development by induction of et al., 2005; Scott et al., 2005).
study (Kinlen et al., 1979) on renal DNA damage (IARC, 2012b). Children in certain regions of
transplant recipients who received Often, milder therapy and less Africa become infected with EBV ear-
azathioprine examined the incidence potently immunosuppressive drugs ly in life, and nearly all have serocon-
of and mortality from different types (e.g. steroids such as prednisone) verted by age 3 years (Biggar et al.,
of cancer compared with the num- are used for autoimmune conditions 1978). EBV is activated when the
bers expected on the basis of the than for maintenance of organ trans- immune system is compromised (re-
incidence and mortality rates for plants. Prednisone and related im- viewed by Hopwood and Crawford,
the relevant country (Australia, New munosuppressive steroid drugs have 2000). Endemic Burkitt lymphoma
Zealand, and the United Kingdom). not been shown to be carcinogenic. (eBL), the most common paediatric
An almost 60-fold increase in the cancer in sub-Saharan Africa, is a
Malaria, a probable human high-grade B-cell lymphoma char-
risk of NHL was observed for all
carcinogen acterized by the consistent presence
countries combined (34 observed,
0.58 expected), as well as a 30-fold of EBV (Epstein et al., 1964, 1965;
In addition to the IARC Group 1
increase in the risk of squamous zur Hausen et al., 1970). eBL occurs
agents that are carcinogenic large-
cell skin cancer in patients from the only where malaria transmission
ly or entirely by an immunosup-
intensity is high, for example in the
United Kingdom (3 observed, 0.13 pressive mechanism, infection with
so-called lymphoma belt of sub-Sa-
expected) (IARC, 2012b). Plasmodium falciparum malaria in
haran Africa and in the high-trans-
Azathioprine is used more often holoendemic areas is probably car-
mission areas of Papua New Guinea.
in individuals with autoimmune con- cinogenic to humans (Group 2A), at
Furthermore, within areas and coun-
ditions than in transplant recipients. least in part by immunosuppression
tries where eBL occurs, it arises only
For example, azathioprine is given (Bouvard et al., 2012; IARC, 2014).
among those living in regions with
for management of the signs and Infection with P. falciparum malar- the highest transmission intensity,
symptoms of rheumatoid arthritis in ia has immunosuppressive effects, the so-called holoendemic or hyper-
adults (IARC, 2012b). Excesses in as reflected by impairment of mac- endemic areas. P. falciparum can
the risk of NHL (relative risk, 10.9) rophage function and antigen pres- disturb the immature immune system
and of squamous cell skin cancer entation (dendritic cell inhibition), in young children by expanding the
(relative risk, 5.0) were found in reduction in specific T-cell response, B-cell pool in which eBL arises, and
non-transplant patients receiving induction of regulatory T cells, and can reactivate latent EBV. Infection
azathioprine, although these ex- high plasma levels of pro-inflamma- with both EBV and P. falciparum is
cesses are smaller than those in tory cytokines (interleukin 6 [IL-6] required for the development of eBL
transplant recipients (Kinlen, 1985). and tumour necrosis factor alpha (Bouvard et al., 2012; IARC, 2012a).
Azathioprine is carcinogenic via [TNF-α]) and regulatory cytokines
two mechanisms: (i) as an immu- (IL-10 and tumour growth factor beta
nosuppressant, it is associated with [TGF-β]) (reviewed by Cunnington
post-transplant lymphoproliferative and Riley, 2010). Impaired humoral

162
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Part 2 • Chapter 16. Immunosuppression 163


part 2.
mechanisms of carcinogenesis

chapter 17.

Inflammation
Agnes B. Kane

CHAPTER 17
PART 2
Introduction has also been associated with devel- Intrinsic and extrinsic
opment of lung cancer and malignant pathways linking
In 1863, Virchow proposed that mesothelioma (Table 17.1). Cancer- inflammation and cancer
cancer originates at sites of inflam- related inflammation has been de-
matory responses (Virchov 1863a, Acute inflammation is a beneficial
fined as “the seventh hallmark of
b; Balkwill and Mantovani, 2001). host response against tissue injury
cancer” (Colotta et al., 2009), and
Based on extensive epidemiologi- and microbial invasion that usually
Hanahan and Weinberg (2011) add-
cal studies in the 20th century (de resolves after killing of the invading
ed “tumour-promoting inflammation”
Martel et al., 2012; Elinav et al., organisms, followed by tissue regen-
as an enabling characteristic of hu-
2013; Okada, 2014), the association eration or repair. Persistent infections
man tumours.
between persistent infections, in- or inadequate resolution of acute in-
This chapter focuses on the con-
flammation, and the development flammatory responses perpetuate
tribution of inflammation to multiple
of human cancers was conclusively tissue injury and lead to prolonged
steps during the evolution of cancer,
established for several types of car- chronic inflammation accompanied
including genetic and epigenetic al- by fibrosis or scarring (Kundu and
cinomas and lymphomas (Table 17.1
and Table 17.2). terations, disruption of tissue organi- Surh, 2012).
Worldwide, infections have been zation and homeostasis, and estab- Persistent infection and inflamma-
linked with 16.1% of human cancers, lishment of a local microenvironment tion disrupt local tissue homeostasis
accounting for 22.9% of cancers in that contributes to tumour growth, and dysregulate cell signalling path-
developing countries (de Martel et al., invasion, and metastasis (Fig. 17.1). ways, leading to recruitment and acti-
2012). Sterile inflammation associat- The key mechanistic pathways and vation of inflammatory cells (Balkwill,
ed with inhalation of crystalline silica mediators involved in inflamma- 2012). Persistent inflammatory con-
or asbestos fibres (Volume 100C of tion-associated carcinogenesis are ditions triggered by infectious or
the IARC Monographs; IARC, 2012a) summarized. environmental agents are defined

Part 2 • Chapter 17. Inflammation 165


Table 17.1. Human carcinomas associated with infections and chronic or Activated inflammatory cells, in-
persistent inflammation cluding neutrophils and macrophag-
es, produce reactive oxygen species
Agent Cancer sites and reactive nitrogen species. These
potent chemical mediators are im-
Hepatitis B and C viruses Liver portant in killing invading pathogens;
Human papillomaviruses Cervix, oral cavity, larynx, vulva, penis, anus
however, their prolonged release can
cause local tissue injury, damage to
Clonorchis sinensis Bile duct
proteins, lipids, and DNA (the DNA
Helicobacter pylori Stomach damage may be mutagenic if not re-
Opisthorchis viverrini Bile duct paired correctly), and upregulation
of signalling pathways, especially of
Schistosoma haematobium Bladder
nuclear factor kappa-light-chain-en-
Asbestos fibres Lung, mesothelium, larynx, ovary hancer of activated B cells (NF-κB),
Crystalline silica Lung that amplify pro-inflammatory re-
sponses (Lu et al., 2006; DiDonato
Erionite fibres Mesothelium
et al., 2012). Key pro-inflammatory
Source: Compiled from de Martel et al. (2012), IARC (2012a, b), and Trinchieri (2012). mediators that initiate and ampli-
fy tumour-associated inflammation
as extrinsic pathways leading to the kinase activates expression of the include prostaglandins, cytokines,
development of cancer (Mantovani chemokines IL-8, chemokine (C-C chemokines, and heat shock pro-
et al., 2010; Multhoff et al., 2011). motif) ligand 2 (CCL2), and CCL20 teins (Fig. 17.1).
Examples of human carcinomas and that attract inflammatory cells, and Prostaglandins are synthesized
lymphomas associated with exoge- induces expression of the chemo- from arachidonic acid and contribute
nous infections or environmental ex- kine receptor CXCR4 by malignant to tumour cell proliferation, surviv-
posures are listed in Table 17.1 and thyroid epithelial cells (Bozec et al., al, angiogenesis, and invasion. The
2010). In addition to CXCR4, which rate-limiting enzyme in prostaglan-
Table 17.2.
is overexpressed in many malignant
Intrinsic pathways driven by ac- din synthesis is prostaglandin-en-
cells as well as in cancer stem cells
tivation of proto-oncogenes and doperoxide synthase 2 (PGHS-2),
(Trautmann et al., 2014), multiple
signalling pathways in pre-neoplas- also known as cyclooxygenase 2
chemokine receptors are expressed
tic and neoplastic cells also recruit (COX-2), induced by NF-κB (Chai
in leukaemias and lymphomas as
host-derived inflammatory cells that et al., 2015). Key cytokines in tu-
well as in cancers of the lung, ova-
stimulate tumour growth and pro- mour-associated inflammation are
ry, stomach, bladder, and prostate,
gression (Grivennikov et al., 2010), IL-1 and tumour necrosis factor alpha
which may contribute to tumour inva-
changes that may be evident in the (TNF-α). IL-1 is produced by tumour,
sion and metastasis (Balkwill, 2012).
absence of any obvious exogenous endothelial, and inflammatory cells
The pathways that are activated
infectious exposure. For example, and activates intracellular signalling
by persistent or repeated episodes
the RAS oncogene is frequently acti- of acute inflammation or by chronic pathways, including those of NF-κB,
vated by point mutation in malignant inflammation and that contribute to activator protein 1 (AP-1), and p38
epithelial cells, which leads to acti- tumour growth, invasion, and me- mitogen-activated protein kinase
vation of intracellular signalling cas- tastasis are illustrated in Fig. 17.1. (MAPK) and c-Jun N-terminal kinase
cades and increased expression of Continued recruitment of host in- (JNK), that stimulate production of
the pro-inflammatory cytokines inter- flammatory cells to the evolving tu- mediators involved in inflammation,
leukin 1 alpha (IL-1α), IL-1β, and IL-6 mour microenvironment in response invasion, and angiogenesis (Multhoff
(Trinchieri, 2012). to persistent infections, ongoing tis- et al., 2011). TNF-α is a pro-inflam-
In papillary thyroid cancer, re- sue injury, or upregulated production matory mediator produced by ac-
arrangement of the RET/PTC (re- of cytokines and chemokines drives tivated macrophages and tumour
arranged during transfection/pa- cancer development (Grivennikov cells, leading to activation of NF-κB
pillary thyroid carcinoma) tyrosine et al., 2010; Chai et al., 2015). and signal transducer and activator

166
Table 17.2. Human lymphomas associated with persistent infections and by macrophages, resulting in acti-
inflammation vation of the NLRP3 inflammasome
(Biswas et al., 2011; Li et al., 2012).
Agent Malignancy Amphibole asbestos fibres, after
inhalation into the lungs, translocate
Borrelia burgdorferi Cutaneous lymphoma to the pleural cavity, where they may
Chlamydia psittaci Ocular adnexal lymphoma
also activate the inflammasome in
the mesothelial cell lining, leading to
Helicobacter pylori Gastric lymphoma
sustained inflammation that contrib-
Epstein–Barr virus Burkitt lymphoma utes to the development of diffuse
Large B-cell lymphoma malignant pleural mesothelioma
Hodgkin lymphoma
(Broaddus et al., 2011; Mossman
Human T-cell lymphotropic virus type 1 Adult T-cell lymphoma et al., 2013). Particles of carbon black
Source: Compiled from de Martel et al. (2012) and IARC (2012b). have also been shown to induce ac-
tivation of the inflammasome and
pyroptosis of alveolar macrophages

CHAPTER 17
of transcription 3 (STAT3) (Lin and sis is usually not associated with an

PART 2
Karin, 2007). IL-6 also promotes inflammatory response, extensive at high doses (Reisetter et al., 2011).
tumour angiogenesis and invasion apoptosis and necrosis do trigger in- After assembly of the inflam-
(Multhoff et al., 2011). Constitutive flammation by releasing damage-as- masome or release of cathepsin B
activation of NF-κB and STAT3 is sociated molecular factors, includ- from lysosomes, pro-caspase-1 is
found in several human tumours, and ing adenosine triphosphate (ATP), activated by proteolytic cleavage
these factors act synergistically to nucleic acids, heat shock proteins, to produce active caspase-1 that
sustain and enhance tumour-associ- S100 proteins, and the high-mobil- cleaves pro-IL-1β and pro-IL-18 to
ated inflammation (Chai et al., 2015). ity group box 1 protein (HMGB1) their active forms, which initiate and
The ultimate impact of intratu- (Pandey et al., 2015). amplify inflammation after release
moural inflammation on tumour Pyroptotic cell death – which is in the local environment (Fig. 17.2).
growth is to stimulate proliferation similar to apoptosis but is dependent Active caspase-1 can also cleave
and survival of tumour cells, resis- on a different set of initiator caspas- pro-caspase-7, triggering cell death
tance to apoptosis, evasion of host es – is associated with necrosis and by pyroptosis (Zitvogel et al., 2012).
immune attack, angiogenesis, and in- depends on activation of the inflam- Active IL-1β and IL-18 can suppress
masome, a multiprotein cytoplasmic immune surveillance in addition to
vasion, which are all major hallmarks
complex assembled in response to promoting growth of tumour stromal
of cancer (Hanahan and Weinberg,
generation of reactive oxygen spe- cells by paracrine signalling path-
2011). These authors identified tu-
cies, potassium ion efflux, or perme- ways (Fig. 17.2). Persistent inflam-
mour-promoting inflammation as an
abilization of lysosomes, resulting mation and pyroptosis may enhance
enabling characteristic of cancer, in
in cytoplasmic release of neutral damage to epithelial barriers and
addition to genomic instability, which
proteases, such as cathepsin B thus contribute to gastric cancer
is discussed later in this chapter.
(Kuraishy et al., 2011; Zitvogel et al., associated with Helicobacter pylori
How do exogenous infectious 2012). infection (Grivennikov et al., 2010).
agents and environmental Environmental exposures to, for Persistent infection with hepatitis B
exposures trigger tumour- example, crystalline silica and as- or C virus can also lead to chronic re-
associated inflammation? bestos fibres also trigger activation lease of pro-inflammatory cytokines
of the inflammasome, which results (Grivennikov et al., 2010; Read and
Exogenous agents responsible in lung epithelial injury, release of Douglas, 2014).
for inflammation cause persistent pro-inflammatory mediators, and Chronic exposure to irritants
tissue injury, aberrant tissue regen- lung fibrosis (Fig. 17.2; Dostert present in tobacco smoke, or to ac-
eration and healing, and a favour- et al., 2008, 2013; Luna-Gomes etaldehyde generated by ethanol
able environment for tumour growth et al., 2015). Crystalline silica and metabolism after consumption of al-
(Trinchieri, 2012). Although apopto- asbestos fibres are phagocytosed coholic beverages, induces epithelial

Part 2 • Chapter 17. Inflammation 167


Fig. 17.1. Pathways linking inflammation to cancer. AP-1, activator protein 1; IL-1, interleukin 1; IL-6, interleukin 6;
NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; STAT3, signal transducer and activator of
transcription 3; TNF-α, tumour necrosis factor alpha. Source: Compiled from Colotta et al. (2009), Grivennikov et
al. (2010), Multhoff et al. (2011), Kundu and Surh (2012), and Cooks et al. (2014).

Chronic or persistent inflammation

Reactive oxygen species,


Inflammatory mediators reactive nitrogen species

Prostaglandins
Cytokines (IL-1, TNF-α, IL-6)
Chemokines DNA and Dysregulated DNA
Heat shock proteins chromosomal repair and epigenetic
damage pathways

Dysregulated signalling pathways


Genomic instability
NF-κB
STAT3
AP-1 Activation of oncogenes;
inactivation of tumour
suppressor genes

Intratumoural inflammation;
tumour cell proliferation and survival
Resistance to apoptosis
Evasion of host immune attack
Angiogenesis
Invasion and metastasis

cell injury in the oral cavity and the Inflammation and genomic which generates excess reactive ni-
upper respiratory tract. These le- instability trogen species (Laskin et al., 2011).
sions synergize with potent chemical Constitutive activation of STAT3 in
carcinogens in tobacco smoke and Activation of the NF-κB and STAT3 cancers maintains NF-κB activation,
smokeless tobacco, thereby increas- signalling pathways and activation creating a self-sustaining amplifica-
ing the risk of cancers of the oral cav- of macrophages by TNF-α are the tion loop (Chai et al., 2015).
ity, larynx, and oesophagus (Smith key contributors to sustained gen- NF-κB activity is prolonged in the
et al., 2006). Persistent chronic in- eration of reactive oxygen species presence of mutant p53 protein and
flammation associated with hepatitis and reactive nitrogen species in upregulates activation-induced cyti-
B viral infection acts synergistically cancer-related inflammation (Colotta dine deaminase, an error-prone DNA
with aflatoxin, a genotoxic carcin- et al., 2009). Activated macrophag- repair enzyme (Cooks et al., 2014).
ogen, in the development of hepa- es release large quantities of re- Reactive oxygen species inactivate
tocellular carcinoma (Kew, 2003; active oxygen species, and NF-κB mismatch DNA repair enzymes by
Cougot et al., 2005). upregulates nitric oxide synthase, inducing oxidative damage; although

168
Fig. 17.2. Crystalline silica and asbestos fibres activate the NLRP3 inflammasome. IL-1β, interleukin 1 beta; IL-
18, interleukin 18. Source: Compiled from Dostert et al. (2008), Biswas et al. (2011), Zitvogel et al. (2012), and
Mossman et al. (2013).

Phagocytosis by macrophages and


generation of reactive oxygen species

Assembly of Release of cathepsin


inflammasome from lysosomes

Pro-caspase-1 Caspase-1

CHAPTER 17
Pro-IL-1β IL-1β

PART 2
Pro-IL-18 IL-18

Autocrine or
paracrine signalling

Amplification of
inflammation;
tumour cell proliferation;
immunosuppression

reactive oxygen species induce the or bromination have been proposed oping tumours. Tumour-associated
base excision repair pathway, over- to induce heritable epigenetic al- inflammation and genomic insta-
expression of these repair enzymes terations due to cytosine methyl- bility drive tumour growth and pro-
enhances microsatellite instability ation (Valinluck and Sowers, 2007). gression, which enable acquisition
(Colotta et al., 2009). Upregulation Chronic infection with Helicobacter of the six core hallmarks of cancer
of NF-κB and sustained production pylori is also associated with hyper- (Hanahan and Weinberg, 2011).
of cytokines cooperate with mutated methylation and silencing of p16 as
Inflammation, fibrosis, and
p53 protein to enhance genomic in- well as E-cadherin (Kundu and Surh,
cancer
stability (Fig. 17.1; Grivennikov et al., 2012).
2010; Cooks et al., 2014). In summary, chronic or persistent In 1986, Dvorak described cancers
Tumour-associated inflammation inflammation favours accumulation of as “wounds that do not heal”, based
is also linked with epigenetic alter- DNA lesions and chromosomal dam- on evidence of inflammatory cells in-
ations that lead to silencing of key age induced by persistent production filtrating into tumours, accompanied
tumour suppressor genes, such as of reactive oxygen species and reac- by angiogenesis and fibrosis, similar
INK4a or p16 in lung cancers and tive nitrogen species, impaired DNA to wound healing (Dvorak, 1986; see
malignant mesothelioma (Blanco repair pathways, and heritable epi- also Dvorak, 2015). Persistent infec-
et al., 2007; Christensen et al., 2009, genetic alterations, leading to activa- tions accompanied by parenchymal
2010; Nelson et al., 2012). Oxidative tion of oncogenes and inactivation of cell injury and chronic inflamma-
damage to cytosine and chlorination tumour suppressor genes in devel- tion, and fibrosis associated with

Part 2 • Chapter 17. Inflammation 169


inhalation of crystalline silica or as- (Hanahan and Coussens, 2012). Resident tissue stem cells in
bestos fibres promote tumorigenesis They are also a source of paracrine adults occupy a specialized niche to
(Kuraishy et al., 2011). For example, growth factors, including insulin-like maintain their polarity, self-renew-
persistent infection with hepatitis B growth factor 1 (IGF-1) and IGF-2, al, and differentiation by anchoring
or C virus can produce fibrotic scar- that promote survival of cancer cells, to receptors in the basement mem-
ring or cirrhosis in the liver, accom- as well as a source of angiogenic brane or in the extracellular matrix.
panied by nodules of regenerating factors that promote tumour angio- Altered extracellular matrix organi-
hepatocytes, leading to development genesis (Lu et al., 2012). zation and stiffness may disrupt this
of hepatocellular carcinoma (El- Matrix metalloproteinases contact, allowing local expansion of
Serag and Rudolph, 2007). Nodular (MMPs), which accelerate the degra- the stem cell pool (Lu et al., 2012).
fibrotic scarring of the lungs is char- dation or remodelling of the tumour These locally proliferating stem cells
acteristic of silicosis (Leung et al., stroma and the release of immobi- may give rise to cancer stem cells or
2012), and inhalation of asbestos lized growth factors and cytokines, tumour-initiating cells. Cancer stem
fibres can cause diffuse fibrosis or are frequently overexpressed in tu- cells express cell surface markers,
asbestosis (Mossman et al., 2011). mours. The gelatinases MMP-2 and including CD24, CD44, and CD133,
Pulmonary fibrosis has been associ- MMP-9 are overexpressed by malig- which are thought to enhance growth
ated with development of lung can- nant tumour cells or stromal cells in and invasion as well as resistance
cer (IARC, 2002; Laskin et al., 2011). a wide range of carcinomas, as well to apoptosis (Keysar and Jimeno,
Several mechanistic links between fi- as by leukaemias and lymphomas. 2010).
brosis and tumour development and Activation of MMPs in the tumour
progression have been proposed microenvironment promotes tumour Summary and conclusions
(Hanahan and Coussens, 2012). cell migration and invasion, release
Tissue fibrosis changes the nor- of sequestered growth factors, and Inflammation has been hypothe-
mal architecture and compliance of activation of latent forms of cytokines sized to contribute to multiple stages
the extracellular matrix, resulting in including IL-1β (Bauvois, 2012). in cancer development (Trinchieri,
dense cross-linked connective tissue Tumour-associated macrophages 2012). Sustained or persistent in-
and increased stiffness (Liu et al., and lymphocytes are major sources flammation releases mediators that
2010). Excess extracellular matrix of cytokines in the tumour environ- can damage DNA and hamper DNA
components, including heparan sul- ment (Laskin et al., 2011; Balkwill, repair, leading to cell transforma-
fate proteoglycans that bind to the 2012). The phenotype of tumour-as- tion; it establishes a local microen-
CD44 receptor, are produced during sociated macrophages is shifted to a vironment that allows the tumour to
cancer development and contribute pro-fibrotic, pro-angiogenic pheno- grow and metastasize, and to avoid
to enhanced growth factor signalling type, M2, characterized by produc- immune destruction, thus prevent-
and cell proliferation (Nasser, 2008). tion of arginase, IL-10, and TGF-β ing an effective immune response
Disrupted assembly and disorgani- (Sica and Mantovani, 2012) as well against the tumour (Mantovani et al.,
zation of the extracellular matrix can as IL-23 (Grivennikov et al., 2010). 2010).
alter polarity and differentiation of The T-lymphocyte subset TH17 pro- Persistent or chronic inflamma-
pre-neoplastic epithelial cells, facil- duces IL-17, which upregulates IL-23 tion, frequently in association with
itating their proliferation, migration, expression in the tumour microenvi- oxidative stress, is considered to be
and invasion through the basement ronment. IL-23 is a key cytokine in tu- an established or likely mechanistic
membrane (Lu et al., 2012). mour growth and invasion; it causes event contributing to human cancers
Resident fibroblasts in connective upregulation of MMP-9 expression associated with exposure to crystal-
tissues and mesenchymal stem cells, and increases angiogenesis and line silica and asbestos fibres (IARC
in local stem cell niches or recruited fibrosis (Langowski et al., 2006). IL- 2002; Shukla et al., 2003; Valavanidis
from the bone marrow, secrete epi- 17 also promotes liver fibrosis and et al., 2013) as well as diesel engine
thelial and fibroblast growth factors activates hepatic stellate cells to emissions and indoor coal com-
as well as pro-inflammatory cy- produce collagen in murine models bustion (Sood, 2012; Carlsten and
tokines and chemokines that contrib- of toxic liver cell injury (Meng et al., Georas, 2014; Vermeulen et al.,
ute to the tumour microenvironment 2012). 2014). These inhalation exposures

170
are associated with lung cancer in contaminant aflatoxin in the devel- occupational, personal, and infec-
humans and experimental animals, opment of hepatocellular carcinoma tious exposures resulting in per-
and asbestos fibres may also induce (Kew, 2003; IARC, 2012b; Simet sistent or chronic inflammation are
cancer at distant sites, including et al., 2012). In combination with po- preventable, and modifying these
the mesothelial lining and the ovary tent carcinogens in tobacco smoke circumstances of exposure would
(IARC, 2012a). and smokeless tobacco (e.g. ar- diminish the worldwide burden of
Persistent bacterial, viral, and ylamines, polycyclic aromatic hydro- cancer.
parasitic infections (IARC, 2012b) carbons, and nitrosamines), ethanol
Acknowledgement
are also associated with a variety of consumption may increase epithelial
human carcinomas as well as leu- permeability and injury, which to- The author’s research is support-
kaemias and lymphomas (Table 17.1 gether with oxidative stress enhance ed by a National Institute of Envi­
and Table 17.2). Persistent or repeat- the development of cancers of the ronmental Health Sciences (NIEHS)
ed episodes of tissue injury and in- oral cavity, larynx, and oesophagus Superfund Research Program grant,
flammation may also synergize with (Bor and Capanoglu, 2009; IARC, P42 ES013660.
viral oncoproteins and the fungal 2012c). Exogenous environmental,

CHAPTER 17
PART 2

Part 2 • Chapter 17. Inflammation 171


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Part 2 • Chapter 17. Inflammation 173


part 2.
mechanisms of carcinogenesis

chapter 18.

Ionizing radiation
Mark A. Hill and Robert L. Ullrich

CHAPTER 18
PART 2
Introduction The rationale for this was that all and international reviews of the liter-
types of ionizing radiation transfer ature on radiation, as well as radia-
The carcinogenic risk associated their energy to biological material in tion risk estimates. These include the
with exposure to ionizing radiation clusters of ionization and excitation publications of the United Nations
has been evaluated previously in the events, primarily through a mecha- Scientific Committee on the Effects
IARC Monographs: radon in Volume nism mediated by free electrons. In of Atomic Radiation (UNSCEAR,
43 (IARC, 1988), X-rays, γ-rays, and addition, DNA damage is a common 2000, 2008, 2010) and the reports
neutrons in Volume 75 (IARC, 2000), biological outcome of exposure to all from the United States National
and some internally deposited radio- ionizing radiation; energy deposition Research Council (NRC, 1999,
nuclides in Volume 78 (IARC, 2001). results in a wide variety of molecular 2006), the United States National
An updated review on all carcinogen- damage, such as base damage and Council on Radiation Protection and
ic types of radiation, also including single- and double-strand breaks, Measurements (NCRP, 1993, 1999,
solar and ultraviolet radiation, was some of which may be clustered to 2001, 2005), and the International
published as Volume 100D (IARC, form complex lesions. Subsequent Commission on Radiologic al
2012). processing of these lesions may lead Protection (ICRP, 2003, 2007;
For certain types of ionizing radi- to chromosomal aberrations and mu- Valentin, 2005).
ation, the evidence of carcinogeni- tations. The generality of induction of Two major issues faced when
city in humans is clear, but in other and response to radiation damage studying radiation carcinogenesis is
cases the data are few or non-exist- is discussed for all types of ionizing that radiation-induced cancers are
ent. However, the overall conclusion radiation in greater depth later in this indistinguishable from those that oc-
reached in Volume 100D of the IARC chapter. cur naturally, and that risk estimates
Monographs was that all types of ion- In addition to the above-mentioned rely on epidemiological data for which
izing radiation should be considered reviews in the IARC Monographs, statistical significance is reached
as carcinogenic to humans (Group 1). there have been many major national only at high doses. The existing data

Part 2 • Chapter 18. Ionizing radiation 175


are not powerful enough to enable directly ionize the medium they are per unit track length. For example,
comment on the shape of the dose– traversing, producing chemical and cobalt-60 γ-rays have an LET of
response curve and the associated biological damage. about 0.25  keV/μm (where 1  eV =
risks at doses associated with typi- The human body can be irradiat- 1.602 × 10−19 J). The ionization and
cal human exposures. Many of the ed either from external sources or excitation events are much closer
in vitro and in vivo studies investi- through internal exposure as a re- together for low-energy charged par-
sult of ingestion, inhalation, dermal ticles, which are considered to be
gating the mechanisms underlying
absorption, or injection of radionu- high-LET radiation. For example, an
cancer risk from exposure to ionizing
clides. The effects of radiation are α-particle with an energy of 2  MeV
radiation have concentrated on low-
directly related to the dose received has an LET of about 180 keV/μm.
dose exposures, typically of 0.1 Gy
by individual cells or organs, and All types of ionizing radiation in-
(= 0.1 J/kg) and below. by the radiation quality. Therefore, duce a wide range of damage and ef-
these effects can vary significant- fects, including DNA damage, chro-
The nature of ionizing
ly, depending on the resulting dose mosomal aberrations, mutations, cell
radiation
distribution or distribution of radionu- transformation, and cell killing (NRC,
clides throughout the body. The dose 1999, 2006; UNSCEAR, 2000;
Ionizing radiation is a term used for
distribution may vary from being es- ICRP, 2003, 2007). The efficiency
any radiation that is capable of ion-
sentially uniform after whole-body in causing damage and subsequent
izing (i.e. removing electrons from)
exposure to being highly heteroge- biological effects is related not only
atoms or molecules of the medium
neous in the case of non-uniform to the amount of energy transferred
being traversed. Ionizing radia- distribution of internal radionuclides per unit mass (the absorbed dose,
tions are usually classified as either that emit short-range α-particles or expressed in units of gray, where
electro­magnetic or particulate. β-particles. Medium- to high-ener- 1 Gy = 1 J/kg) and the rate of energy
X-rays and γ-rays are both gy X-rays, γ-rays, and neutrons are transfer (the dose rate) but also to the
electro­magnetic radiations. They do typically highly penetrating and will microdistribution of energy, which is
not differ in nature, but their designa- traverse the body, whereas α-par- determined by the type of radiation
tion reflects their origin; X-rays are ticles and β-particles typically have and the associated track structure.
produced by extranuclear processes a short range (for α-particles, less The relative biological effective-
and γ-rays by intranuclear process- than 100  μm, and for β-particles, ness is defined as the inverse ratio of
es. These types of radiation are of- from less than 1  μm to several mil- the dose required to produce a given
limetres). In general, the penetra- biological effect to the dose required
ten classified as indirectly ionizing,
tion range of charged particles can by a reference radiation to produce
because the chemical and biological
vary significantly depending on their the same effect. The relative biolog-
damage is dominated by the charged
energy and the type of particle.
particles (mainly electrons) produced ical effectiveness typically increases
as a result of interactions within the with the LET value of the radiation,
Genotoxicity and the
medium. Neutrons are also clas- importance of radiation track and it reaches a peak at about 100–
structure 200 keV/μm for a range of biological
sified as indirectly ionizing. They
end-points. Whereas the absorbed
deposit energy and cause damage
Ionizing radiation interacts within dose unadjusted for attenuation by
through recoil protons, α-particles,
cells and tissues by depositing en- the body is expressed in units of
and nuclear fragments that result
ergy in highly structured tracks of gray (Gy), the weighted organ dose
from neutron interactions.
ionization and excitation events that (the equivalent or effective dose) is
Particulate radiations include expressed in sieverts (Sv) or millisie-
are stochastic in nature. On average,
electrons, positrons, protons, neu- these events are relatively sparsely verts (mSv), which are also the units
trons, α-particles, and other ions. distributed for high-energy X-rays in which radiation exposure limits are
With the exception of neutrons, all of and γ-rays, which deposit energy given.
these particles are charged and are via electrons with relatively low lin- For many biological effects, nu-
classified as directly ionizing (if they ear energy transfer (LET), where clear DNA is a critical target of ion-
have sufficient energy) because they LET corresponds to the energy loss izing radiation (UNSCEAR, 1993).

176
Ionizing radiation can cause DNA (Nikjoo and Goodhead, 1991). These tion, which ultimately leads to stalled
damage either by direct ionization more complex forms of damage are replication forks that may give rise to
of the constituent atoms in the DNA essentially unique to ionizing radia- DSBs or mutations. Therefore, non-
or indirectly by reactions with free tion and are not seen spontaneously DSB clusters are potentially highly
radicals produced by interactions or with other DNA-damaging agents. mutagenic and are likely to play a
with water molecules (most notably The number of DSBs induced in more important role at low doses
the hydroxyl radical, which can in- DNA is approximately 20–40 per
of low-LET radiation; because non-
duce DNA strand breakage or base cell per gray for low-LET X-rays and
DSB damage is produced at a higher
damage), or by a combination of di- γ-rays, and a similar number is ob-
frequency than DSBs at these lower
rect and indirect effects. In the cell, served for α-particles in standard
doses, more cells will contain non-
hydroxyl radicals will typically only assays. However, the percentage
diffuse a few nanometres (< 6 nm), of complex DSBs (with extra strand DSB clustered damage compared
thus preserving the spatial structure breaks and/or associated base dam- with DSBs (reviewed by Eccles et al.,
of the radiation tracks. age within 10 base pairs) is about 2011).
Ionizing radiation can thus induce 30–50% for electrons (similar to the DNA is wrapped around histone
a range of different types of molecular percentage produced by X-rays and proteins to form nucleosomes, which

CHAPTER 18
PART 2
damage in DNA, such as base dam- γ-rays) based on Monte Carlo calcu- are organized into 30 nm chromatin
age (including apurinic/apyrimidinic lations, and this percentage increas- fibres that are typically arranged in
sites), strand breaks, DNA–protein es with increasing ionization den- loops. As a result of the sequence of
cross-links, and combinations of sity (LET) of the radiation, to about ionization events along individual ra-
these within a few base pairs of each 90% for 0.3 MeV protons and about diation tracks, especially in the case
other. Examples are double-strand 96% for high-LET 2  MeV α-parti- of densely ionizing high-LET parti-
breaks (DSBs) and non-DSB clus- cles (Nikjoo et al., 1991; Goodhead,
cles such as α-particles, these tracks
ters (two or more base damages 2006). In addition to this increase in
can lead to multiple correlated DSBs
and/or strand breaks within about the frequency of complex DSBs with
over short sections of DNA arranged
10 base pairs, but not resulting in a increasing LET, there is also an in-
in these structures. Conventional
DSB). The pattern and frequency of crease in the overall complexity of
these lesions are determined by the the damage spectrum produced. DSB assays (e.g. pulsed-field gel
clustering of ionization and excita- Clustering of damage is not con- electrophoresis and γH2AX assays)
tion events on the nanometre scale, fined to DNA but can occur in all are not able to resolve these addi-
which ultimately produces clustering biomolecules. tional DSBs and therefore typically
of damage over the dimensions of Complex non-DSB damage has underestimate the absolute yields
the DNA helix and larger. been shown to be a significant com- (Friedland et al., 2008). However,
Theoretical analyses show that ponent of the lesions induced by experimental and theoretical data
clustered DNA damage that is more radiation, occurring 4–8 times as have demonstrated the existence of
complex than a single-strand break frequently as direct DSB formation. these short fragments for these par-
can occur at biologically relevant Whereas isolated lesions (e.g. base ticles, showing a significant deviation
frequencies with all types of ionizing damage or single-strand breaks) are from a random distribution (Rydberg
radiation (Goodhead 1987, 1994; repaired quickly and generally with
et al., 1998; Friedland et al., 2008).
Brenner and Ward, 1992). Such clus- high fidelity, for non-DSB clusters
Whereas viable radiation-induced
tered damage in DNA is produced the rate of repair is typically impaired
mutations are rarely associated with
mainly within a single track, with a by the presence of additional lesions
visible chromosomal exchanges
probability that increases with in- within the cluster. The delay and the
creasing ionization density (LET). ultimate consequence depend on observed by use of fluorescence in
Calculations show that a dose of the types of lesion and their rela- situ hybridization (FISH), molecular
greater than 10 000 Gy is required tive positions. The longer lifetime of analysis of these sites shows that
for a second track to have a reason- these clusters also results in an in- high-LET particles can induce gene
able chance of contributing to the creased probability that the damage mutations of greater complexity than
local complexity of DNA damage will be present during DNA replica- simple deletions or point mutations,

Part 2 • Chapter 18. Ionizing radiation 177


consistent with the correlation of sion are critical in determining cellu- have been implicated, and these
damage along the radiation track lar function and response. Ionizing typically result in the modulation of
(Singleton et al., 2002). radiation has been shown to modu- reactive oxygen species and reac-
The pattern of energy deposition late protein phosphorylation (Yang tive nitrogen species as a result of
is also important on the cellular or et al., 2006) and gene expression signalling through molecules such
nuclear scale (over distances in the in a dose- and dose rate-dependent as nitric oxide, peroxidase, and the
micrometre range). When an α-parti- manner (Ding et al., 2005; Fachin cytokine transforming growth factor
cle traverses a cell, the dose distribu- et al., 2009). Epigenetic changes beta (TGF-β) and other inflammatory
tion of the energy deposited is highly can also result in modifications in markers (Burdak-Rothkamm et al.,
heterogeneous across the cell, with gene expression, and ionizing ra- 2007; Han et al., 2007; Portess et al.,
a greater probability of correlated diation produces DNA methylation 2007; Coates et al., 2008). Radiation
damage and DSBs within a single (Kovalchuk et al., 2004), histone is capable of perturbing intercellular
chromosome or adjacent chromo- methylation (Pogribny et al., 2005), signalling down to very low doses
somes. Studies with multiplex FISH and chromatin modification (Kim (on the order of 2  mGy for γ-rays
(mFISH) have shown that commonly et al., 2009; Luijsterburg et al., 2009; and 0.3  mGy for α-particles), which
four and up to a maximum of eight Nagarajan et al., 2009; Pandita and are directly relevant to typical human
different chromosomes may be in- Richardson, 2009), along with mod- exposures (Portess et al., 2007).
volved in rearrangements after the ulation of microRNA expression Reactive oxygen species are ex-
nuclear traversal of a human periph- (Templin et al., 2011). pected to be important in initiating
eral blood lymphocyte by an α-parti- and maintaining the inflammatory
cle (Anderson et al., 2002, 2006); a Intercellular communication
process (Barcellos-Hoff et al., 2005;
similar response was seen in human
and the bystander effect
Mantovani et al., 2008). In addition,
CD34-positive haematopoietic stem Within tissues of multicellular organ- radiation can lead to a modification in
cells (Anderson et al., 2007). This is isms, cells do not act in isolation; in- the immune response; at high whole-
in contrast to the production of mainly tercellular signalling is vital for main- body doses, this results in immuno-
simple rearrangements between two taining the multicellular organization suppression, whereas at low doses
chromosomes observed for low dos- of the tissue and for normal func- and dose rates, this can lead to ei-
es of low-LET X-rays. Complex rear- ther suppression or stimulation of
tioning of the constituent cells (Park
rangements have been observed in the immune response (UNSCEAR,
et al., 2003). These cellular inter­
radiation workers with a large body 2008).
actions and the microenvironment
burden of α-particle-emitting pluto- There is increasing evidence to
are also important in influencing the
nium (Anderson et al., 2005). Stable suggest that radiation-induced per-
growth and development of cancer
intrachromosomal rearrangements turbation of intercellular signalling
cells.
were also found in lymphocytes of and of the microenvironment may
Radiation can initiate stress-in-
former nuclear weapons workers play a role in modulating cancer risk.
ducible signals, which can perturb
who were exposed to plutonium However, the relative importance of
this signalling and affect not only
(Hande et al., 2003), although not these effects to cancer induction af-
irradiated cells but also non-irra-
consistently for all cases of in vivo ter human exposure is unclear, and
diated cells. Many studies have
high-LET exposures (reviewed by it is not generally known whether the
shown a wide range of responses
Hada et al., 2011). dominant consequences of these
in non-irradiated “bystander” cells,
including induction of DNA damage, effects are beneficial or detrimental.
Other potential mechanisms
for modifying cancer risk chromosomal aberrations, delayed
Radiation-induced genomic
from radiation exposure genomic instability, mutations, onco-
instability
genic transformation, and cell killing
Ionizing radiation also produces a (Morgan, 2003a, b). In addition to being capable of pro-
whole range of effects with poten- Signalling has been demonstrat- ducing mutations directly in the irra-
tial implications for carcinogenesis ed to occur via intercellular gap diated cell, ionizing radiation can also
(UNSCEAR, 2012). For example, the junctions and media-borne fac- lead to genomic instability, resulting
patterns of gene and protein expres- tors. Several signalling pathways in the cell and its progeny having a

178
reduced ability to replicate the geno- (Kadhim et al., 2004; Barber et al., ination route and rate, and uptake
type faithfully and therefore showing 2009; Filkowski et al., 2010; Rugo and retention in organs. In some cas-
a permanently increased rate of ac- et al., 2011). Genomic instability has es, for example for radioactive heavy
quisition of alterations in the genome also been observed in non-irradiated metals, the health effects and carci-
(Kadhim et al., 1992, 1994; Little, cells that were in the neighbourhood nogenic potential may also be relat-
2000; Morgan, 2003a, b; Barcellos- of irradiated cells, demonstrating the ed to, and potentially dominated by,
Hoff et al., 2005). This may lead to importance of intercellular signalling the chemical properties rather than
an increased probability that the cell in initiating this instability response
the radiation emitted.
and its progeny will undergo the var- (Lorimore et al., 1998). Although
In some cases, a radionuclide
ious genetic and epigenetic changes genomic instability is a plausible
may spread throughout the whole
necessary in multistage carcino- mechanism for cancer induction, its
body; in other cases, it will concen-
genesis. It is thus possible that the precise role, if any, remains to be
trate in specific organs or locations
instability phenotype plays a major proven.
within the body. If the emitted ra-
role in radiation-induced cancer, es-
pecially because genomic instability
The importance of dose diation has a short range (e.g. for
distribution with respect to α-particles and β-particles), this can
is a well-recognized feature in many

CHAPTER 18
tumour sites lead to significant heterogeneity in

PART 2
tumours (Bielas et al., 2006).
Radiation-induced genomic insta- the resulting dose distribution, with
The passage of ionizing radiation
bility typically becomes manifest sev- certain organs receiving a significant
through the body results in the depo­
eral cell generations after irradiation dose while for others the radiation
sition of energy within the irradiat-
and can be detected via a range of dose is minimal. Biokinetic models
ed tissue volume. External irradia-
end-points, including chromosomal (ICRP, 1989, 1993, 1994, 1995a, b,
tion with photons is typically highly
and chromatid aberrations, micro- c, 2001) are used to estimate the
penetrating and will often result in all
nuclei, changes in ploidy, gene mu- cells and tissues in the radiation field spatial and temporal uptake of radio-
tations and amplifications, and mini- being irradiated. In contrast, emis- nuclides as well as their subsequent
and microsatellite instabilities. The sion from internalized radionuclides distribution and ultimate excretion.
frequency of genomic instability was typically occurs from specified lo- Dosimetry models (Eckerman, 1994)
observed to be too high to be ex- cations occupied by the emitting nu- are then used to calculate the result-
plained by the induction of a mutator clide source. This will often lead to a ing dose distribution over the body
genotype. Several mechanisms have non-uniform dose distribution in the and organs, based on the physical
been proposed, including dysfunc- body, especially if the emitted radia- characteristics of the radionuclides.
tional telomeres (Goytisolo et al., tion has only a short range (e.g. for The ability of internal radionu-
2000; McIlrath et al., 2001; Williams α-particles and β-particles). clides to produce a biological re-
et al., 2009) and inflammatory (free The biological effects of deposit- sponse and ultimately cancer in
radical) responses (Barcellos-Hoff ed radionuclides in the body depend various organs is related to the bio-
et al., 2005; Natarajan et al., 2007; on the amount and activity of the distribution of these emitters within
Coates et al., 2008; Lorimore et al., radionuclide deposited, the type of
the body (which will depend on the
2008), along with DNA damage and radiation emitted, the physical half-
chemical and physical properties of
response, for example long-term life of the isotope, the mode of entry,
the particles and the route of entry).
response to directly induced DNA the organs and tissues in which the
Examples are iodine-131, which con-
damage and reduced ability to han- radionuclide is retained, the duration
dle subsequent damage or cell di- centrates largely in the thyroid, and
of retention, and the rate of excretion
vision (Snyder and Morgan, 2005; from the body. The chemical char- strontium-90 and plutonium-239,
Maxwell et al., 2008; Toyokuni et al., acteristics of the radionuclide (or the which are deposited mainly in the
2009). compound in which it is incorporat- bone. The same radionuclide may
Epigenetic modification has been ed) along with its physical properties result in a different range of tumours
implicated as playing an important (such as size and shape) determine if it is delivered in such a way as to
role in the promotion and mainte- its behaviour, including absorption produce a different biodistribution
nance of transmissible instability and transport within the body, elim- pattern. In addition, there may be

Part 2 • Chapter 18. Ionizing radiation 179


confounding factors, such as chem- cles as a result of naturally occurring it energy via lower-energy charged
ical toxicity, that may contribute to or radon gas. With typical residential particles such as protons, deuterons,
even dominate the cancer response. levels of radon gas, the cell nuclei α-particles, and heavy-ion recoils, ul-
in the bronchial epithelium of the in- timately leading to energy deposition
Human exposures to ionizing habitants are estimated to receive on via secondary electrons.
radiation typically occur at average between 0.15 and 0.6 α-par- Therefore, energy deposition by
low dose and low dose rate ticle traversals per year (NRC, 1999). way of electrons is common to all ion-
However, for those cell nuclei that izing radiation. Indeed, isolated track
The effects of radiation are most no-
are occasionally traversed, the dose ends of low-energy electrons (pro-
table at the high doses (above a few
to the traversed nucleus is significant duced by all ionizing radiations) have
gray) that are usually associated with
(on the order of 0.1–0.5 Gy). been shown not only to be capable of
significant radiation accidents and
For the high doses associated affecting a wide range of genotoxic
radiotherapy treatments, and that
with radiotherapy or significant acci- end-points but to do so with a high
are observed in atomic bomb sur-
dental exposures, it is expected that efficiency per unit dose (Goodhead
vivors. These effects include erythe-
classical direct effects of radiation and Nikjoo, 1990; Hill et al., 2001;
ma, oedema, ulceration, necrosis, fi-
are likely to dominate the response, Hill, 2004; HPA, 2007). Because of
brosis, telangiectasia, inflammation,
as a result of radiation-induced their increased local ionization den-
immunosuppression (through bone
DNA damage. However, at the very sity, these track ends of low-energy
marrow depletion), and pneumonitis
low doses associated with typical (0.1–5.0  keV) electrons have been
(HPA, 2007; Stewart et al., 2012).
human exposures, where only a proposed as the biologically critical
Although there is clear evidence from
small fraction of cells have a DNA component of low-LET radiation,
epidemiological data for significant
DSB, it is possible that other mech- rather than the isolated ionization
cancer risks associated with high-
anisms for cancer induction or mod- and excitation events along the path
dose exposures, the existing data for
ulation of cancer incidence (such as of fast electrons (Goodhead and
the low-dose range are limited, such
radiation-induced genomic instability Nikjoo, 1990; Botchway et al., 1997).
that below approximately 0.1 Gy –
or effects associated with perturba- In addition, α-particles emitted by
doses associated with typical human
tion of intercellular signalling) may radionuclides, irrespective of their
exposures – the data are not power-
play a more important role. source, produce the same pattern of
ful enough to enable comment on the
shape of the dose–response curve secondary ionizations and the same
Generality of response after
and the associated risks. pattern of localized damage to bio-
exposure to different types of
For an average annual environ- logical molecules, including DNA,
ionizing radiation
mental background exposure of ap- and ultimately the same biological ef-
proximately 0.001 Gy for low-LET ra- All types of ionizing radiation ulti- fects. Therefore, due to the commu-
diation, individual cells may receive mately lead to clusters of ioniza- nality in their interactions within the
no track at all or only single tracks, tion and excitation events, along body and in the biological responses
well isolated in time. The nucleus of with the production of electrons, induced, all types of ionizing radia-
each cell in a tissue will experience through which energy is deposited. tion have been classified by IARC as
on average one electron track per Interaction of X-rays and γ-rays with carcinogenic to humans (Group 1),
year from background radiation, as- tissues generates fast electrons that even though in some cases direct
suming a spherical nucleus of 8 μm interact with atoms or nuclei, produc- evidence is weak or non-existent,
diameter. Exposure from diagnostic ing additional electrons as they slow with the risk of cancer depending on
procedures can vary from 0.005 Gy down and deposit energy. Charged dose and radiation quality. Although
for dental exposures to approximate- particles such as α-particles and pro- internal radionuclides can vary sig-
ly 0.01 Gy for typical exposures from tons also interact with tissue, produc- nificantly in the range of cancers and
computed tomography (CT), or occa- ing primary ionization and excitation cancer sites observed, the cancer
sionally up to 0.1 Gy for some pro- events, and also a trail of secondary response is ultimately dominated by
cedures over a short period (Brenner electrons along the path of the pri- the biodistribution of these emitters
and Hall, 2007, 2012). Individuals are mary particle. Uncharged neutrons within the body.
also exposed to high-LET α-parti- also interact with tissue and depos-

180
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PMID:18046031

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Part 2 • Chapter 18. Ionizing radiation 183


part 2.
mechanisms of carcinogenesis

chapter 19.

Host susceptibility
Jane C. Caldwell, Ronald L. Melnick, and Lauren Zeise

CHAPTER 19
PART 2
Introduction experimental design of animal bio- within species, and how host sus-
assays, in methods used to identify ceptibility factors may affect evalua-
For several tumour sites (i.e. lung, concordance, and in the degree to tions of tumour site concordance.
lymphoid tissue, and digestive tract), which the animal model captures the It can be difficult to parse out
concordance has often been ob- range of potential human response reasons for lack of tumour site con-
served among different species after to the particular agent tested. An cordance (i.e. lack of response or
exposure to a given IARC Group 1 integral consideration for the devel- common responses between spe-
human carcinogen (see Chapter 21, opment and use of these models is cies). The factors alluded to above
by Krewski et al.). When reported in host susceptibility – the intrinsic and that are not strongly associated with
epidemiological studies, these tu- extrinsic factors that have an impact host susceptibility include the follow-
mour sites are also noted in some on variable response to carcinogens: ing. Competing causes of mortali-
or all of the animal species tested genetic variation, health status, life ty may prevent the development of
experimentally. There are several stage, lifestyle, sex, and the impact late-developing tumours, or studies
other tumour sites with fewer, or less of co-exposures. The microbiome may lack statistical power to detect
common, tumour site concordances can also play a critical part in host an increase in tumour incidence at
among the species studied. susceptibility. sites with high background rates.
Evaluation of concordance of This chapter focuses on exam- Limitations in how the database on
cancer development in specific ples in the IARC Monographs and in tumour site concordance was con-
target organs between and within recent literature on how well animal structed may affect the types of re-
species is dependent on several fac- models reflect the range of human sponses observed (e.g. some stud-
tors. There are various limitations in susceptibility, how host susceptibility ies may focus only on specific tumour
epidemiological studies (e.g. statis- factors may modulate the impact of outcomes or may not be designed to
tical power, exposure assessment, mechanistic events leading to tumour detect some types of tumours). Also,
follow-up, and misclassification), in development between species and when the concordance database

Part 2 • Chapter 19. Host susceptibility 185


was constructed, the identification of an approach based on mode of best collected in a way that provides
of a site in animals required a signif- action to assess risk of leukaemia only partial evidence on differences
icant response in multiple species or (McHale et al., 2012). in host susceptibility.
in both sexes even though one sex The mechanistic database as- Advances in the understanding of
may be more susceptible at a par- sembled for IARC Group 1 carcino- host susceptibility in tandem with the
ticular site (i.e. the mammary gland gens contains information on mecha- evolution of the knowledge on the
in females). In addition, there may be mechanisms of carcinogenesis allow
nistic characteristics, one or more of
mechanistic concordance between for greater understanding of both.
which are commonly exhibited, that
The mechanistic data for all types of
species in how an agent elicits ef- can be used to identify and organize
ionizing radiation (IARC, 2012f) are
fects (e.g. is able to induce genotox- mechanistic information related to
particularly informative with regard to
icity or affect similar pathways), but cancer induction. The database is or- mechanisms of genetic damage (mu-
host susceptibility factors may result ganized in terms of whether an agent tation and epigenetic changes, and
in different site-specific neoplastic displays these key characteristics bystander effects; see Chapter 18,
responses. Thus, host susceptibil- of carcinogens: (1) is electrophilic by Hill and Ullrich), as well as other
ity may determine how and wheth- or can be metabolically activated to host susceptibility factors from this
er specific individual sites or target
electrophiles, (2) is genotoxic, (3) al- large and rich database. The classic
organ systems are influenced by mutation theory of cancer no longer
ters DNA repair or causes genomic
mechanistic events associated with fully encompasses the mechanistic
instability, (4) induces epigenetic
cancer induction. data for several carcinogens (e.g.
alterations, (5) induces oxidative
Although host susceptibility benzene) that induce not only muta-
stress, (6) induces chronic inflam-
factors have a modulating role in tions but also a variety of epigenetic
mation, (7) is immunosuppressive,
carcinogenesis and can affect un- changes. With the present state of
(8) modulates receptor-mediated
derlying mechanistic events, they knowledge, the carcinogenic pro-
effects, (9) causes immortalization,
should not be confused with con- cess cannot be confidently attributed
and/or (10) alters cell proliferation,
cepts such as modes of action and to either a purely genetic or epige-
cell death, or nutrient supply (see
adverse outcome pathways. A mode netic process but probably involves
Chapter 10, by Smith; see also Smith both (see Chapter 12, by DeMarini).
of action is a well-defined and bi-
et al., 2016). Differences and similarities in
ologically plausible series of key
These characteristics are not apparent tumour types and targets
events leading to an adverse effect
in themselves sufficient to explain between rodents and humans can
(EPA, 2005a); an adverse outcome
all aspects of carcinogenesis (see result from a variety of factors that af-
pathway is a construct that attempts
Chapter 13, by Caldwell) but are in- fect absorption, distribution, metabol-
to link an initiating event with an ad-
dicative of multiple mechanisms and ism, and elimination (ADME) of the
verse outcome at a biological level of
associated biological changes ob- agent, as well as the wide range of
organization relevant to risk assess-
served after exposure to carcinogen- inherent susceptibility elements as-
ment (Ankley et al., 2010). Both the
ic agents. Similar to the limitations sociated with toxicodynamic factors.
mode of action and the adverse out-
mentioned above for the database on Differences in the expression of
come pathway concepts can have
genes coding for enzymes that reg-
limitations for the determination of tumour site concordance, the mech-
ulate these processes can contribute
mechanistic concordance between anistic database may present what
to differences in cancer susceptibility
species. For example, leukaemia was studied and reported, and re-
and tumour targets within a species,
induced by exposure to benzene ap- flect the depth to which an agent was
between species, and at various life
pears to result from multiple mecha- treated in each review in the IARC stages when exposure occurs. In
nistic events, some of which are not Monographs. Identification and cate- some instances, the apparent dis-
well characterized and are difficult to gorization of mechanistic data by use cordance between cancer outcomes
quantitate or quantify, and they do of these key characteristics cannot in different species may be explained
not occur in an ordered sequence; always predict tumour site concor- when these genetic factors are iden-
these features limit the applicability dance, because the information is at tified, when relevant animal models

186
are tested, and when more suscep- hypotheses for the inherited basis of well as 730 novel non-synonymous
tible human populations are studied complex genetic traits are that they alleles with uncommon deleterious
for carcinogenic effects. result from “common disease–com- variations that, although individually
mon variant” (i.e. many common al- rare, were present in 7.6–11.7% of
Analogous transgenic, strain- leles of small effect) or “common dis- the population studied (Gordon et al.,
specific, or species-specific ease–multiple rare variants” (i.e. few 2014). Genetic variability in cell sig-
animal models rare alleles of large effect). Although nalling and gene expression may be
genome-wide association study the result of the variants in regulato-
Human and animal studies on cancer
(GWAS) approaches have been ry regions of the genes, rather than
may be more effective in discerning
based on the “common disease– being a consequence of variants in
tumour responses when those most
common variant” hypothesis, they the genetic code that instructs how
at risk are studied, such as a suscep-
have not been successful in explain- to build proteins, or in the regulato-
tible subgroup within a human cohort
ing genetic predisposition to disease ry code itself (see Chapter 13, by
and susceptible animal species and
(Zhang et al., 2011). Caldwell).
strains in a bioassay. Concordance
The understanding of the scope Just as there is genetic variabili-
of response between species may
of human genetic variability now ty in the human population that has

CHAPTER 19
increase for a particular agent when

PART 2
shows that although there is a great an impact on host susceptibility to
mechanisms of carcinogenesis and
deal of similarity in the DNA se- cancer, there is also variability within
subpopulations at risk are identified
quences between individuals, rare rodent strains and species. Genetic
and more analogous transgenic or
gene mutations are abundant, are heterogeneity resulting from cross-
strain-specific animal models are ex-
geographically localized across the ing different mouse strains has long
amined. However, these are general-
world, are difficult to catalogue, and been recognized as an issue of con-
ly not known before an animal study
are possibly a consequence of the cern in the development of experi-
is conducted. There may be gaps in
rapid spread and growth of the hu- mental mouse models, and has been
the understanding of the impact of
man population and weak purifying used as an argument to create ge-
inherent variability on tumorigenesis,
selection (Nelson et al., 2012). The netically inbred strains. Transgenic
in the identification of susceptible
breadth and scope of rare mutations mice carrying exogenous DNA and
human populations, and in the devel-
have also been illustrated through gene-targeted knockout mice have
opment of adequate animal models studies of asthma that have attempt- both been used as models for stud-
to detect a carcinogenic risk from an ed to discover the role of rare muta- ies on cancer and for identifying car-
agent or exposure condition. tions in the “missing heritability” of cinogenic properties of chemicals
Inherent variability the genetic contributions to disease. (Lunardi et al., 2014).
Although common variants at many Two examples illustrate the com-
Most animal studies used to identify loci have been associated with asth- plexity and usefulness of studying
a potential carcinogenic risk in hu- ma, they do not account for overall cancer susceptibility with such mod-
mans are conducted in rodents un- genetic risk. A study of rare and els. Although they carried exactly
der standard conditions (e.g. 2-year low-frequency variants has reported the same mutation in K-Ras, mouse
cancer bioassays) with one particu- ethnic specificity but was unable to lung tumours that resulted from car-
lar agent. However, humans are ex- account for the missing heritability cinogen-induced versus genetical-
posed throughout their lifetime to a of the disease (Igartua et al., 2015), ly engineered models appeared to
mixture of agents (see below), and which is relevant to that associated develop along different mechanistic
inherent biological variability among with cancer risk. pathways (Westcott et al., 2015).
individuals is due to epigenetic and Whole-genome (i.e. exome) se- Exposure of two strains of mice of
genetic variance (Zeise et al., 2013). quencing of common (i.e. minor al- different susceptibility (i.e. A/J and
The contribution of the inherited lele frequency > 5%) and rare (i.e. BALB/cBy) to the same treatment
predisposition to diseases, such as minor allele frequency < 1%) alleles (3-methylcholanthrene, a polycyclic
cancer, has been an active area of across 12 cytochrome P450 genes aromatic hydrocarbon found in to-
research and has an impact on sus- has identified many polymorphisms bacco smoke, and butylated hydrox-
ceptibility analyses. Two proposed with pharmacogenetic effects, as ytoluene) produced lung tumours

Part 2 • Chapter 19. Host susceptibility 187


with different K-Ras mutations in induced by benzene was observed of pathways to activate the parent
codon 12, one resembling human in Diversity Outbred mice compared compound and detoxify reactive me-
tumours from smokers and the oth- with the inbred B6C3F1 mice (French tabolites at different rates in different
er resembling human tumours from et al., 2015). Other groups have used tissues (IARC, 2012c).
non-smokers (Fritz et al., 2010). genetically diverse panels of inbred Polymorphism in the human al-
Thus, the same carcinogenic treat- mice to better predict liver toxicity dehyde dehydrogenase enzyme is
ment given to different strains of (Bradford et al., 2011) and kidney related to risk of cancer from alco-
mice produced tumours at the same toxicity (Harrill et al., 2012). hol consumption in a complex way.
site but with different K-Ras muta- Individuals who do not express the
Genetic polymorphisms
tions. These examples highlight the enzyme at all may have lower risk
utility of using different animal mod- There are several examples of organ- of cancer, because the acute effects
els to understand the mechanistic ic compounds where polymorphisms they experience from alcohol intake
basis for tumour induction in diverse in metabolizing genes in the human (e.g. facial flushing and physical dis-
human populations, but they also population may cause an increased comfort) cause them to abstain from
demonstrate that studies in multiple risk of cancer within certain subpop- alcohol consumption, whereas indi-
animal models are needed. ulations exposed to such agents (see viduals with reduced expression of
Concerns have been raised about Chapter 1, by Bond and Melnick). the enzyme would be able to drink
the sensitivity and design of accel- Inherited mutations in cancer-relat- alcohol and consequently would
erated cancer bioassays that use ed genes (e.g. TP53, BRCA1, APC, have a higher blood concentration
genetically modified mice. Design and mismatch repair enzymes) have of acetaldehyde (IARC, 2012d). The
features (e.g. sample size, study du- a low frequency in the population but development of animal models to
ration, reproducibility, and genetic can confer a high individual risk of reflect this response is dependent
stability of the animals), pathway de- cancer (Melnick, 2001). In such cas- on recognition of the role of these
pendency of effects, and potentially es there may be more concordance metabolic polymorphisms in forming
different carcinogenic mechanisms of response between species when or eliminating cancer-causing inter-
render their utility for predicting hu- analogous transgenic or strain-spe- mediates. Cancers related to alco-
man health risks uncertain, espe- cific animal models are also tested hol consumption were first detected
cially in terms of dose–response with that carcinogenic agent. without consideration of enzyme
(Eastmond et al., 2013). Transgenic mice that lack a func- polymorphisms. Later on, differential
Although the use of isogenic mice tional epoxide hydrolase gene are risks were associated with specific
to detect carcinogenicity of an agent more susceptible to the mutage- polymorphisms.
should reduce the within-group var- nicity of butadiene, as are workers The main focus in pharmacoge-
iance and the number of animals with low-activity epoxide hydrolase netics has been on polymorphisms of
required to detect a response, such polymorphisms (see Chapter 1, by genes encoding drug-metabolizing
mice fail to model the influence of Bond and Melnick). Thus, a trans- enzymes, based on the supposition
genetic diversity. The genetically genic mouse model with a reduced that inter-individual differences in
diverse inbred Collaborative Cross ability to eliminate a mutagenic response were determined by such
mouse strains and the heteroge- metabolite more closely simulates genetic differences and that the main
neous Diversity Outbred mice derived a susceptible human subpopu- genomic hazard was mutagenesis
from the same eight founder strains lation. Polymorphisms in genes or physical damage to DNA (Szyf,
as the Collaborative Cross were de- that encode enzymes involved in 2007). However, human variability
veloped to more accurately capture metabolism of aromatic amines and susceptibility are influenced not
the impact of human variability on (N-acetyltransferases: NAT1 and only by genetic polymorphisms but
tumour responses (Threadgill et al., NAT2) have also been noted to have also by differences in the epigenome
2011; Churchill et al., 2012; French an impact on inter- and intraspecies and its regulatory features.
et al., 2015). Because they more differences in risk for cancer of the Time-dependent changes in
accurately reflect human suscepti- bladder; conflicting findings be- global DNA methylation have been
bility, an order of magnitude greater tween studies are potentially a con- demonstrated in the same individu-
sensitivity to chromosomal damage sequence of the interdependence als in separate populations in widely

188
separated geographic locations, on ADME considerations and spe- DNA from electrophilic metabolites.
with familial clustering for both in- cies-specific vulnerabilities to biolog- Cancer of the bladder is associated
creased and decreased methylation ical agents. with exposure to 2-naphthylamine in
(Bjornsson et al., 2008). The same For example, with regard to in- humans, rats, dogs, hamsters, and
study also showed considerable in- duction of cancer of the bladder by monkeys, as well as with exposure
ter-individual variation with age, with aromatic amines, increased risks to o-toluidine in humans and rats.
differences in DNA methylation ac- are consistently found in humans In mice, however, tumours are seen
cruing over time among individuals and in dogs exposed to, for exam- in other tissues but not in the blad-
who would be missed by studies that ple, 4-aminobiphenyl, benzidine, der. Exposure to benzidine is asso-
apply group averaging. Thus, a focus 4,4′-methylenebis(2-chloroaniline), ciated with cancer of the bladder in
only on genetic polymorphisms does and 2-naphthylamine. Several aro- humans, but in rodents liver tumours,
not consider the fact that epigenetic matic amines (e.g. o-toluidine and not bladder tumours, are observed.
programming plays an equally impor- 2-naphthylamine) induce bladder tu- Conflicting findings between studies
tant part in generating inter-individu- mours in rats (IARC, 2012c; see also are potentially a consequence of the
al differences in phenotype (Szyf, Chapter 2, by Beland and Marques). interdependence of pathways to ac-
Multiple organ site carcinogenicity tivate the parent compound and de-

CHAPTER 19
2007), and that it should be taken

PART 2
of aromatic amines in experimental toxify reactive metabolites at differ-
into account in the analysis of such
animals is associated with metabolic ent rates in different tissues (IARC,
phenotypic diversity. Such inter-indi-
activation of these agents to DNA- 2012c).
vidual differences would also not be
reactive intermediates via multiple Human exposure to asbestos
readily observed with conventional
pathways in target organs. For dogs, has resulted in lung cancer, pleu-
rodent models, for several reasons
lack of N-acetylation of aromatic ral and peritoneal mesothelioma,
(see below).
amines reduces elimination of the and cancer of the larynx and ovary
Strain- and species-specific parent compound via a detoxification (IARC, 2012a). The targets of this
differences in ADME and pathway (IARC, 2010), and their abil- carcinogen are associated with its
susceptibility to biological ity to store urine – as humans do – distribution. After inhalation, fibres
agents increases exposure to urinary me- may penetrate into the interstiti-
tabolites that are hydrolysed in the um and translocate to the pleura or
Most cancer bioassays have
bladder lumen epithelium to reac- peritoneum or more distant sites.
been conducted in rodents (see
tive electrophilic metabolites (IARC, Asbestos has been shown to accu-
Chapter 21, by Krewski et al.). The
2012c). Indeed, infrequent voiding mulate in the ovary in women (IARC,
genetic code has been described
has been associated with increased 2012a). Bronchial carcinomas and
as conserved between humans and
DNA adduct formation in the bladder pleural mesotheliomas have been
mice in terms of genome size, struc- in dogs (Kadlubar et al., 1991). Thus, observed in rats after exposure to
ture, and sequence composition, similarities between the metabolism asbestos fibres, with no consistent
and although candidate regulatory of aromatic amines in dogs and met- increases reported for tumours at
sequences have been conserved abolic polymorphisms in susceptible other sites. However, the Working
and the chromatin landscape in humans, and physiological similari- Group for Volume 100C of the IARC
cell lineages is relatively stable, ties (i.e. the ability to store urine) be- Monographs (IARC, 2012a) noted
there are interspecies differences tween dogs and humans contribute that in many studies complete histo-
in gene expression and regulation to a stronger correspondence with pathology was not done, so it was not
(see Chapter 13, by Caldwell), which respect to the target organ. possible to observe a similar tumour
may account for some apparent dif- The mechanism of tumour induc- pattern associated with carcinogen
ferences in susceptibility or specific tion by aromatic amines is similar distribution.
tumour site concordance after expo- between humans and rodents, but The complexity of developing an
sure to a carcinogenic agent or con- the target organ is not always the appropriate animal model that takes
dition. Other factors described for same; in rodents, there are multi- into account similar distribution fac-
some agents in Volume 100 of the organ targets for exposure to these tors is further illustrated by the exam-
IARC Monographs focus primarily agents through similar effects on ple of cancer induced by asbestos.

Part 2 • Chapter 19. Host susceptibility 189


After inhalation of asbestos or tion of lymphoproliferative disease also renders the analysis of tumour
synthetic fibres, Syrian golden ham- associated with Epstein–Barr virus site and mechanistic concordance
sters are more susceptible than rats (EBV), the use of surrogate hosts problematic.
to induction of malignant pleural has not proven useful for assessing
Life stage
mesothelioma. More rapid transloca- the carcinogenicity of human tumour
tion of synthetic vitreous fibres to the viruses, and for several of them (e.g. The timing of exposure to an agent
pleural space of hamsters compared EBV, Kaposi sarcoma-associated during one’s lifetime can affect the
with that in rats has been proposed herpesvirus, and human papillo- specific type of tumour that may
as the reason for interspecies differ- mavirus), there is no understanding arise, as well as the degree of can-
ences in susceptibility (Gelzleichter of cancer etiology in the context of cer risk from such an exposure. Life
et al., 1999). Rats and hamsters natural viral infection (IARC, 2012b). stage as a susceptibility factor has
are equally susceptible after direct Thus, determinations of interspecies been recognized and included in
intrapleural or intraperitoneal injec- concordance are hampered by the guidelines used by regulatory agen-
tion of the fibres, which circumvents species specificity of most human cies in assessing cancer hazards
differences in distribution. tumour viruses. and risks (EPA, 2005a, b). Although
Because rodents do not smoke, In addition, human susceptibili- puberty and its associated biologi-
it is difficult to develop rodent mod- ty and the identification of tumour cal changes could lead to changes
els that mimic human smoking pat- in cancer susceptibility, exposures
targets of virally induced cancers
terns and exposure to mainstream during that critical period and in that
involve many factors. The type of
tobacco (see Chapter 5, by Hecht age group are seldom the subject of
tumour induced is not only associ-
and DeMarini). However, rodents epidemiological studies; historically,
ated with the age of the subject but
and other species have been used the focus on cancer has been as a
also related to stages of latency of
to study some of the carcinogenic disease associated with ageing after
the viral agent and the presence of
components in cigarette smoke (e.g. extended exposure duration, with
susceptibility cofactors (e.g. vari-
polycyclic aromatic hydrocarbons, prolonged latency periods before the
ants or subtypes of the virus, gene
nitrosamines, aromatic amines, ben- cancers appear (EPA, 2005b).
polymorphisms and the immune sta-
zene, and butadiene). Although stud- Cancer studies in rodents are
tus of the host, and environmental
ies of the individual components of generally designed to last some-
co-exposures that may lead to viral
cigarette smoke have demonstrated what less than a lifetime (2 years),
reactivation) (IARC, 2012b).
genotoxicity, the development of mu- beginning in early adulthood, and
For EBV, specific latency tran-
rine models that reflect the induction to mimic mostly occupational expo-
scription programmes that arise at
of analogous forms of human lung sure circumstances (Melnick et al.,
cancer from smoking involves not specific stages in the viral life-cycle
2008). With the exception of biolog-
only ADME considerations but also have been associated with specif-
ical agents, radiation, or household
strain susceptibility (see above). ic tumours, i.e. latency I with EBV-
exposures (IARC, 2012b, d, f), many
Standard animal cancer bio- related Burkitt lymphoma (BL), la-
data in cancer epidemiology come
assays (i.e. 2-year testing in rats and tency II with Hodgkin lymphoma
from exposures that occur in the
mice) are not used to study biological and T-cell non-Hodgkin lymphoma,
workplace or upon the use of cer-
agents that are specific to humans. and latency III in immunocompro- tain pharmaceuticals. Thus, these
Biological agents have evolved to mised individuals with lymphoprolif- studies may not reveal the potential
preferentially target specific host erative disorders. In addition, three of exposures during the sensitive
species, specific organs or cell types subtypes of BL are associated with early-life period to induce childhood
within those species, and cell types EBV (endemic, sporadic, and im- tumours, nor do they detect tumours
with a specific differentiation status. munodeficiency-associated), two of with long latency periods.
There are data on the development which primarily involve children (i.e. Although similarities between
and use of transgenic models to study endemic and sporadic BL) (IARC, childhood and adult cancers have
biological agents with critical mech- 2012b). Thus, the complexity of iden- been noted, childhood cancers gen-
anistic evidence (see Chapter 9, by tifying target sites and susceptibility erally are embryonic cell tumours
Lambert and Banks). With the excep- factors for these agents in humans (i.e. leukaemias, tumours of the brain

190
and the central nervous system, lym- response (i.e. potency) may also may enhance fixation of mutations,
phomas, bone cancers, soft-tissue be increased. There are examples and the absence of key DNA repair
sarcomas, kidney cancers, eye can- of IARC Group 1 carcinogens for enzymes in some embryonic cells,
cers, and adrenal gland cancers), which potency is greatly increased in such as brain cells. In addition, in-
whereas adults generally develop the young. For example, vinyl chlo- creased risk may result from lack of
more carcinomas (i.e. cancers of ride is an agent for which young ro- fully functional components of the
the skin, prostate, breast, lung, and dents are more susceptible for the immune system during development,
colorectum). In addition, some tu- target site and cell types (i.e. rare different functional operation of hor-
mours appear to be unique to the liver angiosarcomas and more com- monal systems during different life
young, for example tumours of the mon hepatocellular carcinomas) that stages, and induction of develop-
kidney (Wilms tumour) or eye (ret- are also observed in humans (see mental abnormalities that can result
inoblastoma) (EPA, 2005b). Thus, Chapter 1, by Bond and Melnick). in a predisposition to carcinogenic
another aspect of tumour site con- The literature on cancer induced by effects later in life (e.g. diethylstilbes-
cordance between species is the dif- exposure of animals to vinyl chlo- trol) (EPA, 2005b). However, several
ference in tumour types that may be ride is extensive and includes trans- other factors may also increase sus-
observed in children versus adults. placental and perinatal exposures ceptibility in the young. The develop-

CHAPTER 19
A full assessment of cancer risks

PART 2
(IARC, 2012c). Barton et al. (2005) mental origins of health and disease
from childhood exposure to chemi-
estimated the increase in potency of (DOHaD) hypothesis posits that
cals in the environment has been im-
vinyl chloride for liver angiosarcomas environmental exposures during de-
peded by the relative rarity of child-
to be 30-fold and for hepatomas to velopment increase susceptibility to
hood cancers, the lack of studies of
be about 50-fold in female rats after cancer in adulthood through epige-
the late effects of childhood expo-
early-life exposure compared with nomic reprogramming (Walker and
sure with sufficiently long follow-up,
exposure as adults. Ho, 2012).
and the lack of relevant animal test-
As noted above, exposure to ben- In some cases, the newborn or
ing guidelines and assays focused
zidine is associated with bladder young rodent may be a better model
on early-life or perinatal exposures
cancer in humans and liver tumours to assess human cancer risk for ei-
(EPA, 2005b). However, some hu-
in mice (IARC, 2012c). The ratio of ther children or adults. Components
man carcinogens listed in Volume
potency after early-life versus adult of diesel exhaust, an IARC Group 1
100 of the IARC Monographs have
exposure in studies with repeat ex- carcinogen (Benbrahim-Tallaa et al.,
been specifically identified as asso-
posures of juvenile and adult ani- 2012; IARC, 2013), appear to be
ciated with increased risk of child-
hood cancer (i.e. radiation and cer- mals to benzidine is about 100 for metabolized in a similar fashion in
tain pharmaceutical agents used in liver cancer induction in male mice rodents and humans at different
chemotherapy), as well as cancers (Barton et al., 2005). This example stages of development. Nitroarenes
occurring later in life after exposure illustrates an increased susceptibility (and, by extension, diesel exhaust)
during childhood. in the young but an apparent lack of are activated to mutagens in humans
In animals, several agents in- site concordance between humans and young rodents. Concordance
duce a higher incidence of tumours and mice. of lung cancer risk is observed
occurring later in life after perinatal For most of the IARC Group 1 hu- between young rodents and hu-
exposure, for example diethylnitros- man carcinogens, there are data indi- mans (see Chapter 5, by Hecht and
amine, benzidine, polybrominated cating genotoxicity as defined by the DeMarini). Specifically, 1-nitropyr-
biphenyls, and dichlorodiphenyl- toxicological end-point of DNA dam- ene (a component of diesel exhaust)
trichloroethane (DDT). For vinyl chlo- age (see Chapter 12, by DeMarini, is a compound that lacks evidence
ride, there appears to be greater and Chapter 22, by Krewski et al.). of carcinogenicity when exposure
susceptibility of weanling animals to DNA damage has been noted to po- occurs in adult rodents, but it is
the formation of DNA adducts (EPA, tentially exhibit a greater effect after carcinogenic in young adult or new-
2005b). early-life versus later-life exposure; born rodents because of its more
Along with the potential for more this increased susceptibility has extensive metabolism to mutagens.
tumour types occurring after ear- been attributed to more frequent cell Metabolism of 1-nitropyrene by adult
ly-life exposure, the strength of the divisions during development, which humans resembles that of newborn

Part 2 • Chapter 19. Host susceptibility 191


rodents. Accordingly, examination of olar carcinomas and liver hepato- anti-carcinogenic effects, and the
bioassay data for exposures of adult cellular carcinomas in the offspring same regimens may have markedly
rodents only would miss any similar- when they become adults; continu- different effects in different organs
ity of cancer response between the ous exposure during adulthood was and at different stages of women’s
two species. not required, and exposure only in lives.”
In addition to the difficulty of de- adulthood did not induce these tu- To date, there are no mouse mod-
veloping adult rodent models that mours (see Chapter 3, by Waalkes; els for ovarian cancer that reflect
mimic human adult smoking pat- IARC, 2004, 2012a). However, a re- the genetics and histology of hu-
terns, the use of rodent models ex- cent study in male mice reported that man serous ovarian cancer, which
posed in adulthood may not reflect low doses of arsenic in drinking-wa- is most often diagnosed in postmen-
susceptibility. Lung tumours can be ter given according to a scheme opausal women (Smith et al., 2014).
induced in Swiss mice if exposure to that more closely resembles human Tamoxifen has been given to these
mainstream cigarette smoke begins exposure (i.e. to parents before con- women to treat metastatic breast
within 12 hours after birth, but not ception and throughout pregnancy cancer, or to women who are at high
if exposures are delayed (Balansky and lactation, and to offspring after risk of developing the disease. There
et al., 2007; IARC, 2012d). weaning and throughout adulthood) is a concordant decrease in risk of
Diethylstilbestrol is an important caused lung cancer, a response that breast cancer in such women and
example of a transplacental car- has also been reported for in utero in female rodents treated chronically
cinogen where in utero exposure or early-life exposures in humans as adults. However, tamoxifen treat-
causes vaginal and uterine cancer in (Waalkes et al., 2014). ment also causes an increase in risk
daughters but not in exposed moth- Bladder cancer has been induced of endometrial cancer in postmeno-
ers (IARC, 2012e; see Chapter 20, in adult rats after chronic exposure to pausal women. In female mice and
by Rice and Herceg). The effects arsenic. The skin, kidney, and blad- rats, perinatal exposure to tamoxifen
of diethylstilbestrol on the develop- der have been reported as cancer is required to produce tumours of the
ing reproductive tract of rodents are targets in multiple rodent studies of reproductive tract (IARC, 2012e).
species- and strain-specific; neo- inorganic arsenic upon co-exposure In children, several types of ion-
natal exposure to diethylstilbestrol with other carcinogens, via drink- izing radiation show life stage-relat-
results in uterine adenocarcinomas ing-water or transplacentally (IARC, ed differences in susceptibility that
in CD1 mice but not in C57BL/6 2004, 2012a). There is no identified affect target sites and cancer risk
mice. Increased incidence of uterine rodent model for arsenic-induced later in life (see Chapter 18, by Hill
tumours is seen in Eker rats (i.e. a cancer of the skin or lung after ex- and Ullrich; IARC, 2012f). Low-dose
strain that is tumour-prone because posure by inhalation. Thus, arsenic radiation at background levels has
of a germline defect in the Tsc2 tu- is an example where carcinogenicity recently been reported to contribute
mour suppressor gene) but not in and tumour site concordance are de- to the risk of leukaemia and tumours
wild-type rats. CD1 mice exposed to pendent on experimental design, and of the central nervous system in chil-
diethylstilbestrol also exhibit perma- especially on the impact of early-life dren (Spycher et al., 2015). Children
nent estrogen imprinting, morpho- exposures. exposed to ionizing radiation from
logical changes in the reproductive For some carcinogens, there may the atomic bombs in Japan and from
tract, and persistent expression of not be an appropriate animal model the accident with the Chernobyl
the Ltf (lactoferrin) and c-Fos genes for human cancer risk from later-life Nuclear Power Plant in Ukraine had
(Cook et al., 2005). exposures. The timing of exposure an increased risk of thyroid cancer
In humans, exposure to inorganic determines tumour patterns and is attributable to iodine-131 and its
arsenic compounds causes cancer of critical for tumour concordance rela- accumulation in the thyroid.
the lung, bladder, and skin, with limit- tionships for estrogens. The Working Ultraviolet radiation from tan-
ed evidence for cancer of the kidney, Group for Volume 100A of the IARC ning beds increases the risk of skin
liver, and prostate (IARC, 2012a). Monographs (IARC, 2012e) cau- cancer, especially when exposure
Transplacental exposure to arsenic tioned that “estrogen products given occurs at a younger age, i.e. an in-
from oral intake by pregnant female with or without a progestogen have creased risk for malignant melanoma
mice induces lung bronchiolo-alve- markedly different carcinogenic or when first exposure occurs before

192
age 30 years, and for squamous cancer induced by aromatic amines. genitors overlap in haematopoietic
cell carcinoma when first exposure Epidemiological studies of aromatic cancers and complicate determina-
occurs before age 20 years (IARC, amines have not considered breast tions of “target organ or target cell”.
2012f). In mice exposed to this type cancer, because industrial cohorts For studies in humans, changes in
of radiation, squamous cell carcino- were generally small and the relevant classification schemes for haemato-
ma is regularly observed, but no ma- poietic cancers can present diffi-
workforce did not include women.
lignant melanoma has been report- culties in target organ identification
The lack of studies involving female
ed. However, transgenic mice that from different studies. Modern clas-
subjects not only affects species
spontaneously develop malignant sifications of leukaemia and other
concordance but also influences the
melanomas or that have melanocyte lymphatic and haematopoietic ma-
hyperplasia can develop early-onset weight of evidence of an effect when
lignancies are based on cytogenetic
malignant melanoma if exposure to data on both sexes are required to and molecular principles (Swerdlow
ultraviolet radiation occurs neonatal- identify a target site in experimental et al., 2008) that do not always co-
ly, but not after the age of 6 weeks animals. For example, in the con- incide with those of the ICD (IARC,
(IARC, 2012f). This example illus- struction of the animal database to 2012f). Although there may be con-
trates the complexity of developing assess tumour site concordance cordance of haematopoietic cancers

CHAPTER 19
an animal model that mimics human

PART 2
(see Annex 1, by Grosse et al.), the between or within species, the mani-
susceptibility. Target site suscepti- same neoplastic effect is required in festation of disease may differ. Thus,
bility as well as age at which expo- two animal species or in both sexes the determination of tumour site con-
sure occurs must be taken into ac- cordance can be dependent on the
of one species. Breast cancer is rare
count when evaluating tumour site definition and level of specificity of
in male rats as well as in men. Thus,
concordance between species. the target.
limitations in epidemiological stud-
The highest likelihood of identify-
Influence of study design ies and sex differences in cancer
ing a human cancer risk may come
on determination of site response can also account for lack
from the study of sensitive subgroups
concordance of tumour site concordance between
with increased susceptibility to an
humans and experimental animals.
Host susceptibility as well as the agent or groups of agents. If multiple
Inaccurate diagnoses of disease disease categories are lumped to-
type of information collected in either
or incorrect entries on death certif- gether and sensitive subpopulations
human or animal studies influence
icates can affect concordance de- are not distinguished, it may be diffi-
the degree of tumour site concor-
dance that can be identified and terminations, especially for myelo- cult to detect a subtle but real cancer
evaluated. Epidemiological research proliferative and lymphoproliferative response in epidemiological studies.
is often done in men, especially for disorders, which can be described For example, taking into account the
occupational exposures. This can as extranodal or predominantly nod- influence of genetic polymorphisms
limit or preclude the detection of fe- al, precursor or mature neoplasms, or the heterogeneity of tumour phe-
male-specific cancers in humans and which may have multiple cellular notypes will improve the ability to
and thus site concordance between phenotypes. Changing codes in determine the risk to specific sub-
species. the International Classification of populations for colon cancer after
The Working Group for Volume excessive alcohol consumption
Diseases (ICD) can make it difficult
100F of the IARC Monographs (Schernhammer et al., 2010).
to develop a conclusion from hu-
(IARC, 2012c) specifically noted Similarly, designing animal stud-
man studies. However, a multipo-
that many plausible tumour sites ies in such a way that rare tumours
tent haematopoietic stem cell is the
identified in rodents have not been can be detected may increase the
precursor of myeloid or lymphoid ability to determine a response and
reported in humans, and gave the
example of rats treated with aromat- progenitors that further give rise to establish site concordance or mech-
ic amines that developed tumours in several cell types (Greaves, 2004). anistic concordance between spe-
the mammary gland, an organ that Although the disease induced by an cies. In epidemiology, rare tumours
has not been studied adequately as agent may be considered a “lympho- are considered a special type of find-
a potential target site in humans for ma or leukaemia”, the common pro- ing, and they constitute a data set

Part 2 • Chapter 19. Host susceptibility 193


that is different from the tumours that observed when rare tumours were is consistent with the identification
occur more commonly. For example, detected in multiple strains of rats of cigarette smoking as a germ cell
asbestos-induced mesothelioma is and mice, and when genetic poly- mutagen in humans (IARC, 2012d).
a rare tumour associated with expo- morphisms were taken into account Like in humans, hepatocellular
sure to a specific agent. In addition in human studies (Guha et al., 2012; adenomas and carcinomas occur in
to the role of organ distribution of Chiu et al., 2013; IARC, 2014). aged rodents, and background oc-
asbestos fibres in the determination In some cases, tumour site con- currence rates depend on species,
of tumour site concordance, asbes- cordance between humans and ex- strain, and sex. However, hepato-
tos carcinogenicity also provides perimental animals may be more evi- blastoma is extremely rare in rodents
an example of the importance of tu- dent when studies use rodent strains and, unlike in humans, this tumour
mour rarity for the determination of in which there is a lower background usually occurs in aged rather than in
a response after exposure, either in rate of more common tumours as well young animals (Turusov et al., 2002).
humans or in animal bioassays: un- (e.g. the use of mice with lower body Therefore, at the organ level the liver
treated controls from lifetime studies weight and decreased background is a similar target for cancer induction
of asbestos exposure in five strains tumour rates; see the discussion be- from exposure to cigarette smoke for
of rats and Syrian hamsters showed low), but with enough sensitivity to adults and children. However, inter-
zero incidence of mesothelioma in detect a response. species tumour site concordance is
1175 rats and 253 hamsters (IARC, As illustrated by the example of more difficult to demonstrate if cellu-
2012a). exposure to ultraviolet radiation, a lar phenotype, life stage susceptibil-
Many reports of animal bioassay specific cell type (i.e. the melano- ity, and age and timing of exposure
data only highlight statistical signifi- cyte) at the origin of skin cancer in are not taken into account.
cance to identify a positive tumour humans may not lead to skin can-
finding. Because of the relatively cer in wild-type mice (IARC, 2012f). Lifestyle, disease status, and
small number of animals involved in Different cell types within a target co-exposures
rodent bioassays, these studies may organ may have different mechan-
lack statistical power to identify rare isms of tumour development, sus- Cancer susceptibility involves not
tumours induced by a specific agent. ceptibilities, and cancer phenotypes only genetic predisposition but also
As noted above, the use of a genet- that depend on the life stage at which the myriad of exposures experi-
ically heterogeneous strain of mice exposure occurs. The determination enced over a lifetime, at home and
increased the ability to determine of tumour site concordance between at work, and the various other mi-
a genotoxic response to benzene species can depend on the degree croenvironments in which voluntary
(French et al., 2015). of specificity of the target description and involuntary choices affect can-
Use of multiple strains of rats and (i.e. cellular vs organ) in addition to cer risk. Genetic and environmental
mice in chronic studies of trichloro- cancer phenotype. interactions involving complex path-
ethylene enhanced the likelihood of The risk of liver cancer from cig- ways, multiple genes, and multiple
observing increases in the incidence arette smoking illustrates how tim- exposures have been suggested to
of rare kidney tumours and improved ing of exposure and interspecies provide an explanation for the ina-
the probability of showing concor- differences in susceptibility are bility of GWAS approaches to ac-
dance with the finding of increased related to specific phenotypes of count for the missing heritability of
risk of kidney cancer through ep- hepatocellular tumours. There is an most complex diseases, and for the
idemiology. The epidemiological increased risk of hepatocellular car- failure of analyses of rare mutations
database for the current Scientific cinoma in adult humans who smoke to account for asthma (Schadt and
Publication also includes a study cigarettes. However, the strongest Björkegren, 2012).
showing lower risk of kidney cancer risk of hepatoblastoma (an embryo- The “exposome” concept en-
among individuals with genetic poly- nal hepatocellular tumour) is asso- compasses the totality of exposures
morphisms that reduced their ability ciated with paternal smoking before from conception onwards, comple-
to produce mutagenic metabolites conception and a median age at di- menting the genome, instead of fo-
from trichloroethylene. Thus, tumour agnosis of 12 months; the timing of cusing on single exposure–health
site concordance was more easily exposure for the cancer response effect relationships (Vrijheid et al.,

194
2014). The exposome includes three genetic control and cell signalling few decades. Type 2 diabetes,
broad domains of non-genetic ex- can affect cancer susceptibility. In non-alcoholic fatty liver disease, car-
posures: the internal environment addition, exposures to preceding diovascular disease, and increased
(e.g. endogenous hormones, the gut generations have been identified body mass index are risk factors for
microflora, and ageing), specific ex- as affecting susceptibility to cancer. liver cancer, and diabetes induces
ternal exposures (e.g. chemical con- In experimental studies, transgen- synergistic actions with other var-
taminants, lifestyle factors such as erational endocrine effects have iables, such as viral hepatitis and
tobacco use, and occupation), and been identified in the third gener- alcohol consumption (Fan et al.,
the general external domain (which ation of mice after the exposure 2009).
includes influences such as stress, to diethylstilbestrol (Ziv-Gal et al., Immune system status can af-
the urban–rural environment, and 2015). fect human responses to carcino-
climate) (Wild, 2012). Obesogens have not been eval- gen exposures, and thus influence
Tumour site concordance can be uated for carcinogenicity by the the ability to determine site concor-
affected by the inherent nature of the IARC Monographs. However, pre- dance between species (e.g. lack of
conditions under which each spe- natal exposure to obesogens that concordance in responses because
cies is studied to assess cancer risk. activate the constitutive androstane animal models are used that do not

CHAPTER 19
PART 2
Human study subjects have a wide receptor in neonates may affect also take immunosuppression into
and varied range of co-exposures, susceptibility by causing permanent account). With the increasing surviv-
whereas studies in experimental changes in enzyme expression and al of patients with the acquired im-
animals involve relatively uniform metabolism of environmental agents mune deficiency syndrome (AIDS),
exposures in highly controlled envi- encountered as adults (Caldwell, associated cancers in West Africa
ronments. As noted above, chang- 2012). Consequently, obesity asso- have been reported to be Kaposi
es in expression levels of metab- ciated with prenatal environmental sarcoma, non-Hodgkin lymphoma,
olizing enzymes through genetic exposures may render the subject cervical cancer, anogenital cancer,
polymorphisms have been a focus more susceptible to cancer later in and liver cancer (Tanon et al., 2012).
of research, but metabolism is also life. Increased background levels Infection with human immunodefi-
affected by environmental co-expo- of all cancers have been observed ciency virus (HIV) or immunosup-
sures, which are less well studied. in conjunction with increased body pression causes a higher risk for
Also, many solvents have similar ex- weight and obesity in rodent bio- lymphomas, i.e. a 400-fold increase
posure targets, and in humans these assays (Rao et al., 1987; Leakey in risk of non-Hodgkin lymphoma in
exposures often occur together with et al., 2003). the presence of HIV infection (Bassig
co-exposures that have the potential Such changes in background et al., 2012). However, the increas-
to increase the effects of solvents tumour incidence and altered sus- es in the incidence of non-Hodgkin
(Caldwell et al., 2008). However, ceptibility will affect the detection of lymphoma can only be partially ex-
studies of solvents in general may site concordance, especially across plained by the HIV epidemic (Bassig
mask effects of specific agents, data sets that span many decades et al., 2012).
for example trichloroethylene of research. Site concordance may An example of a common co-ex-
(Vermeulen et al., 2012). Lifestyle be more difficult to detect, because posure that affects cancer risk is that
and co-exposures (e.g. obesity, al- exposure-induced and background of aflatoxin B1 contamination of food
coholism, nutritional status, a com- tumours are harder to distinguish in supplies, which tends to be highest
promised immune system, and viral small groups of animals. Site con- in areas with high prevalence rates of
infections) can affect environmental cordance may be detected more fre- infection with hepatitis B and C virus-
cancer risk, but they are often not quently between animal models and es. While aflatoxin B1 and particularly
considered in animal models of car- humans when obesity status is taken its epoxide metabolite are potent mu-
cinogenicity, nor are they typically into account. Such is the case with tagens by themselves, infection with
addressed in human studies. liver cancer (Caldwell, 2012). hepatitis B virus greatly amplifies the
In humans, lifestyle choices and The proportion of the population risk of liver cancer from aflatoxin ex-
previous exposure during develop- that is overweight or obese has in- posure (IARC, 2012b), i.e. from 4-fold
ment that may change set points in creased substantially over the past with aflatoxin alone to 60-fold in the

Part 2 • Chapter 19. Host susceptibility 195


presence of infection with hepatitis B bly produce carcinogenic synergies 2014) and other multifactorial diseas-
virus (Wu-Williams et al., 1992; Yu (Goodson et al., 2015). The modifi- es. Microbial imbalance (dysbiosis)
and Yuan, 2004). As discussed pre- cation of human responses to carcin- usually involves shifts in the relative
viously, aflatoxin metabolism and the ogens from co-exposures would not abundance of commensal microbes.
attendant risk are also affected by be reflected in current animal cancer Inter-individual differences in arse-
polymorphisms of detoxification or bioassays of individual agents, and nic-induced disease are associated
activation pathways (IARC, 2012c). would thus affect the demonstration with differences in arsenic metabol-
Aflatoxin is one of several agents of tumour site concordance. ism; disturbances of the gut microbi-
for which carcinogenicity in experi- ome phenotype have also been re-
Microbiome effects
mental animals was established or ported to affect the biotransformation
highly suspected before epidemio- Included in the exposome concept of arsenic (Lu et al., 2014).
logical studies confirmed its carci- is a more recently described compo- Shifts in the microbiome have also
nogenicity in humans (IARC, 2012d). nent of gene–environment interac- been associated with several types
Aflatoxin B1 is a liver carcinogen in tions that influence cancer suscep- of cancer, and two dominant phy-
humans, rats, tree shrews, trout, and tibility in humans and experimental la normally associated with healthy
several types of transgenic mice, but animals: the contribution to cancer individuals (i.e. the gram-negative
not in wild-type mice (IARC, 2012c). risk of the microbiota living on and Bacteroidetes and the gram-positive
The resistance of adult mice to afla- in humans. These microbiota include Firmicutes) were underrepresented
toxin carcinogenesis has been sug- 100 trillion (1014) microbial cells, out- in colorectal tumour tissue compared
gested to result from constitutive numbering human somatic and germ with adjacent normal colonic tissue
hepatic expression of an α-class glu- cells combined by 10-fold (Bultman, from the same individuals (Bultman,
tathione-S-transferase, mGSTA3-3, 2014), and a quadrillion (1015) virus- 2014).
a detoxifying enzyme with a high es that interact with one another Modulation of microbiota in mouse
affinity for aflatoxin B1 8,9-epoxide and with the host immune system models of cancer has demonstrated
(IARC, 2012c). However, aflatox- in ways that influence disease out- that cancer susceptibility and pro-
in is a liver carcinogen in newborn come. As humans age and develop, gression are affected by concurrent
mice (Vesselinovitch et al., 1972). so do their microbiota. These micro- changes in inflammation, the geno-
Therefore, risk of aflatoxin-induced biota and the genes they encode are mic stability of the host cell, and the
liver cancer serves as an exam- collectively known as the microbi- production of metabolites that func-
ple not only of the effects of co-ex- ome (Clemente et al., 2012). The mi- tion as histone deacetylase inhibitors
posure but also of the influence crobiome differs across species and to epigenetically regulate host gene
of genetic polymorphism and age as individuals, and its effects on tumour expression. Specific diets associat-
susceptibility factors. site concordance have yet to be de- ed with changes in cancer suscepti-
Finally, using the framework of termined. However, its potential ef- bility (e.g. increased consumption of
the hallmarks of cancer (Hanahan fect on human susceptibility to many red meat and higher intake of dietary
and Weinberg, 2011), a task force of chronic diseases, as well as cancer, fibres) have also been associated
174 scientists from 28 countries who is an emerging subject of research. with corresponding changes in the
participated in the Halifax Project The composition of the microbi- microbiome (Bultman, 2014).
(“Getting to know cancer”) published ome varies across anatomical sites; Altering the composition of the mi-
a series of reviews that evaluated the gut microbiome is highly en- crobiota in transgenic mice prone to
exposures to mixtures in the envi- riched in genes involved in carbohy- colorectal cancer led to a lower can-
ronment that may have the potential drate metabolism, in contrast to the cer incidence as a result of reduced
to contribute to cancer risk (Harris, relatively few genes in the human ge- provision of carbohydrate-derived
2015). Cumulative effects of indi- nome that encode carbohydrate-me- metabolites that fuel hyperprolifera-
vidual chemicals that had not been tabolizing enzymes (Bultman, 2014). tion of colon epithelial cells, without
identified as carcinogens were re- The microbiome not only alters met- changes in inflammation or DNA
viewed for actions on key pathways abolic pathways in the human gut but damage induction (Belcheva et al.,
and mechanisms related to carcino- is also linked to host susceptibility 2014). Microbiota have also been im-
genesis and were reported to plausi- to metabolic diseases (Suez et al., plicated in the activation of the innate

196
immune response against tumours its attendant co-exposures to exoge- to obtain the same precision as with
(Iida et al., 2013; Viaud et al., 2013). nous chemicals and its influences on isogenic mice (French et al., 2015).
The interplay between human carci- the microbiome. Other issues to be considered for
nogenic pathogens and the microbi- The analyses of tumour site con- Diversity Outbred models would be
ome as well as the linkage between cordance are dependent on the the percentage survival, the tumour
dysbiosis and carcinogenesis have types of information and databases rates in the controls, and the limited
available at present. Such analyses historical database that is used in the
recently been reviewed (Dzutsev
are limited by the underlying avail- interpretation of data from current
et al., 2015).
able studies, which may not provide animal models. When these models
Conclusions adequate coverage of host suscep- are applied to address these issues,
tibilities. Animal models that cannot they may prove to be an invaluable
This chapter discusses the impor- reflect the intrinsic and extrinsic fac- resource for determining the impact
tance of considering host suscep- tors that have an impact on biological of host susceptibility and of the intrin-
variability in humans may not have sic and extrinsic factors on variable
tibility factors and their modulation
adequate sensitivity to detect all tar- responses to carcinogens.
of tumour response in interpreting
gets of carcinogenicity occurring in

CHAPTER 19
findings of tumour site concordance
humans. Similarly, limitations in epi- Acknowledgements

PART 2
between species, or lack thereof.
demiological studies affect their abil-
Examples are given of how discor- The authors wish to thank D.M.
ity to detect many tumour responses
dance can result from lack of studies observed in animals. DeMarini, M. Marty, S. Vulimiri, V.J.
covering sensitive sexes, subgroups, Transgenic animal models as well Cogliano, and N. Keshava for their
or life stages. Examples are also as highly diverse outbred mouse helpful comments on this manuscript.
provided in which polymorphisms strains and panels of diverse inbred
in metabolizing genes were associ- strains have been developed as an Disclaimer
ated with sensitive subpopulations, approach to model the genetical-
ly highly diverse human species. This article was reviewed and
and where experimentally sensitive
Diversity Outbred mouse models approved for publication by the
rodent strains were studied that also
may be used for future bioassays National Center for Environmental
had sensitivity because of similar
to obtain a better direct estimate Assessment, United States
capacity for increased activation or
of genetic contribution to variance, Environmental Protection Agency.
reduced detoxification (e.g. in the
and these assays may detect po- Approval does not signify that the
case of butadiene, aromatic amines,
tential human tumour sites missed contents reflect the views of the
and alcohol consumption). For aro-
by studies in genetically homoge- agency, nor does mention of trade
matic amines, anatomical and physi- names or commercial products con-
neous strains. These models may
ological similarity (infrequent voiding stitute endorsement or recommen-
also provide a platform to study
of the bladder) between humans other susceptibility factors, such as dation for use. The views expressed
and dogs increases DNA adduct co-exposures or obesity. However, are those of the authors and do
formation and ultimately tumour de- the use of such mouse models will not necessarily reflect those of the
velopment at the same site, i.e. the involve greater expense; heritability California Office of Environmental
bladder. More challenging in study estimates suggest that sample sizes Health Hazard Assessment.
design is to account for lifestyle, with should be increased by a factor of 3

Part 2 • Chapter 19. Host susceptibility 197


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200
part 2.
mechanisms of carcinogenesis

chapter 20

Age and susceptibility


Jerry M. Rice and Zdenko Herceg

CHAPTER 20
PART 2
Introduction with chemical agents. Experimental fetus or infant than that experienced
evidence for susceptibility in utero by the mother.
There is abundant experimental evi- and during infancy to chemical car- However, in some cases the tu-
dence from studies in animals, espe- cinogens and, to a lesser extent, to mours that result from prenatal or
cially rats and mice, that susceptibili- various sources of ionizing radiation perinatal exposures are different
ty to certain chemical carcinogens is has been summarized in reviews and from those that occur in exposed
higher, and sometimes much higher, symposium proceedings (Tomatis adults. Tumours induced prenatally
during prenatal and early postnatal and Mohr, 1973; Rice, 1979; Napal­ become manifest only during adult
life than in adulthood. There is also an kov et al., 1989; Rice, 2004). life in rats and mice, except in certain
extensive epidemiological literature At least in experimental animals, genetically modified strains, because
on the differential effects of a wide greater susceptibility to chemical in these species the interval between
variety of carcinogens in humans at carcinogens in utero and during birth and sexual maturity is only a
different stages of life, including var- early postnatal life is usually man- few weeks. Therefore, the types of
ious forms of radiation, carcinogen- ifested as a higher incidence of the tumours that occur during childhood
ic infectious agents, and chemicals
same kinds of tumours that occur in in humans, including various embry-
and chemical mixtures. This chapter
exposed adults, with a shorter laten- onal solid tumours, are observed as
summarizes the literature that docu-
cy period from the time of exposure tumours of adult life in conventional
ments this high susceptibility of the
to the carcinogen until the appear- rodents. An example is the develop-
fetus, infant, and child to many po-
ance of the tumour. In bioassays for ment of nephroblastomas – embryo-
tentially carcinogenic exposures.
carcinogenicity in adult rodents, the nal kidney tumours that correspond
Studies in experimental incidence and multiplicity of tumours to Wilms tumour in humans – in the
animals increase and the latency period de- adult rat after perinatal exposure to a
creases with increasing dose. Thus, chemical carcinogen. Such tumours
Most experimental studies of the predominant results of early-life do not develop in rats exposed to the
carcinogenesis during prenatal life exposure are what would be expect- same carcinogen during adult life
and infancy have been conducted ed from a higher effective dose to the (Diwan and Rice, 1995).

Part 2 • Chapter 20. Age and susceptibility 201


Chemical carcinogens that reach contribute to carcinogenic effects in when organogenesis starts. Tumour
the fetus via the maternal circulation utero, but when the reactive metab- multiplicity rises to a maximum in
must have crossed the placenta, and olites formed in maternal tissues are offspring exposed at approximate-
consequently are generally referred too unstable to circulate in the mater- ly 21 days of gestation, a few days
to as transplacental carcinogens. All nal bloodstream, cross the placenta, before birth. The susceptibility of
but a few known transplacental car- and reach the fetus, a carcinogenic the fetus relative to that of adult rats
cinogens are organic compounds chemical may have no transplacen- is measured as the incidence and
that act principally or entirely by a ge- tal carcinogenic activity or may only multiplicity of tumours that develop
notoxic mode of action. Factors that cause a low incidence of tumours in offspring after birth, compared
contribute to fetal susceptibility to near the end of gestation, in offspring with the incidence and multiplicity of
these agents include maternal, pla- that were exposed trans­placentally. the same types of tumours in their
cental, and fetal metabolism, the im- This pattern can be seen in the directly exposed mothers. By that
mature state of fetal DNA repair ca- transplacental carcinogenicity of sin- measure, the susceptibility of the rat
pability, the high rate of cell division gle doses of N-nitrosodimethylamine fetus to induction of brain tumours by
during prenatal development, and (NDMA) in rats (Alexandrov, 1968). ENU during the final week of gestat-
the rapidly changing patterns of gene In both the fetus and the pregnant ion is approximately 50 times that of
expression in fetal target tissues, female rat, the target organ for single the mother.
which may render the genetic mate- doses of NDMA is the kidney, but a Transplacental carcinogenesis
rial of fetal cells highly accessible to much lower incidence of tumours is stu­dies with ENU in non-human pri-
carcinogens. observed in the offspring. For many mates, although far less extensive
Different organs and tissues are other compounds whose reactive than studies in rats, also indicate that
not equally susceptible to transpla- metabolites are longer-lived in vivo, the susceptibility of the fetus is great-
cental carcinogens: in experimental the maternal contribution of reactive er than that of the mother. Tumours
animals, some fetal organs, nota- metabolites to fetal tissue burden have been induced in the offspring of
bly the developing nervous system, may be substantial, and the result- rhesus and patas monkeys exposed
are exceptionally susceptible to a ing susceptibility of the fetus may be to ENU during the first trimester of
wide range of agents. Although dif- greater than that of the mother. pregnancy (Rice et al., 1989).
ferences between mother and fetus Short-chain alkylnitrosourea com- Ionizing radiation, both from exter-
in absorption and distribution of a pounds are chemically highly reactive nal sources and from internalized ra-
carcinogen may well exist and may and are extremely potent, direct-act- dionuclides, is capable of producing
contribute to greater apparent ef- ing transplacental carcinogens. In mutations, mainly by large-scale
fects of an administered dose in the rats, a single exposure to one of gene deletions, as well as gross
offspring, these differences are likely these short-lived agents can cause chromosomal damage, and thus is
to be less important than certain oth- a high incidence and multiplicity similar in its effects to direct-acting
er physiological differences between of tumours of the nervous system genotoxic chemical carcinogens,
mother and fetus, including differ- in the offspring of females treated because there is no metabolic bio-
ences in metabolic competence and during the second half of gestat- transformation of the agent (IARC,
in DNA repair capacity. ion (Ivankovic and Druckrey, 1968). 2012e). There are many examples
Most chemicals that have a geno­ These substances are direct-acting, of carcinogenesis by different forms
toxic mode of action must be bio- and the simplest members of this of ionizing radiation, in experimen-
transformed to chemically reactive chemical class, especially N-ethyl- tal animals and in humans and at
metabolites to initiate carcinogeni- N-nitrosourea (ENU), have been all ages, from prenatal life to adult-
city. The requisite enzymes are often used to probe changing susceptibility hood. Solar and ultraviolet radiation
expressed in fetal tissues only late to carcinogenesis during prenatal life also are carcinogens that directly
in gestation, and then at low levels due to factors other than carcinogen damage DNA, but they are less pen-
of activity; thus, only small amounts metabolism. Tumours are induced in etrating than the more highly ener-
of reactive metabolites are gener- offspring exposed once transplacen- getic forms of ionizing radiation (e.g.
ated in fetal tissues. Metabolites of tally to ENU, beginning at approxi- X-rays), and thus exert their carcino-
maternal or placental origin may mately 12 days of gestation in the rat, genic effects primarily on the skin,

202
causing distinctive mutations in DNA mice exposed to transplacental car- increasingly effective and in many
(Agar et al., 2004; IARC, 2012e). cinogens, presumably because the cases is curative, but it imposes a
Results from experiments with ge- probability of such a combination of long-term risk of second cancers in
netically engineered transgenic and events without concomitant lethal survivors. The most intense expo-
knockout mice, especially double genetic damage is immeasurably sure of children to ionizing radiation
knockouts, provide information about low. and to genotoxic chemicals most
the significance of individual genes Although the importance of spe- commonly occurs in the context of
and gene combinations in suscepti- cific genetic events, including muta- anticancer therapy. Carcinogenic
bility to and pathogenesis of specific tions and chromosomal alterations, effects resulting from early-life expo-
tumours, including embryonal neu- in the genesis of cancers is clear, sures are most clearly seen among
rogenic tumours of childhood such evidence is accumulating that many the long-term survivors of childhood
as medulloblastoma (reviewed in carcinogens also cause intracellu- cancers who were successfully treat-
Rice, 2004), and offer some insight lar changes that may contribute to ed with high doses of radiation and/
into why embryonal tumours appear the carcinogenic process but do not or chemotherapy. The examples
relatively later in life in mice than in involve carcinogen-induced alter- given here and in the next section
humans. are representative rather than

CHAPTER 20
ations in genetic sequences. These

PART 2
For example, the gene PTCH1, the changes, which may occur several comprehensive.
human homologue of the Drosophila cell generations after exposure to the The risk of acute myeloid leu-
segment polarity gene patched, is a carcinogen, are termed epigenetic kaemia, non-Hodgkin lymphoma,
tumour suppressor gene associated and can be caused by ionizing radi- and solid cancers of the breast, thy-
with nevoid basal cell carcinoma syn- ation, chemicals, and ultraviolet light. roid, bone, central nervous system,
drome. Patients with this syndrome They include genomic instability, a colorectum, and stomach increased
are predisposed to develop primi- reduced ability to replicate the geno- significantly in survivors of Hodgkin
tive neuroectodermal tumours of the type faithfully (Barcellos-Hoff, 2005), lymphoma diagnosed before age
central nervous system, including and various other effects (IARC, 16 years and successfully treated
medulloblastomas, and mutations 2012e). It is not yet clear how epige- with radiation, chemotherapy with
in PTCH1 have been identified in netic events in carcinogenesis may alkylating agents, or both. Breast
a subset of sporadic primitive neu- vary with age at time of exposure to cancer occurred only in women
roectodermal tumours. Genetically the carcinogen. who had received X-radiation alone
engineered knockout mice with only or chemotherapy and X-radiation
a single normal allele of Ptc1, the Epidemiological findings in combined to treat Hodgkin lympho-
mouse homologue of PTCH1, devel- humans ma. Breast cancers developed usu-
op medulloblastoma-like cerebellar ally within the radiation field, and the
tumours (7–14% incidence). Neo- The consequences of environmen- risk of breast cancer was 75 times
natal exposure of these Ptc1+/− mice tal exposures to chemicals and ra- as great as that in the general pop-
to 3 Gy X-radiation increased this diation during childhood for the risk ulation. Second cancers occurred
incidence to 50%, but irradiation in of cancer later in life have been at increased rates in patients orig-
adulthood had no effect on medullo- reviewed (Carpenter and Bushkin- inally treated with chemotherapy
blastoma incidence (Pazzaglia et al., Bedient, 2013). In patients who re- alone, X-radiation alone, or chemo-
2002). ceive anticancer therapies, the ex- therapy and X-radiation combined,
Dramatically, 95% of Ptc1+/− mice posures are much more intense, and but at different sites; breast cancers
that had also been genetically engi- consequently the risk of cancer is occurred only in patients who had
neered to remove both alleles of the higher. received X-radiation with or with-
tumour suppressor gene p53 devel- Anticancer therapy out chemotherapy, and leukaemia
oped medulloblastomas, and did so was observed only in patients who
very early in life, at younger than Non-surgical therapy for cancer in had received chemotherapy (Bhatia
12 weeks (Wetmore et al., 2001). childhood and adolescence – by ion- et al., 1996). In survivors of childhood
This combination of inactivating gene izing radiation, combination chemo- cancers overall, the risk of gastroin-
mutations is not seen in conventional therapy, or both – has become testinal second cancers increased

Part 2 • Chapter 20. Age and susceptibility 203


significantly with abdominal radia- Medical radiation in survivors of various childhood
tion and after high-dose chemother- cancers who had received radio-
apy with procarbazine and platinum People who were exposed to di-
therapy for their first malignancy in-
drugs (Henderson et al., 2012a). agnostic X-rays in utero and in
creased linearly with radiation dose
childhood during the 1950s are at
to the thyroid up to 20 Gy; the relative
Radiation from nuclear increased risk of cancer, as doc-
weapons and nuclear reactor risk peaked at 14.6-fold (Bhatti et al.,
umented in the Oxford Survey of
accidents 2010).
Childhood Cancers (Wakeford and
Therapeutic anti-tumour X-radia­
There is a statistically significant ex- Little, 2003). However, a more recent
tion to the chest during childhood or
cess risk of solid cancers in people meta-analysis (Schulze-Rath et al.,
adolescence for Hodgkin lymphoma,
who were exposed to ionizing radia- 2008) of studies published after 1990
and to a lesser extent for non-Hodg-
tion from the atomic bombs in Japan did not find any association between
kin lymphoma, Wilms tumour, leu-
either in utero or during early child- in utero exposure to medical radia-
kaemia, bone cancer, neuroblasto-
hood (age < 6 years). Cancers devel- tion and the risk of childhood cancer,
ma, and soft tissue sarcoma, greatly
oped in both children and adults (age probably because in utero diagnos-
increased the risk of breast cancer
12–55 years at the time of diagnosis) tic doses for single-film X-rays are
in female survivors, who tended to
and included leukaemia and a vari- now substantially lower than those
develop the second malignancy at
ety of solid tumours (Preston et al., that were used previously and that
a comparatively early age, during
2008; IARC, 2012e). Cancers of the formed the database for earlier
young adulthood (Henderson et al.,
thyroid are notable in this cohort in reports of increased cancer risk.
2010). Secondary sarcomas are as-
the context of an exceptional sus- Computed tomography (CT) diag-
sociated in a dose-dependent fash-
ceptibility to develop cancer during nostic scans, for which much higher
ion with radiation therapy for child-
early life, because they occurred doses of radiation are used than for
hood tumours; radiation exposure
almost exclusively in survivors who single-film X-rays, have come into
was the most important factor for
were younger than 14 years at the common use for diagnostic proce-
development of secondary sarco-
time of the bombings. dures in both adults and children. CT
mas in survivors of childhood cancer
Short-lived radionuclides of io- scans have recently been reported
(Henderson et al., 2012b).
dine, especially iodine-131, were re- to increase the risk of leukaemia and
leased into the atmosphere in enor- brain tumours in a dose-dependent Solar radiation
mous quantities during the accident fashion in patients who received
with the Chernobyl Nuclear Power their first scan when younger than Solar radiation and sunburn during
Plant in Ukraine, in 1986. Children in 22 years (Pearce et al., 2012). childhood are significant risk factors
Ukraine and in neighbouring coun- Therapeutic X-radiation of the for malignant melanoma of the skin.
tries who were exposed to this radia- head and neck during childhood Duration of residence in Australia –
tion at an early age developed papil­ for non-neoplastic conditions, most and the associated exposure to in-
lary adenocarcinoma of the thyroid commonly to treat fungal infections tense solar radiation – is associated
later in childhood, beginning only a of the scalp, caused a statistically with the risk of developing malig-
few years after the event (Bennett significant increase in the incidence nant melanoma, and childhood is
et al., 2006; IARC, 2012e). Thyroid of intracranial meningiomas and an especially vulnerable life stage
cancer has also been observed in nerve sheath tumours and a small- (Holman and Armstrong, 1984). A
children from highly contaminated er increase in the incidence of brain history of sunburn, especially dur-
areas who were in utero at the time tumours (Ron et al., 1988; Sadetzki ing childhood, is also correlated with
of the accident (Hatch et al., 2009). et al., 2005). Thyroid carcinoma the risk of cutaneous melanoma. A
Children exposed to radioisotopes also occurred in irradiated children, study in England concluded that the
of iodine from the Chernobyl ac- who were much more sensitive to strongest association with elevated
cident were at much higher risk of X-ray-induced thyroid cancer than melanoma risk was for sunburn that
thyroid cancer than adults who were were adults (Ron et al., 1995; IARC, occurred in children aged 8–12 years
similarly exposed. 2012e). The risk of thyroid cancer (Elwood et al., 1990).

204
Chemicals and chemical These changes were found in spe- although the weight of evidence
mixtures other than cytotoxic cific genes (Fos and Ltf [lactoferrin]) favours the greater importance of
anti-tumour agents and persisted even after cessation prenatal exposures. Also, a posi-
of treatment. Interestingly, chang- tive association has been observed
Diethylstilbestrol (DES) is a synthet-
es in gene expression were asso- between parental smoking and risk
ic non-steroidal estrogen that was
ciated with epigenetic alterations: of childhood leukaemia (particular-
administered to pregnant women
specifically, the genes that were ly acute lymphoblastic leukaemia)
during the 1950s and 1960s in an
differentially expressed in animals (IARC, 2012c).
effort to maintain high-risk pregnan-
treated with DES also exhibited ab- A plethora of experimental studies
cies. Although DES is rarely used
normal DNA methylation (Newbold indicate that chemical components
now, it has been estimated that
et al., 2000, 2006). These findings, in tobacco smoke induce a wide
5–10 million women in the USA were
although limited in genome cover- range of genetic changes (Hainaut
treated with DES during pregnancy
age, strongly suggest that exposure and Pfeifer, 2001; Pfeifer et al.,
or were exposed to the drug in utero
to DES may have a significant and 2002; Wistuba et al., 2002; Lea et al.,
(Giusti et al., 1995).
long-term effect on gene expression 2007). More recent studies also im-
Female offspring of women treat-
through epigenetic mechanisms. plicate epigenetic events in human

CHAPTER 20
ed with DES developed an unusual
cancer associated with tobacco

PART 2
More recent studies that used mi-
cancer of the vagina and cervix, clear
croarray-based transcriptome anal- exposure (Herceg, 2007; Lin et al.,
cell adenocarcinoma, which became
ysis in both rats and mice identified 2010; Huang et al., 2011).
clinically evident during adolescence
DES-induced changes in expression In a study of the methylome in
and early adulthood (Herbst et al.,
of a wide range of genes (Hsu et al., cord blood of newborns in connec-
1971). DES caused breast cancer
2009; Warita et al., 2010; Lee et al., tion with maternal smoking during
and is positively associated with the
2011). Whether these changes are pregnancy, differential DNA methyl-
risk of endometrial cancer in women
caused by epigenetic deregulation ation changes in a specific set of
who were exposed while pregnant.
In addition, a positive association has not been tested. genes were associated with tobacco
has been observed between prena- Another interesting feature of exposure (Joubert et al., 2012). A ge-
tal exposure to DES and squamous exposure to DES is its potential im- nome-wide methylomics approach
cell carcinoma of the cervix in female pact on cancer incidence in subse- and measurement of cotinine (a
offspring and cancer of the testis in quent generations. In addition to validated and objective biomarker
male offspring (IARC, 2012d). DES is an increased cancer susceptibility of smoking) were used to identify
the only chemical carcinogen known associated with epigenetic chang- methylation alterations in newborn
to have caused cancer in humans by es in parents treated with DES, an cord blood samples from a mother–
transplacental exposure. epigenetic mechanism may operate child cohort in relation to maternal
The mechanism of action of DES in subsequent generations of mice smoking. Maternal smoking during
as a carcinogen is complex. DES is a (the second generation) (Newbold pregnancy influenced methylation
potent estrogen, and some of its ef- et al., 2006). These findings further changes in specific genes. CYP1A1
fects are mediated, at least in large support the notion that DES-induced and AHRR, which encode proteins
part, by estrogen receptor alpha. carcinogenesis may operate in part involved in the detoxification of
DES can also undergo oxidative through an epigenetic mechanism, chemicals in tobacco smoke, were
metabolism. In fetal mouse tissues, although studies extending to the among the differentially methylated
it causes aneuploidy, chromosomal third generation are needed to es- genes (Joubert et al., 2012), sug-
breaks, and other chromosomal tablish a true transgenerational gesting a potential epigenetic mech-
aberrations; it binds covalently to epigenetic inheritance. anism involved in adverse effects
DNA and thus probably acts in part Parental cigarette smoking caus- associated with in utero exposure to
through a DNA-reactive genotoxic es hepatoblastoma, an embryonal tobacco smoke.
mechanism. In mice, neonatal expo- tumour of the liver, in children. The Various forms of inorganic arsenic
sure to DES also causes persistent effects of prenatal and postnatal ex- have been collectively classified as
changes in gene expression in tar- posures to parental cigarette smoke carcinogenic to humans (Group 1).
get tissues (Newbold et al., 2006). cannot be evaluated separately, These compounds cause cancer of

Part 2 • Chapter 20. Age and susceptibility 205


the skin, bladder, and lung, and pos- infections in young children, setting ages 5–9 years. As a result of this
sibly of the liver and kidney, in ex- in motion pathogenic processes that combined infection, they are at high
posed humans. Arsenic compounds may lead to overt cancer develop- risk of developing endemic Burkitt
have been notoriously difficult to ment during childhood. Examples are lymphoma (eBL) during that period
evaluate in conventional animal bio- Epstein–Barr virus (EBV) and hepa- (IARC, 2012b). eBL is a high-grade
assays for carcinogenicity. However, titis B virus (HBV). Other oncogenic B-cell lymphoma characterized by
during the past decade sodium ar- pathogens, including Kaposi sarco- the consistent presence of EBV
senite has been shown in several ma-associated herpesvirus (KSHV), (zur Hausen et al., 1970) and is the
studies to be a transplacental car- the bacterium Helicobacter pylori, most common paediatric cancer in
cinogen for the lung, liver, ovary, and the bladder fluke Schistosoma sub-Saharan Africa (Greenwood
and adrenal cortex in mice (Waalkes haematobium, establish prima- et al., 1970). The determining factors
et al., 2007; IARC, 2012a; see also ry infection during childhood, but that bring about eBL are, as far as
Chapter 3, by Waalkes). Sodium the resulting cancers appear after is now known, the malaria parasite
the paediatric age (> 18 years) and P. falciparum and EBV.
arsenite is unique in this respect
may require cofactors, especially HBV readily infects young chil-
among inorganic carcinogens.
immunosuppression. Suppression dren by percutaneous and permu-
Also, recent epidemiological
of the immune response may result cosal exposure to infected blood and
studies indicate that early-life expo-
either from co-infection with a sec- other body fluids. The infection caus-
sure of humans to inorganic arsenic,
ond agent, generally the malarial es chronic active hepatitis that leads
most commonly in drinking-water
parasite Plasmodium falciparum or to a high incidence of hepatocellular
but also in contaminated food prod-
human immunodeficiency virus type carcinoma (HCC) in young children
ucts (Yorifuji et al., 2011), can lead to
1 (HIV-1), or by iatrogenic immuno- in Asian and African countries where
liver cancer during childhood (Liaw
suppression before and after organ the prevalence of HBV infection is
et al., 2008), to lung cancer in young
or tissue transplantation. high. Perinatal transmission from
adulthood (Smith et al., 2006), and to
EBV is a ubiquitous oncogenic HBV surface antigen-positive moth-
kidney cancer decades later (Yuan gamma herpesvirus that infects and ers to their newborn babies, or trans-
et al., 2010). persists for life in more than 90% mission from one child to another,
The possible transplacental ef- of the adult population worldwide. is a major source of HBV infection
fects of other inorganic Group 1 Children in certain regions of Africa in many areas of the world (WHO,
agents, such as nickel, cadmium, and become infected with EBV early in 2001). In utero transmission is rela-
chromium(VI), in animals or humans life, and nearly all have serocon- tively rare. Most (80–90%) of infect-
have not been well established. verted by age 3 years, whereas in ed infants and 30–50% of children
affluent countries primary infection infected at ages 1–4 years develop
Infectious agents
is often delayed until adolescence a chronic infection, and about 25%
The factors that underlie the high (Biggar et al., 1978a, b). Primary of those who become chronically
susceptibility to oncogenic infectious EBV infection in early childhood, un- infected during childhood develop ei-
agents during early life are different like that in adolescence, is usually ther cirrhosis or HCC. HCC can be-
from those that govern susceptibility asymptomatic (Chan et al., 2001). come clinically evident in chronically
to chemical carcinogens and radia- EBV coexists for a lifetime in HBV-infected children during – or
tion. Lack of immunity to the agents most human hosts without caus- even before – adolescence (IARC,
in infants and immature immune re- ing overt disease, but viral replica- 2012b).
sponses to infection in infancy and tion can be reactivated in several KSHV is transmitted primarily by
during childhood are major contribu- ways, including malaria infection, saliva; in geographical areas where
tors to susceptibility to these agents specifically P. falciparum malaria. the virus is highly prevalent, infec-
early in life, in common with the well- Children living in areas endemic for tion occurs during childhood, and
known susceptibility of children to malaria, notably in tropical regions of the peak age of acquisition is gener-
other, non-oncogenic infections. sub-Saharan Africa, have an elevat- ally 6–10 years (Whitby et al., 2000;
Several oncogenic infectious ed EBV viral load and a diminished Dedicoat et al., 2004; Malope et al.,
agents readily establish persistent EBV-specific immunosurveillance at 2007). KSHV infection is necessary

206
but not sufficient to cause Kaposi Children whose mothers are in- leukaemia, non-Hodgkin lymphoma,
sarcoma or other cancers in the fected with HIV-1 can be infected dur- and solid cancers of the breast, thy-
absence of severe immunosup- ing gestation and at birth, and during roid, bone, central nervous system,
pression, for example by co-infection infancy by nursing (IARC, 2012b). colorectum, and stomach. Certain
with HIV-1 (IARC, 2012b). In the absence of any intervention, tissues are extremely radiosensitive
The bacterium H. pylori typically transmission of HIV-1 in utero and during childhood and adolescence,
establishes infection of the human during birth is estimated to occur in including the thyroid and the female
stomach during childhood, and un- approximately 25% of infants born breast. Cancers of these and other
treated infections may persist for to HIV-1-positive women (Connor tissues occur at increased frequency
life (Malaty and Graham, 1994; et al., 1994). The risk of mother-to- not only among survivors of child-
Goodman et al., 1996; Brown, 2000). child transmission increases steadily
hood cancer but also in individuals
The infection evolves to cause chron- towards the late stages of pregnan-
exposed as children and adoles-
ic atrophic gastritis, a pre-neoplastic cy; almost 80% of new HIV-1 infec-
cents to diagnostic X-rays (including
condition that leads to development tions occur during the period from
CT scans) and to ionizing radiation
of gastric adenocarcinoma and gas- 36 weeks of pregnancy to delivery
from nuclear weapons and nuclear
tric mucosa-associated lymphoid

CHAPTER 20
(Kourtis et al., 2006).
reactor accidents.

PART 2
tissue lymphoma later in life (IARC, In summary, infants and chil-
2012b). Other high-dosage circumstanc-
dren are exceptionally susceptible
Infestation with the bladder fluke es early in life that pose increased
to many carcinogenic infectious
S. haematobium causes squamous cancer risks include transplacental
agents. Some infections can result
cell carcinoma of the bladder as a in the onset of malignancy within the exposure to the non-steroidal estro-
result of chronic inflammation. The first decade of life. In children, HBV gen DES, which causes distinctive
parasite has a complex life-cycle infection causes HCC, and EBV ac- carcinomas of the reproductive tract
that includes an infective cercar- companied by P. falciparum malaria in female offspring of women treated
ia form present in freshwater bod- infection results in eBL. Infections with DES during pregnancy. Intense
ies in sub-Saharan Africa, the Nile with KSHV, H. pylori, and S. haema- and repeated exposures to solar ra-
valley in Egypt and Sudan, and the tobium typically occur within the first diation during childhood, including
Arabian Peninsula. Infections are few years of life but result in devel- sunburn, predispose to development
percutaneous and result from direct opment of cancer – Kaposi sarcoma, of cutaneous malignant melanoma.
contact with contaminated water. gastric adenocarcinoma and gastric Evidence is beginning to accumu-
Maintenance of transmission of the mucosa-associated lymphoid tissue late that exposure to inorganic ar-
infection depends on contamination lymphoma, and bladder carcinoma, senic in utero and during childhood
of fresh water with excreta contain- respectively – decades later. On a can cause cancer of the liver during
ing schistosome eggs, the presence global scale, in terms of the numbers childhood and of the lung or kidney
of snails as intermediate hosts, and of children exposed and the numbers decades later.
human contact with contaminated of cancer cases that result, oncogen- The consequences of exposures
water (Jordan and Webbe, 1993). ic infectious agents pose the greatest to lower doses or concentrations of
Children start to accumulate worms cancer risks during childhood. other carcinogens during prenatal
as soon as they are old enough to
and early postnatal life have been
have contact with water, and they Summary
more difficult to establish (Carpenter
may be continuously reinfected and
remain infected throughout their Treatment of childhood cancers with and Bushkin-Bedient, 2013). Parental
lives (IARC, 2012b). The incidence high doses of ionizing radiation and cigarette smoking can cause hepato-
of schistosome-related bladder combinations of cytotoxic drugs, blastoma in children, an extreme
cancer in Africa peaks at ages 40– many of which are carcinogenic to case of the danger of second-hand
49 years, whereas infection with S. humans (Group 1), has been very tobacco smoke. Possible environ-
haematobium begins as early as age successful in recent years, but sur- mental causes of other embryonal
6 months and usually peaks at ages vivors are at high risk of second tumours of childhood continue to be
5–15 years (Mostafa et al., 1999). cancers, including acute myeloid investigate.

Part 2 • Chapter 20. Age and susceptibility 207


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210
part 3.
statistical analyses of concordance and key characteristics

chapter 21.

Analysis of tumour site


concordance
Daniel Krewski, Jerry M. Rice, Michael Bird, Brittany Milton, Brian Collins, Pascale Lajoie, Mélissa Billard, Yann Grosse,
Vincent J. Cogliano, Jane C. Caldwell, Ivan I. Rusyn, Christopher J. Portier,
Ronald L. Melnick, Julian Little, and Jan M. Zielinski (deceased)
in collaboration with other participants (see the Contributors list) in the Workshop on Tumour Site Concordance
and Mechanisms of Carcinogenesis, which was convened by IARC in April and November 2012 in Lyon

CHAPTER 21
PART 3
Introduction Monographs (Cogliano et al., 2004; evaluations involve classifying the
IARC, 2006), are used to evaluate data from both the human and the
Since its establishment in the ear- and integrate the evidence provided animal studies as providing suffi-
ly 1970s, the IARC Monographs by human epidemiological studies, cient evidence of carcinogenicity,
Programme has evaluated more than animal cancer bioassays, and infor- limited evidence of carcinogenicity,
1000 agents with evidence of human mation on possible biological mech- inadequate evidence of carcinogen-
exposure for which some suspicion anisms of action, to classify agents icity, or evidence suggesting lack of
exists of an increased cancer risk into one of the following categories: carcinogenicity. The information on
to humans. The IARC Monographs carcinogenic to humans (Group 1), biological mechanisms of action may
Programme has developed detailed probably carcinogenic to humans be evaluated as strong, moderate, or
criteria against which to evaluate (Group 2A), possibly carcinogenic to weak, and is taken into consideration
the available scientific evidence on humans (Group 2B), not classifiable in the overall evaluation.
the carcinogenic potential of such as to its carcinogenicity to humans To date, IARC has developed 119
agents. These criteria, which are de- (Group 3), and probably not carcino- Monographs Volumes on more than
scribed in the Preamble to the IARC genic to humans (Group 4). These 1000 agents for which there exists

Part 3 • Chapter 21. Analysis of tumour site concordance 211


some evidence of cancer risk to hu- PCBs, which were determined to be animals, and includes internationally
mans; of these, 120 agents met the Group  1 agents in Volume 107. For peer-reviewed and published data
criteria for Group  1. Volume 100 of the purposes of the present anal- from studies in humans and experi-
the IARC Monographs provided a re- ysis, PCBs and dioxin-like PCBs mental animals to support analyses
view and update of the 107 Group 1 were considered as a single group of tumour sites seen in humans and
agents identified as of 2009. Volume of PCBs, resulting in 113 – 2 = 111 animals. Although the database
100 is divided into six parts, focus- distinct agents for analysis. Including also includes human tumour sites
ing on pharmaceuticals (Volume for which there is limited evidence
the five Group 1 agents identified
100A; IARC, 2012e); biological of carcinogenicity of the agent,
since Volume 100, there are 23, 11,
agents (Volume 100B; IARC, 2012b); such sites were not systematically
10, 18, 12, and 37 Group 1 agents in
arsenic, metals, fibres, and dusts
Volumes 100A to 100F*, respectively. identified in the IARC Monographs.
(Volume 100C; IARC, 2012a); radi-
Because both animal and human Likewise, animal tumour sites were
ation (Volume 100D; IARC, 2012f);
data are considered in evaluating the generally not identified in the case of
personal habits and indoor combus-
weight of evidence for human car- limited evidence of carcinogenicity in
tions (Volume 100E; IARC, 2012d);
cinogenicity, the degree of concor- animals.
and chemical agents and related
dance between species for tumour The next section describes how
occupations (Volume 100F; IARC,
induction by carcinogenic agents is information was retrieved and as-
2012c). Since the publication of
sembled from the data set compiled
Volume 100, five additional agents important. A high degree of site con-
by Grosse et al., as well as the ap-
had been added to Group 1 at the cordance between species supports
proach used to evaluate tumour site
time the present analysis was under- the ability of studies in experimental
concordance between animals and
taken: (i) diesel engine exhaust (re- animals to predict not only a potential
humans. A detailed description of the
viewed in Volume 105; IARC, 2013), cancer risk to humans but also the
results of the analysis of these data is
(ii) trichloroethylene (TCE) (evalu- specific sites of cancer induction ex-
then presented both in the text of this
ated in Volume 106; IARC, 2014), pected from human exposure to car- chapter and in online supplemental
(iii) polychlorinated biphenyls (PCBs) cinogenic agents. In contrast, lack of material (see below). A discussion
and dioxin-like PCBs (reviewed in concordance may indicate the need of the results of these analyses and
Volume 107; IARC, 2016b), and
for further research to make sure the conclusions drawn from this work
(iv) outdoor air pollution and (v) par-
that all cancer sites have been iden- are presented in the last two sections
ticulate matter in outdoor air pollu-
tified in sensitive human subpopula- of this chapter.
tion (both evaluated in Volume 109;
tions or in appropriate experimental
IARC, 2016a). Had these five agents Methods
animal models, and to identify the
been evaluated within Volume 100,
underlying mechanisms that differ- Tumour nomenclature in
they would have been included in
Volume 100F; for ease of reference, ent species may or may not have in animals and humans
these agents are included in an ex- common.
Although human tumours can be
panded group of chemical agents This chapter uses the data set as-
coded in a standardized manner by
and related occupations, denoted by sembled by Grosse et al. (Annex 1)
use of the International Classification
Volume 100F*. derived from the available informa-
of Diseases coding system (WHO,
The 113 agents classified by tion on the agents classified by IARC
1977, 2011), a comparable nomen-
IARC as known causes of cancer in as carcinogenic to humans (Group 1)
clature system does not exist for
humans up to and including Volume in Volume 100 to Volume 109, the
animal tumours. To render the ani-
109 of the IARC Monographs are list- last Monograph for which final data mal and human tumours identified
ed in Table 21.1. Note that although were available at the time this anal- in the IARC Monographs compa-
3,3′,4,4′,5-pentachlorobiphenyl (PCB ysis was conducted. This database rable, a taxonomy of tumour sites
126) was evaluated as a separate includes all tumour sites identified was constructed (Table 21.2). As
Group 1 agent in Volume 100F, it is in the IARC Monographs for which detailed in Supplemental Material
included within the group of agents agents presented sufficient evidence I (online only; available from:
consisting of PCBs and dioxin-like of carcinogenicity in humans and/or http://publications.iarc.fr/578), this

212
Table 21.1. Group 1 agents included in Volumes 100A–F, 105, 106, 107, and 109a

Volume Type of agent Number Agents


of
agents

100A Pharmaceuticals 23 Aristolochic acid; Aristolochic acid, plants containing; Azathioprine; Busulfan; Chlorambucil; Chlornaphazine; Ciclosporin;
Cyclophosphamide; Diethylstilbestrol; Estrogen-only menopausal therapy; Estrogen–progestogen menopausal therapy
(combined); Estrogen–progestogen oral contraceptives (combined); Etoposide; Etoposide in combination with cisplatin and
bleomycin; Melphalan; Methoxsalen in combination with UVA; MOPP; Phenacetin; Phenacetin, analgesic mixtures containing;
1-(2-Chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (Methyl-CCNU); Tamoxifen; Thiotepa; Treosulfan

100B Biological agents 11 Clonorchis sinensis (infection with); Epstein–Barr virus; Helicobacter pylori (infection with); Hepatitis B virus; Hepatitis C virus;
Human immunodeficiency virus type 1; Human papillomavirusesb; Human T-cell lymphotropic virus type 1; Kaposi sarcoma-
associated herpesvirus; Opisthorchis viverrini (infection with); Schistosoma haematobium (infection with)

100C Arsenic, metals, 10 Arsenic and inorganic arsenic compounds; Asbestos (all forms, including actinolite, amosite, anthophyllite, chrysotile, crocidolite,
fibres, and dusts and tremolite); Beryllium and beryllium compounds; Cadmium and cadmium compounds; Chromium(VI) compounds; Erionite;
Leather dust; Nickel compounds; Silica dust, crystalline, in the form of quartz or cristobalite; Wood dust

100D Radiation 18 Fission products including strontium-90; Haematite mining with exposure to radon (underground); Ionizing radiation (all types);
Neutron radiation; Phosphorus-32, as phosphate; Plutonium-239; Radioiodines, including iodine-131; Internalized radionuclides
that emit α-particles; Internalized radionuclides that emit β-particles; Radium-224 and its decay products; Radium-226 and
its decay products; Radium-228 and its decay products; Radon-222 and its decay products; Solar radiation; Thorium-232 (as
Thorotrast); UV radiation (bandwidth 100–400 nm, encompassing UVC, UVB, and UVA); UV-emitting tanning devices; X- and
γ-radiation

100E Personal habits and 12 Acetaldehyde associated with consumption of alcoholic beverages; Alcoholic beverages; Areca nut; Betel quid with tobacco;
indoor combustions Betel quid without tobacco; Coal, indoor emissions from household combustion of; Ethanol in alcoholic beverages;
N′-Nitrosonornicotine (NNN) and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone NNK); Salted fish, Chinese-style; Second-hand
tobacco smoke; Tobacco smoking; Tobacco, smokeless

100F Chemical agents 32 Acid mists, strong inorganic; Aflatoxins; Aluminium production; 4-Aminobiphenyl; Auramine production; Benzene; Benzidine;
and related Benzidine, dyes metabolized to; Benzo[a]pyrene; Bis(chloromethyl)ether; Chloromethyl methyl ether (technical grade);
occupations 1,3-Butadiene; Coal gasification; Coal-tar distillation; Coal-tar pitch; Coke production; Ethylene oxide; Formaldehyde; Iron
and steel founding, occupational exposure during; Isopropyl alcohol manufacture using strong acids; Magenta production;
4,4′-Methylenebis(2-chloroaniline) (MOCA); Mineral oils, untreated or mildly treated; 2-Naphthylamine; ortho-Toluidine; Painter,
occupational exposure as a; 3,3′,4,4′,5-Pentachlorobiphenyl (PCB 126)a; 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF); Rubber
manufacturing industry, occupational exposures in the; Shale oils; Soot (as found in occupational exposure of chimney sweeps);
Sulfur mustard; 2,3,7,8-Tetrachlorodibenzo-para-dioxin; Vinyl chloride

Part 3 • Chapter 21. Analysis of tumour site concordance


213
PART 3
CHAPTER 21
Table 21.1. Group 1 agents included in Volumes 100A–F, 105, 106, 107, and 109a (continued)

214
Volume Type of agent Number Agents
of
agents

105c Diesel and gasoline 1 Engine exhaust, diesel


engine exhausts and
some nitroarenes

106c Trichloroethylene 1 Trichloroethylene


and some
chlorinated agents

107c Polychlorinated 1 Polychlorinated biphenyls (PCBs) and dioxin-like PCBsa


biphenyls and
polybrominated
biphenyls

109c Outdoor air pollution 2 Outdoor air pollution; Particulate matter in outdoor air pollution

UV, ultraviolet.
a Although 113 Group 1 agents have been identified up to and including Monographs Volume 109, the present analysis is based on 111 distinct agents remaining after considering PCBs

and dioxin-like PCBs within the broader category of PCBs, and including PCB 126 within the broader category of PCBs.
b Human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 were evaluated as carcinogenic to humans.

c During the concordance analyses, the Group 1 agents in these Volumes were included with “chemical agents and related occupations” in Volume 100F*.
Table 21.2. Anatomically based taxonomy of tumour sites/organ systems in animals and humans

Organ system Sites coded from Volume 100 (A, B, C, D, E, and F*)a

Upper aerodigestive tract Nasal cavity and paranasal sinuses


Nasopharynx
Oral cavity
Pharynx
Tongue
Tonsil
Salivary gland

Respiratory system Larynx


Lung
Lower respiratory tract

Mesothelium Mesothelium

Digestive tract Oesophagus


Stomach
Intestine (including colon and rectum)

Digestive organs Liver parenchyma and bile ducts


Pancreas NOS
Gallbladder

Nervous system and eye Brain and spinal cord (CNS)


Eye

Endocrine system Thyroid, follicular epithelium


Adrenal gland (medulla, cortex, NOS)
Pituitary gland

Kidney Kidney (renal cortex, renal medulla, kidney NOS)

Urothelium Urothelium (renal pelvis, ureter, or bladder)

Lymphoid and haematopoietic tissues Haematopoietic tissue

CHAPTER 21
Lymphoid tissue

PART 3
Skin Skin and adnexae
Cutaneous melanocytes

Connective tissues Soft connective tissue


Blood vasculature (endothelium)
Hard connective tissue (bone, cartilage)

Female breast, female reproductive organs, and Breast


female reproductive tract Ovary
Uterine cervix
Uterus
Vulva/vagina

Other groupings All cancers combined


All solid cancers
Exocrine glands NOS
CNS, central nervous system; NOS, not otherwise specified.
a These sites are derived from all site descriptors used in IARC Monographs to describe human and experimental animal cancer data

(see Supplemental Table 1. Animal and human tumour sites for 111 Group 1 agents identified up to and including Volume 109 of the
IARC Monographs).

Part 3 • Chapter 21. Analysis of tumour site concordance 215


taxonomy is anatomically based and and haematopoietic tissues, and animal species are also noted.
includes 47 tumour sites grouped bone and cartilage can arise wherev- Information on the histology of ani-
within 15 organ and tissue systems. er in the body their progenitor tissues mal lesions, when available, is also
There are 39 distinct animal and occur, and are aggregated according recorded in Table 21.3; however, be-
human tumour sites specified for to tissue of origin without regard to cause this information is not general-
Group 1 agents in Volume 100A–F*, anatomical location. Likewise, skin ly available in the IARC Monographs
and eight additional tumour sites cancers are aggregated irrespective for human studies, it was not consid-
were considered to be important, of anatomical location, with the ered in the comparative analyses re-
even though they did not appear in exception that malignant melanoma ported here.
the tumour site concordance data set as it occurs in humans is unknown Although tumour sites for which
developed by Grosse et al. (Annex 1). in rats or mice; cutaneous melano- agents show limited evidence of car-
The individual tumour sites seen in cytes are thus included separately in cinogenicity in humans are included
either animals or humans up to and Table 21.2 as a human tumour site in Table 21.3, this information is not
including Volume 109 of the IARC only for the sake of completeness. considered in the present analysis.
Monographs are listed in Table 21.2. Estrogen-producing and estrogen- In fact, although the original intent
The category “other groupings” in- responsive tissues are aggregated was to consider tumour sites with
cludes the three sites (“all cancers in the organ system “female breast, sufficient or limited evidence in hu-
combined”, “all solid cancers”, and female reproductive organs, and fe- mans when evaluating concordance
“exocrine glands not otherwise spec- male reproductive tract”. In contrast with animal tumour sites with suffi-
ified”) that do not fit into any of the to the female reproductive system, cient evidence, there are only two
other 14 groupings of organ and tis- no carcinogens are known with suf- Group 1 agents with limited, but not
sue systems. All analyses reported ficient evidence for the male repro- sufficient, evidence of carcinogeni-
in this chapter are based on the 39 ductive system in humans, despite city in humans.
individual tumour sites within the 14 the high prevalence in humans of
Effects of sex, strain, and
organ and tissue systems listed in prostate and testicular germ cell
route of administration
Table 21.2 (excluding tumours of the cancers.
male reproductive tract, for which the The last column in Table 21.3 pro-
Retrieval of data on tumour
data do not show sufficient evidence vides details on animal studies rele-
occurrence from the IARC
in both humans and animals). vant to the evaluation of the agent of
Monographs
Aggregation of tumour sites with- interest, including the sex and strain
in an organ and tissue system was Grosse et al. (Annex 1) extracted of the test animals and the route
guided by several factors, including data from Volumes 100, 105, 106, of administration of the test agent.
anatomical and functional related- 107, and 109 on tumour sites report- Although this information has been
ness. The specialized epithelia of the ed in humans or animals for the 111 recorded where available, it is diffi-
upper aerodigestive tract, respiratory distinct Group 1 agents considered cult to examine concordance with re-
system, digestive tract, and digestive here. This information is illustrated in spect to these important factors for a
organs are found for the most part Table 21.3, with one compound from variety of reasons, as outlined below.
in a single or a few anatomical sites, each of Volumes 100A–F, as well Because many epidemiological
which are precisely captured by the as diesel engine exhaust (Volume studies are based on predominant-
available epidemiological and experi- 105), TCE (Volume 106), PCBs ly male occupational cohorts, men
mental data. In contrast, both the kid- (Volume 107), and particulate matter tend to be over-represented in the
ney and the urothelium are data-rich in outdoor air pollution (Volume 109). human studies on Group 1 agents.
sites, and carcinogenic agents for ei- Table 21.3 gives the tumour sites Other agents, such as hormonal oral
ther site display little or no overlap in for which the agents provide suffi- contraceptives, are evaluated only
target organ. Accordingly, the kidney cient evidence of carcinogenicity in in women. Certain lesions, notably
and the urothelium were analysed humans, as well as sites for which breast cancer and prostate cancer,
separately rather than being aggre- there is limited evidence. Tumour are largely sex-specific. Also, some
gated as “urinary tract”. Cancers of sites for which sufficient evidence animal studies use only one sex, and
soft connective tissues, lymphoid of carcinogenicity exists in specific others do not specify whether male

216
Table 21.3. Information on animal and human tumours and tumour sites for Group 1 agents in the IARC Monographs (adapted from Annex 1, by Grosse et al.)

Volume Agent Sites with sufficient Site with limited Agent tested in Species Histology Study/sex/strain/ Comments
Agent evidence in evidence in experimental Site exposure route
number humans humans animals

100A Azathioprine Non-Hodgkin   Azathioprine Mouse Lymphoma Mitrou et al. (1979a)


3 lymphoma, skin Lymphoid (Volume 26), F, New
(squamous cell tissue Zealand Black and New
carcinoma) Zealand White, s.c.;
Mitrou et al. (1979b)
(Volume 26), F, New  
Zealand Black and New
Zealand White, s.c.; Ito
et al. (1989), F, B6C3F1,
p.o.; Brambilla et al.
(1971), MF, Swiss, i.p.

100B Epstein–Barr Burkitt lymphoma, Lymphoepithelioma-         No data on


25 virus immunosuppression- like carcinoma, animal studies
related non-Hodg- gastric carcinoma listed; humans
kin lymphoma, are the only
extranodal NK/T-cell natural hosts for
lymphoma (nasal Epstein–Barr
type), Hodgkin virus
lymphoma,
nasopharyngeal
carcinoma

100C Arsenic and Lung, bladder, skin Kidney, liver, Dimethylarsinic Mouse Bronchiolo- DMA(V): Tokar et al.
35 inorganic arsenic prostate acid [DMA(V)], Lung alveolar (2012a), M, CD1, d.w.;
compounds Monomethylarsinous carcinoma Sodium arsenite:
acid [MMA(III)], Waalkes et al. (2003),
Sodium arsenite F, C3H/HeNCr, in utero;
Waalkes et al. (2006),
M, CD1, in utero; Tokar  
et al. (2011), MF, CD1,
in utero + p.o.; Tokar
et al. (2012a), M, CD1,
in utero; MMA(III): Tokar

Part 3 • Chapter 21. Analysis of tumour site concordance


et al. (2012b), M, CD1,
in utero

217
PART 3
CHAPTER 21
Table 21.3. Information on animal and human tumours and tumour sites for Group 1 agents in the IARC Monographs (adapted from Annex 1, by Grosse et al.)

218
(continued)

Volume Agent Sites with sufficient Site with limited Agent tested in Species Histology Study/sex/strain/ Comments
Agent evidence in evidence in experimental Site exposure route
number humans humans animals

100D Fission products Solid cancers,   Strontium-90 Mouse Osteosarcoma Nilsson (1970, 1971), M,
45 including leukaemia Bone CBA, i.p.; Nilsson et al.  
strontium-90 (1980), F, CBA, i.p.

100E Coal, indoor Lung   Coal smoke Mouse Bronchiolo- Liang et al. (1988), MF,
68 emissions from Lung alveolar Kunming, inh.; Lin et al.
 
household carcinoma (1995), MF, Kunming,
combustion of inh.

100F Benzene Acute myeloid Acute lymphoblastic Benzene Mouse Lymphoma Snyder et al. (1980), M,
80 leukaemia, acute leukaemia, chronic Thymus C57BI/6J, inh.; Cronkite
non-lymphoblastic lymphocytic leu- et al. (1984), F, C57BI/6
 
leukaemia kaemia, multiple BNL, inh.
myeloma, non-
Hodgkin lymphoma

105 Engine exhaust, Lung Bladder Whole diesel engine Rat Bronchiolo- Ishinishi et al.
107 diesel exhaust Lung alveolar (1986),MF, F344, inh.:
carcinoma Mauderly et al. (1986,
1987), MF F344, inh.:
Iwai et al. (1986), F,
F344, inh.: Heinrich  
et al. (1995), F, Wistar,
inh.: Nikula et al..
(1995), F, F344, inh.:
Iwai et al. (2000), F,
F344, inh.

106 Trichloroethylene Kidney Non-Hodgkin Trichloroethylene Rat Renal cell National Toxicology
108 lymphoma, liver Kidney carcinoma Program (1990), M,
F344/N, g.; National
Toxicology Program
 
(1988), M, Osborne-
Mendel, g.; National
Toxicology Program
(1988), F, ACI, g.
Table 21.3. Information on animal and human tumours and tumour sites for Group 1 agents in the IARC Monographs (adapted from Annex 1, by Grosse et al.)
(continued)

Volume Agent Sites with sufficient Site with limited Agent tested in Species Histology Study/sex/strain/ Comments
Agent evidence in evidence in experimental Site exposure route
number humans humans animals

107 Polychlorinated Skin (melanoma) Non-Hodgkin Aroclor 1260 Rat Hepatocellular Mayes et al. (1998),
109 biphenyls lymphoma, breast Liver carcinoma F, Sprague-Dawley,
p.o.; Norback and
Weltman (1985), F,  
Sprague-Dawley, p.o.;
Kimbrough et al. (1975),
F, Sherman, p.o.

109 Particulate Lung           Sufficient


111 matter in outdoor evidence in
air pollution experimental
animals, but
no organ sites
identified due
to the absence
of two (or more)
studies of
adequate design
and quality
pointing at the
same organ site
(with a similar
histological
origin) in the
same species

F, female; d.w., drinking-water; g., gavage; inh., inhalation; i.p., intraperitoneally; M, male; MF, male and female; NK, natural killer; p.o., orally; s.c., subcutaneously.

Part 3 • Chapter 21. Analysis of tumour site concordance


219
PART 3
CHAPTER 21
or female animals – or both – were species. These distributions are of extensive mechanistic data on B[a]P,
used. For these reasons, separate value in demonstrating the spectrum suggesting that the mechanisms by
analyses of species concordance of tumours caused by these agents in which this agent causes tumours in
across the spectrum of Group 1 different species, including the identi- animals would also be expected to
agents are difficult to conduct. fication of the most common tumours operate in humans; no data in hu-
Separate concordance analyses by caused in humans. Human tumours mans on B[a]P alone were available
strain are also difficult, because of caused by the human tumour virus- for evaluation (IARC, 2010). An im-
the sparseness of studies on spe- es reported in Volume 100B were portant aspect of such mechanistic
cific strains of experimental animals. included in these distributions, so upgrades for purposes of the present
Indeed, in many cases information that these results reflect the tumours analysis is the general lack of identi-
on strain is unavailable, preclud- caused by all 111 distinct Group 1 fication of a human tumour site.
ing the possibility of strain-specific carcinogens considered here. Of the nine agents in Table 21.4
analyses. placed in Group 1 on the basis of
Human exposure to carcinogens Organization of concordance mechanistic upgrades, all but one –
can occur by oral ingestion, inhala- analyses
etoposide – demonstrated sufficient
tion, or dermal absorption, as well as evidence of carcinogenicity in ani-
Analytical results are presented first
via other routes, such as injection of mals. In the assignment of etoposide
for the 39 tumour sites and then for
pharmaceutical agents for therapeu- to Group 1 in the absence of sufficient
the 14 organ and tissue systems.
tic purposes. Animal studies may evidence in animals, the Monograph
Because the present database in-
involve other routes of exposure, noted the limited evidence of carci-
volves only a moderate number of
such as intraperitoneal injection or nogenicity in humans on the basis
agents with comparable data in an-
intratracheal instillation. In many of the induction of acute myeloid
imals and humans, results aggregat-
cases, the route of exposure used in leukaemias with distinctive chro-
ed by organ and tissue system may
animal studies may not correspond mosomal translocations by drugs,
be expected to be more stable.
to the predominant route by which including etoposide, that target topo-
humans are exposed; in such cases,
Results isomerase II (IARC, 2012e). Of the
the dose of the reactive metabolite
nine mechanistic upgrades, three
reaching critical target tissues may The concordance data set assem- showed limited evidence in humans,
be quite different, depending on the bled by Grosse et al. (Annex 1) and and six had inadequate evidence
route of administration. Differences summarized in Table 21.1 includes in humans or no epidemiological
in routes of exposure between ani-
111 distinct Group 1 agents iden- data were available, for example for
mals and humans could thus contrib-
tified in the IARC Monographs up B[a]P and 2,3,4,7,8-pentachlorodi-
ute to lack of concordance between
to and including Volume 109. Nine benzofuran (PeCDF).
tumour sites observed in animals
of these 111 agents were placed in Apart from the nine Group 1
and humans. However, because
Group 1 in the absence of sufficient mechanistic upgrades for which no
data on cancer outcomes for a giv-
evidence of carcinogenicity in hu- human tumour sites were identified,
en route of exposure are not avail-
mans (Table 21.4). These determi- there are four other agents for which
able across the entire set of Group 1
nations were made on the basis of the same is true (Table 21.5): ion-
agents, a systematic evaluation of
mechanistic upgrades according to izing radiation (all types), internalized
concordance for specific exposure
the evaluation criteria outlined in the radionuclides that emit α-particles,
routes is not possible.
Preamble to the IARC Monographs internalized radionuclides that emit
Species-specific tumour site (IARC, 2006). For example, benzo[a] β-particles, and ultraviolet (UV) ra-
profiles pyrene (B[a]P) was placed in Group 1 diation (bandwidth 100–400 nm, en-
Before the concordance analyses on the basis of epidemiological data compassing UVC, UVB, and UVA).
were conducted, the organ distribu- on exposure to mixtures of polycy- These were generic evaluations
tion was examined of the tumours clic aromatic hydrocarbons (PAHs) across a range of agents falling in
caused by the 111 distinct Group 1 containing B[a]P that provided suf- these categories. In addition, no hu-
carcinogens identified by IARC to ficient evidence for cancer of the man tumour site was specified for
date, both in humans and in animal lung or skin in humans, coupled with the agents areca nut and ethanol in

220
alcoholic beverages, because no ep- judged as providing inadequate evi- which was found difficult to interpret,
idemiological data were available for dence of carcinogenicity in animals. whereas in another study busulfan,
areca nut alone or for ethanol in alco- The available studies with wood dust when given to rats during gestation,
holic beverages alone (see Annex 1, originally considered in Volume 62 affected the incidence of uterine ad-
by Grosse et al.). (IARC, 1995) did not show signifi- enocarcinomas in the offspring upon
No animal tumour sites were cant carcinogenic or co-carcinogen- intrauterine treatment with N-ethyl-
identified for 38 of the 111 agents ic potential of beech wood dust, but N′-nitro-N-nitrosoguanidine (IARC,
considered here (Table 21.6). These these studies were subject to several 2012e). As a second example, sulfur
included 20 agents with inadequate limitations as well as inadequacies in mustard significantly increased the
evidence in animals: seven agents data reporting. Upon re-evaluation of incidence of lung tumours (not oth-
representing occupational expo- wood dust in Volume 100C (IARC, erwise specified) in mice after ex-
sures that would be difficult to rep- 2012a), it was concluded that most of posure by inhalation for 15 minutes,
licate in the laboratory; two pharma- the studies conducted with wood dust and of pulmonary tumours (not oth-
ceutical agents used in combination (nearly all with beech wood dust) had erwise specified) after intravenous
for which no animal data were avail- small numbers of animals or were of injection; a significant increase in the
able on the mixture; seven biologi- short duration, thus providing inade- incidence of mammary tumours was
cal agents (all viruses) for which the quate evidence of carcinogenicity in seen after subcutaneous injection
selection of an appropriate animal animals. These considerations sug- of sulfur mustard in rats, relative to
model was problematic; two agents, gest that neither etoposide nor wood an external control group, whereas
etoposide and wood dust, for which dust have been subject to adequate forestomach tumours were numer-
the available animal tests were con- animal testing, therefore precluding ically, but not significantly, elevated
sidered inadequate; and two agents, a determination of their carcinogenic in rats treated by oral gavage (IARC,
treosulfan and leather dust, for potential in animals. 2012c). The exposure by subcutane-
which no animal data were available. Ten agents, including six pharma- ous and intravascular injection was
Although the two agents that lack ceutical products (busulfan, chlor- considered to be of limited relevance
any animal test data – treosulfan and naphazine, cyclosporine; combined to the most common human routes of
leather dust – clearly do not permit estrogen–progestogen menopausal exposure. Although not meeting the
an evaluation of concordance be- therapy, 1-(2-chloroethyl)-3-(4-meth- stringent criterion for sufficient evi-
tween animals and humans, the two ylcyclohexyl)-1-nitrosourea [methyl- dence of carcinogenicity in animals,
agents for which inadequate animal CCNU], and analgesic mixtures con- the limited evidence provided by

CHAPTER 21
data were available – etoposide and taining phenacetin), three biological busulfan, as well as by the other six

PART 3
wood dust – warrant some further agents (infections with Clonorchis chemicals with only limited evidence
discussion to distinguish between sinensis, Opisthorchis viverrini, and of carcinogenicity in animals, does
the case in which well-conducted Schistosoma haematobium), and suggest that these agents have the
animal studies have failed to demon- one chemical agent (sulfur mus- potential to cause cancer in animals.
strate carcinogenicity and the case tard), provided limited, but not suf- No tumour sites were specified
in which the animal data are largely ficient, evidence of carcinogenicity for eight agents demonstrating suf-
uninformative because of inadequate in animals. As mentioned above, ficient evidence of carcinogenicity
testing: Volume 76 (IARC, 2000) tumour sites are not specified in the in animals, because reproducible
and Volume 100A (IARC, 2012e) of IARC Monographs for agents that results were unavailable in two or
the IARC Monographs noted that demonstrate only limited evidence in more studies of adequate design in
etoposide was tested in only one animals. the same species for any of these
experiment with wild-type and het- The reasons that these 10 agents agents. Although melphalan showed
erozygous neurofibromatosis type 1 were judged as providing only limited evidence of a statistically significant
(Nf1) knockout mice that were treat- evidence of carcinogenicity in ani- increase in the incidence of tumours
ed by gastric intubation for 6 weeks mals varied. For example, treatment of the forestomach, skin, and lung
with etoposide at 100 mg/kg body with busulfan resulted in a significant in mice, as well as lymphosarcoma,
weight/week (Mahgoub et al., 1999). increase in the incidence of thymic these results were not replicated in
This single short-duration study was and ovarian tumours in BALB/c mice, a second, independent study (IARC,

Part 3 • Chapter 21. Analysis of tumour site concordance 221


Table 21.4. Agents placed in Group 1 on the basis of mechanistic upgradesa

222
Agent Level of evidence Human tumour Basis for mechanistic upgrade
in humans/ site
animals

Aristolochic acid Limited/ Not specified Herbal remedies containing aristolochic acid provide sufficient evidence for
Sufficient upper urinary tract cancer in humans; genotoxic mechanistic data

Benzo[a]pyrene (B[a]P) [No epidemio- Not specified PAH mixtures containing B[a]P provide sufficient evidence for lung or skin
logical data]/ cancer in humans; extensive mechanistic data on B[a]P linking animal and
Sufficient human biology

Dyes metabolized to benzidine Inadequate/ Not specified Benzidine provides sufficient evidence of being a human bladder
Sufficient carcinogen

Ethylene oxide Limited/ Not specified Limited evidence for non-Hodgkin lymphoma, breast cancer in humans;
Sufficient genotoxic mechanistic data

Etoposide Limited/ Not specified Limited evidence of acute myeloid leukaemia in humans, with distinctive
Inadequate chromosomal translocations

4,4′-Methylenebis(2-chloroaniline) (MOCA) Inadequate/ Not specified Bladder cancer expected in humans, based on mechanistic data and
Sufficient human case report

Neutron radiation Inadequate/ Not specified Biophysics of radiation damage induction similar across different types of
Sufficient radiation

N′-Nitrosonornicotine (NNN) and Inadequate/ Not specified Target sites correspond to those of smokeless tobacco; mechanistic data
4-(Methylnitrosamino)-1-(3-pyridyl)- Sufficient on tobacco smoke
1-butanone (NNK)

2,3,4,7,8-Pentachlorodibenzofuran [No epidemio- Not specified Sufficient evidence in experimental animals combined with strong
(PeCDF) logical data]/ mechanistic support for receptor-mediated mechanism, with biological
Sufficient activity identical to that of 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD)
for every mechanistic step

PAH, polycyclic aromatic hydrocarbon.


a Although dioxin-like PCBs evaluated in Volume 107 were also upgraded to Group 1 on the basis of support for receptor-mediated mechanisms and analogies with TCDD (IARC, 2016b),

dioxin-like PCBs have been subsumed within the broader category of PCBs for the purposes of the present analysis of 111 distinct Group 1 agents, and are therefore not included in this
table.
Table 21.5. Group 1 agents with no human tumour sites specified (15 agents)

Nature of evidence in humans Volume: Agent(s)


(number of agents)

Mechanistic upgrades

Mechanistic upgrade with no human tumour Volume 100A: Aristolochic acid; Etoposide. Volume 100D:
site specified (9 agents) Neutron radiation. Volume 100E: N′-Nitrosonornicotine (NNN) and
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Volume
100F: Benzo[a]pyrene (B[a]P); Dyes metabolized to benzidine;
Ethylene oxide; 4,4′-Methylenebis(2-chloroaniline) (MOCA);
2,3,4,7,8-Pentachlorodibenzofuran (PeCDF)

Generic evaluations

Generic evaluation, of all types of ionizing Volume 100D: Ionizing radiation (all types); Internalized radionuclides
radiation; internalized radionuclides that emit that emit α-particles; Internalized radionuclides that emit β-particles; UV
α-particles; internalized radionuclides that radiation (bandwidth 100–400 nm, encompassing UVC, UVB, and UVA)
emit β-particles; and the UV region (100–
400 nm) of the electromagnetic spectrum
(4 agents)

Absence of epidemiological data on the agent alone

No epidemiological data available for agent Volume 100E: Areca nut; Ethanol in alcoholic beverages
alone (2 agents)

2012c). In rats, melphalan also pro- the PAHs detected in air samples evidence of carcinogenic activity of
duced mammary gland tumours and from such plants, and previous- PeCDF, based on increased inci-
peritoneal sarcoma, but these find- ly evaluated in Volume 92 (IARC, dences of hepatocellular adenoma
ings were again not replicated in in- 2010). Had the animal evidence for and cholangiocarcinoma of the liver
dependent studies. Phosphorous-32 the agents mentioned above been el- and gingival squamous cell carcino-
caused leukaemia in mice and os- igible for inclusion in the tumour site ma of the oral mucosa. The occur-

CHAPTER 21
teogenic sarcomas in rats in single concordance database, additional rence of cystic keratinizing epithe-

PART 3
studies. Similarly, acetaldehyde in concordant results would have been lioma of the lung, neoplasms of the
drinking-water induced pancreatic noted, including concordance be- pancreatic acinus, and carcinoma
adenomas, combined lymphomas tween lymphoid and haematopoietic of the uterus may have been related
and leukaemias, uterine and mam- tissues in mice and humans for both to administration of PeCDF. There
mary gland adenocarcinomas, and melphalan and phosphorous-32, and were also three rat studies of PeCDF
head osteosarcomas in rats, but concordance between tumours of in combination with N-methyl-N′-
without replication. Betel quid with to- the upper aerodigestive tract in ham- nitro-N-nitrosoguanidine (MNNG)
bacco produced malignant forestom- sters and humans for betel quid with and N-nitrosodiethylamine (NDEA),
ach and cheek pouch tumours in a tobacco. where increased tumour multiplicity
single study in hamsters. Sufficient Although PeCDF provided suffi- was observed in each case (IARC,
evidence of carcinogenicity in ani- cient evidence of carcinogenicity in 2012c). These observations led to
mals of aluminium refining was based animals, no animal site was iden- the conclusion that there is sufficient
on a single limited skin application tified. PeCDF was tested by the evidence for the carcinogenicity of
study in mice with PAH-containing United States National Toxicology PeCDF in animals, although there
particulates from aluminium pro- Program in a 2-year animal bioassay is no specific organ site that can be
duction plants, in conjunction with (female rats only) with exposure by designated as responsible for this
sufficient evidence of carcinogenicity oral gavage (National Toxicology sufficient evidence. Because of the
in experimental animals for many of Program, 2006). There was some absence of a specific tumour site in

Part 3 • Chapter 21. Analysis of tumour site concordance 223


Table 21.6. Group 1 agents with no animal tumour sites specified (38 agents)

224
Nature of evidence in animals Volume: Agent(s)
(number of agents)

Agents with inadequate evidence in animals

Occupational exposures are complex Volume 100F: Acid mists, strong inorganic; Auramine production; Iron and steel founding, occupational exposure during; Isopropyl
and probably could not be reliably alcohol manufacture using strong acids; Magenta production; Painter, occupational exposure as a; Rubber manufacturing industry,
replicated in the laboratory (7 agents) occupational exposures in the.

Used in combination; no animal data Volume 100A: Etoposide in combination with cisplatin and bleomycin; MOPP.
available on mixture (2 agents)

Use of animal models problematic Volume 100B: Infection with Epstein–Barr virus; Hepatitis B virus; Hepatitis C virus; Human immunodeficiency virus type 1; Human
because of species specificity and papillomaviruses; Human T-cell lymphotropic virus type 1; Kaposi sarcoma-associated herpesvirus.
other limitations (7 agents)

Animal tests conducted but considered Volume 100A: Etoposide. Volume 100C: Wood dust.
inadequate (2 agents)

No animal data available (2 agents) Volume 100A: Treosulfan. Volume 100C: Leather dust.

Agents with limited evidence in animals

Evidence of carcinogenicity in animals Volume 100A: Busulfan; Chlornaphazine; Ciclosporin; Estrogen–progestogen menopausal therapy (combined); 1-(2-Chloroethyl)-
judged as limited for various reasons 3-(4-methylcyclohexyl)-1-nitrosourea (Methyl-CCNU); Phenacetin, analgesic mixtures containing. Volume 100B: Clonorchis
(10 agents) sinensis (infection with); Opisthorchis viverrini (infection with); Schistosoma haematobium (infection with). Volume 100F: Sulfur
mustard.

Agents with sufficient evidence in animals

Sufficient evidence in animals, but no Volume 100A: Melphalan. Volume 100D: Phosphorus-32, as phosphate. Volume 100E: Acetaldehyde associated
tumour sites specifieda (8 agents) with the consumption of alcoholic beverages; Betel quid with tobacco. Volume 100F: Aluminium production;
2,3,4,7,8-pentachlorodibenzofuran (PeCDF); Volume 109: Outdoor air pollution; Particulate matter in outdoor air pollution.
aSufficient evidence in experimental animals, but no organ sites identified due to the absence of at least two studies of adequate design and quality showing tumours at the same organ
site with a similar histological origin in the same species.
animals, PeCDF is not included in or limited evidence (nine agents) of pharmaceuticals account for half (9
the concordance analyses. carcinogenicity in animals. This ob- of 18) of the agents that cause tu-
A component of four Group 1 servation provides support for the mours in haematopoietic tissues.
agents, but not the agents them- use of animal data in human cancer The number of agents that induce
selves, demonstrated sufficient risk assessment. tumours in one or more animal spe-
evidence of carcinogenicity in an- To further explore the correspon- cies at each of the 39 tumour sites is
imals. These are: fission products dence between sites where tumours shown in Fig. 21.2 by type of agent.
including strontium-90, where stron- are seen in animals and humans As in humans, lung tumours are the
tium-90 demonstrated sufficient ev- among the 111 distinct Group 1 most common tumour in animals,
idence of carcinogenicity in animals agents considered here, descriptive with 29 of the 111 known human car-
(IARC, 2012f); haematite mining with statistics are presented on tumour cinogens inducing lesions at this site,
exposure to radon (underground), site profiles by species, followed by mostly from the categories of chem-
where radon demonstrated sufficient an evaluation of concordance be- ical agents and related occupations
evidence of carcinogenicity in ani- tween tumour sites seen in animals (10 agents), arsenic, metals, fibres,
mals (IARC, 2012f); acetaldehyde and humans. Results are presented and dusts (7 agents), and radiation
associated with consumption of al- first for the 39 tumour sites included (7 agents). After the lung, the ani-
coholic beverages, where acetal- in the anatomically based tumour mal sites associated with the largest
dehyde demonstrated sufficient ev- nomenclature system seen in either number of carcinogenic agents are
idence of carcinogenicity in animals animals or humans, followed by the the liver parenchyma and bile ducts
(IARC, 2012d); and occupational data for the 14 organ and tissue (19 agents), the skin and adnexae (18
exposures during aluminium produc- systems. agents), lymphoid tissue (14 agents),
tion, where airborne particulate poly- the breast (12 agents), and soft con-
Tumour site profiles by
nuclear organic matter from alumin- nective tissue (11 agents). Separate
species
ium production plants demonstrated tumour profiles are shown for
sufficient evidence of carcinogenicity The number of agents that induce tu- agents that cause tumours in mice
in animals (IARC, 2012c). Although mours in humans at each of the 39 (48 agents) and rats (49 agents) in
this animal evidence is consistent tumour sites is shown in Fig. 21.1 by Fig. 21.3 and Fig. 21.4, respectively.
with the sufficient evidence for the type of agent (pharmaceuticals; bio- In rodents (mice and rats combined),
carcinogenicity of these four agents logical agents; arsenic, metals, fibres, the lung is the site associated with
in humans, the animal evidence rep- and dusts; radiation; personal habits the largest number of carcinogens.

CHAPTER 21
resents only a component of these and indoor combustions; and chemi-
Organ and tissue system

PART 3
agents. cal agents and related occupations).
profiles by species
Excluding the 20 agents in Lung tumours are the most common
Table 21.5 that lack appropriate an- tumour seen in humans, with 28 of The number of agents that induce
imal data, i.e. seven occupational the 111 known human carcinogens tumours in humans in each of the 14
exposures not reproducible in the inducing lesions at this site; of these, aggregate organ and tissue systems
laboratory, two agents used in com- 13 are associated with exposure to is shown in Fig. 21.5 by type of agent.
bination with no animal data avail- chemical agents and related occupa- Tumours of the respiratory system
able on the mixture, seven agents tions and seven are in the category are caused by 31 of the 111 human
where the use of animal models of arsenic, metals, fibres, and dusts. carcinogens, mostly from the cate-
is problematic because of species Tumours of the haematopoietic tis- gories of chemical agents and relat-
specificity or other limitations, and sues are associated with exposure to ed occupations (14 agents), arsenic,
four agents for which animal tests 18 agents, urothelial tumours with 18 metals, fibres, and dusts (7 agents),
were inadequate (two agents) or un- agents, skin tumours with 12 agents, and personal habits and indoor com-
available (two agents), all 91 distinct and liver and bile duct tumours with bustions (5 agents). After the res-
Group 1 agents identified by IARC 11 agents. The category chemical piratory system, the organ and tissue
up to and including Volume 109 of agents and related occupations ac- systems associated with the largest
the IARC Monographs provided ei- counts for half (9 of 18) of the agents number of agents are lymphoid and
ther sufficient evidence (82 agents) that cause urothelial tumours, and haematopoietic tissues (26 agents),

Part 3 • Chapter 21. Analysis of tumour site concordance 225


Fig. 21.1. Number of agents that induce tumours in humans in each of 39 tumour sites, by type of agent.

the urothelium (18 agents), and the the digestive organs are caused by chemical agents and related occupa-
upper aerodigestive tract (16 agents). 19 agents, mostly from the catego- tions (5 agents).
Pharmaceuticals are the largest ries of chemical agents and related
Qualitative assessment of
group of agents associated with tu- occupations (12 agents) and radi-
concordance
mours of the lymphoid and haemato- ation (4 agents). Skin tumours are
poietic tissues (11 of 26 agents), and caused by 18 agents, mostly from Of the 111 distinct Group 1 agents
chemical agents and related occupa- the category of chemical agents identified up to and including Volume
tions are most often associated with and related occupations (12 agents). 109 (see Table 21.1), for 60 agents
tumours of the urothelium (9 of 18 Connective tissue tumours are as- both a human tumour site and an ani-
agents). Personal habits and indoor sociated with 17 agents, mostly from mal tumour site have been identified,
combustions are most commonly as- the categories of radiation (8 agents) 15 agents had no human tumour site
specified (Table 21.5), and 38 agents
sociated with tumours of the upper and chemical agents and related oc-
had no animal tumour site identified
aerodigestive tract (7 of 16 agents). cupations (5 agents).
(Table 21.6). Because two agents –
The number of agents that induce In mice (Fig. 21.7), tumours of
etoposide and PeCDF – have neither
tumours in one or more animal spe- the skin and connective tissues are
a human nor an animal tumour site
cies at each of the 14 organ and tis- caused by 29 agents, consisting
specified, there are 111 − 15 − 38 +
sue systems is given in Fig. 21.6 by mostly of tumours caused by chem-
2 = 60 agents with at least one tu-
type of agent. Tumours of the res- ical agents and related occupa- mour site identified in both humans
piratory system are caused by 29 of tions (14) and radiation (10). In rats and animals. These 60 agents have
the 111 agents, mostly from the cate- (Fig. 21.8), tumours of the respirato- been used to evaluate concordance
gories of chemical agents and relat- ry system are caused by 19 agents, between tumour sites seen in ani-
ed occupations (10 agents), arsenic, including those in the categories of mals and humans, because at least
metals, fibres, and dusts (7 agents), arsenic, metals, fibres, and dusts one tumour site has been identified
and radiation (7 agents). Tumours of (6 agents), radiation (6 agents), and in both.

226
Fig. 21.2. Number of agents that induce tumours in animals in each of 39 tumour sites, by type of agent.

Fig. 21.3. Number of agents that induce tumours in mice in each of 39 tumour sites, by type of agent.

CHAPTER 21
PART 3

Part 3 • Chapter 21. Analysis of tumour site concordance 227


Fig. 21.4. Number of agents that induce tumours in rats in each of 39 tumour sites, by type of agent.

The overlap between human Nine agents cause tumours of the humans or animals, as in the phar-
and animal tumour sites targeted by upper aerodigestive tract in humans, ynx, tongue, and salivary gland.
these 60 agents is summarized in and nine agents cause tumours in The lung is the most common site
Table 21.7 by organ and tissue system this organ and tissue system in ani- at which tumours are observed, with
and tumour site. The category “other mals; four agents cause tumours in 62% overlap among the 26 agents
groupings” of tumours – which com- this system in both humans and ani- that cause lung tumours in humans
prises “all cancers combined”, “all mals. There are 9 + 9 − 4 = 14 distinct or animals. Among the 10 agents
solid cancers”, and “exocrine glands agents that cause tumours in this that cause tumours in the urotheli-
not otherwise specified” – was creat- system in either humans or animals, um (renal pelvis, ureter, or bladder),
ed to accommodate tumour sites re- for an overlap of 4 of 14, or 29%. there is 70% overlap between agents
ported in the IARC Monographs that Within the upper aerodigestive tract, that cause tumours in humans or
did not fall into any of the other cate- there are three agents that cause tu- animals.
gories in Table 21.2. The only human mours in the nasal cavity and para- Because results for individual tu-
site identified for 2,3,7,8-tetrachlo- nasal sinuses in humans and three mour sites are often based on small
rodibenzo-para-dioxin (TCDD) is “all agents that cause tumours at this numbers, emphasis is placed on in-
cancers combined”; fission products site in animals, with no overlap. Of terpretation of results at the organ
including strontium-90 are associat- the three agents that induce tumours and tissue system level, where the
ed with “all solid cancers” in humans, in the nasopharynx, one agent sample size is generally larger than
but also with tumours in haemato- causes tumours in both humans and for individual tumour sites within
poietic tissue. Because this category animals, for an overlap of 33%. In the organ and tissue systems. Overlap
lacks biological cohesiveness, “other oral cavity, overlap is 25%. Overlap varies among the organ and tissue
groupings” of tumours were not con- is not calculated when there are no systems, ranging from 20% (based
sidered in the concordance analysis. agents that cause tumours in either on 10 agents) in the digestive tract

228
Fig. 21.5. Number of agents that induce tumours in humans in each of 14 organ and tissue systems, by type of
agent.

CHAPTER 21
PART 3
to 100% in the mesothelium. Overall, clusion in the concordance analysis in this system in animals (and not in
high overlap is seen for some or- caused tumours at this site. humans), and four agents cause tu-
gan and tissue systems but not for The results in Table 21.7 are de- mours in this system in both humans
others. Some caution is needed in picted in graphical form in Fig. 21.9. and animals, for an overlap of 29%.
interpreting concordance at sites As noted above, of the 14 Group 1 Of the 27 agents that cause tumours
where the sample size is particularly agents that cause tumours of the of the respiratory system in either
small: although 100% concordance upper aerodigestive tract in either humans or animals, 21 agents cause
was noted for agents that cause humans or animals, nine agents respiratory tumours in humans, 22
tumours of the mesothelium, only cause tumours in the upper aerodi- agents cause respiratory tumours
two Group 1 agents – asbestos and gestive tract in humans (and not in in animals, and 16 agents cause
erionite – meeting the criteria for in- animals), nine agents cause tumours respiratory tumours in both humans

Part 3 • Chapter 21. Analysis of tumour site concordance 229


Fig. 21.6. Number of agents that induce tumours in animals in each of 14 organ and tissue systems, by type of
agent.

and animals, for an overlap of 59%. between tumour sites seen in hu- ysis, 40, 38, 8, 7, and 3 agents cause
Although they present the same data mans and all animal species test- tumours in mice, rats, hamsters,
as shown in Table 21.7, the graphical ed, reflecting the interest in evalu- dogs, and monkeys, respectively.
representations of these results in ating the extent to which tumours Therefore, including only mice and
Fig. 21.9 for all organ and tissue sys- caused by Group 1 agents occur in rats in the analysis yielded results
tems also illustrate the large varia- similar organ and tissue systems in similar to those in Table 21.7 (see
tion in sample size among the organ humans and in animals. The animal details in Supplemental Material
and tissue systems; the area of the data included in this analysis are II [online only; available from:
circles is proportional to sample size. dominated by results obtained in http://publications.iarc.fr/578], where
The results presented in Ta- studies with rats and mice: of the 60 Supplemental Table 6 presents re-
ble 21.7 are based on concordance Group 1 agents included in the anal- sults for all animal species tested

230
Fig. 21.7. Number of agents that induce tumours in mice in each of 14 organ and tissue systems, by type of agent.

CHAPTER 21
PART 3
and Supplemental Table 7 presents As detailed in Supplemental Material tumours in that system in either
results for mice and rats only). II (online only; available from: http:// humans or animals, providing an
Fig. 21.10 shows the percentage publications.iarc.fr/578), it is impor- overall measure of overlap between
of Group 1 agents that cause tu- tant to note that the measures of animal and human carcinogens in
mours in specific organ and tissue concordance presented in Fig. 21.10 a specific organ and tissue system.
systems in humans that are also differ from those in Table 21.7. The The percentage overlap in panel A of
associated with tumours in animals percentage overlap in Table 21.7 Fig. 21.10 provides a measure of the
(panel A), as well as the percent- (and Fig. 21.9) reflects the number overlap between agents that cause
age of agents that cause tumours in of agents that cause tumours in a tumours in a specific organ and
specific organ and tissue systems specific organ and tissue system in tissue system in animals with agents
in animals that are also associated both humans and animals, relative that cause tumours in that system in
with tumours in humans (panel B). to the number of agents that cause humans. Conversely, the percentage

Part 3 • Chapter 21. Analysis of tumour site concordance 231


Fig. 21.8. Number of agents that induce tumours in rats in each of 14 organ and tissue systems, by type of agent.

overlap in panel B of Fig. 21.10 pro- panel A, where human carcinogens mours in those organ and tissue sys-
vides a measure of the overlap be- constitute the reference set against tems in animals. Overlap of at least
tween agents that cause tumours in which animal carcinogens are com- 50% is observed for all other organ
a specific organ and tissue system pared, will differ from those in panel and tissue systems, with the excep-
in humans with agents that cause B, where animal carcinogens consti- tion of the upper aerodigestive tract
tumours in that system in animals. tute the reference set for comparison (44%) and the digestive tract (33%).
Note that unless the numbers of with human carcinogens. Conversely, there is less overlap
agents that cause tumours in hu- As indicated in panel A of between agents that cause tumours
mans and animals in a specific organ Fig. 21.10, all agents (100%) that in specific organ and tissue systems
and tissue system are the same (as cause tumours of the mesothelium, in animals with results in humans
is the case for tumours of the upper endocrine system, and connective (Fig. 21.10, panel B), possibly re-
aerodigestive tract), the results in tissues in humans also cause tu- flecting the larger number of studies

232
Table 21.7. Concordance between tumours seen in humans and animals for 60 Group 1 agents by organ and tissue
system and tumour site

Number of agents Overlapb (%)


Organ and tissue systema
Tumour site a
Humans Animals Both

Upper aerodigestive tract 9 9 4 29

Nasal cavity and paranasal sinuses 3 3 0 0

Nasopharynx 3 1 1 33

Oral cavity 4 6 2 25

Pharynx 2 0 0 N/A

Tongue 0 1 0 N/A

Salivary gland 1 0 0 N/A

Respiratory system 21 22 16 59

Larynx 3 1 1 33

Lung 20 22 16 62

Mesothelium 2 2 2 100

Mesothelium 2 2 2 100

Digestive tract 6 6 2 20

Oesophagus 5 0 0 N/A

Stomach 3 5 1 14

Intestine (including colon and rectum) 3 1 0 0

Digestive organs 8 14 4 22

Liver parenchyma and bile ducts 7 14 4 24

CHAPTER 21
PART 3
Pancreas NOS 2 0 0 N/A

Gall bladder 1 0 0 N/A

Nervous system and eye 2 0 0 N/A

Brain and spinal cord (CNS) 1 0 0 N/A

Eye 1 0 0 N/A

Endocrine system 2 3 2 67

Thyroid, follicular epithelium 2 2 2 100

Adrenal gland (medulla, cortex, NOS) 0 1 0 N/A

Pituitary gland 0 1 0 N/A

Kidney 3 5 2 33

Kidney (renal cortex, renal medulla, kidney NOS) 3 5 2 33

Urothelium 10 7 7 70

Urothelium (renal pelvis, ureter, or bladder) 10 7 7 70

Part 3 • Chapter 21. Analysis of tumour site concordance 233


Table 21.7. Concordance between tumours seen in humans and animals for 60 Group 1 agents by organ and tissue
system and tumour site (continued)

Number of agents Overlapb (%)


Organ and tissue systema
Tumour site a
Humans Animals Both

Lymphoid and haematopoietic tissues 12 10 7 47

Haematopoietic tissues 10 2 2 20

Lymphoid tissue 2 10 1 9

Skin 11 16 7 35

Skin and adnexae 9 16 6 32

Cutaneous melanocytes 3 0 0 N/A

Connective tissues 6 14 6 43

Soft connective tissue 0 9 0 N/A

Blood vasculature (endothelium) 1 0 0 N/A

Hard connective tissue (bone, cartilage) 5 5 4 67

Female breast, female reproductive organs, and


8 9 4 31
female reproductive tract

Breast 4 8 2 20

Ovary 3 1 0 0

Uterine cervix 3 2 1 25

Uterus 2 2 1 33

Vulva/vagina 1 0 0 N/A

Other groupings 2 4 0 0

All cancers combined 1 0 0 N/A

All solid cancers 1 0 0 N/A

Exocrine glands NOS 0 4 0 N/A

CNS, central nervous system; N/A, not applicable: assigned to sites/systems when overlap is not possible (positive data are available
in either humans or animals, but not in both); NOS, not otherwise specified.
a Systems/sites in the anatomically based tumour nomenclature system (see Table 21.2) that lack sufficient evidence in both humans

and animals not shown. For example, there were insufficient data on tumours of the male reproductive tract in both humans and
animals.
b Percentage overlap calculated as [N /(N + N − N )] × 100%, where N , N , and N denote the number of agents with sufficient
b h a b h a b
evidence of carcinogenicity in humans, animals, or both humans and animals, respectively.

234
Fig. 21.9. Concordance between tumour sites seen in humans and animals for 60 Group 1 agents by organ and
tissue system.

2 4 2 4
5 4 5 5 16 6

Upper aerodigestive tract


(14 agents) Respiratory system (27 agents) Mesothelium (2 agents) Digestive tract (10 agents)

4 4 10 2 2 1 2 3

Digestive organs (18 agents) Nervous system and eye (2 agents) Endocrine system (3 agents) Kidney (6 agents)

7 3 5 7 3 4 7 9 6 8

Lymphoid and haematopoietic


Urothelium (10 agents) tissues (15 agents) Skin (20 agents) Connective tissues (14 agents)

Legend
4 4 5
2 4
Humans only Animals only Both

Female breast and reproductive


organs/tract (13 agents) Other groupings (6 agents)

CHAPTER 21
conducted in animals compared with tissue system level only, because in at least one tumour site in both hu-

PART 3
humans, the broader spectrum of tis- results for individual tumour sites mans and animals, 52 (87%) cause
sues (potential tumour sites) exam- are too sparse to support meaningful tumours within at least one of the
ined in animal studies than in human comparisons. The human data are same organ and tissue systems in
studies, or the limitations associated presented in the column on the left, Table 21.8.
with the conduct of human studies at the animal data in the column on the To permit a more complete com-
environmental exposure levels. As is right, and the overlap in the middle parison between animal and human
the case with the concordance re- column. With this display, potential tumour sites, tumour sites with only
sults focusing on overall overlap, as relationships among agents that limited evidence in humans are in-
presented in Table 21.7, caution is cause tumours within the same or- cluded in Table 21.8 (in italics). For
needed in interpreting results where gan and tissue system can be exam- agents such as diethylstilbestrol (a
there are few agents for comparison ined. Overlap between human and synthetic non-steroidal estrogen
in Fig. 21.10 (both panels A and B). animal carcinogens acting within the that was widely prescribed in the
The 60 agents included in the same organ and tissue system can USA between the 1940s and the
present concordance analysis are also be examined both for individu- 1970s but is rarely used now), there
listed in Table 21.8. This table pre- al agents and for groups of agents. is difficulty in generating newer data
sents the tumour site data for hu- Of the 60 agents for which there is on human exposure. Because men
mans and animals at the organ and sufficient evidence of carcinogenicity exposed to diethylstilbestrol in utero

Part 3 • Chapter 21. Analysis of tumour site concordance 235


Fig. 21.10. Overlap between Group 1 agents with sufficient evidence of carcinogenicity in humans and animals that cause tumours in specific organ and tissue

236
systems. (A)  Overlap between animals and humans; the number of Group 1 agents that cause tumours in specific organ and tissue systems in humans is shown.
(B) Overlap between humans and animals; the number of Group 1 agents that cause tumours in specific organ and tissue systems in animals is shown.

B
Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in
animals in specific organ and tissue systemsa

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Upper aerodigestive tract (29% overlapd)

Chromium(VI) compounds (100C) Alcoholic beverages (100E) Chromium(VI) (100C)


Nickel compounds (100C) Salted fish, Chinese-style (100E) Alcoholic beverages (100E)
Radium-226 and decay products (100D) Tobacco, smokeless (100E) Salted fish, Chinese-style (100E)
X- and γ-radiation (100D) Formaldehyde (100F) Tobacco, smokeless (100E)
Radioiodines, including iodine-131(100D) Chromium(VI) compounds (100C) Formaldehyde (100F)
Betel quid without tobacco (100E) Benzene (100F)
Alcoholic beverages (100E) 2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F)
Salted fish, Chinese-style (100E) Polychlorinated biphenyls (100F)
Second-hand tobacco smoke (100E) Bis(chloromethyl)ether; Chloromethyl methyl ether (100F)
Tobacco, smokeless (100E)
Tobacco smoking (100E)
Formaldehyde (100F)

Part 3 • Chapter 21. Analysis of tumour site concordance


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Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in

238
animals in specific organ and tissue systemsa (continued)

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Respiratory system (59% overlap)

Arsenic and inorganic arsenic compounds (100C) Arsenic and inorganic arsenic compounds (100C) Cyclophosphamide (100A)
Asbestos (all forms, including actinolite, amosite, Asbestos (all forms, including actinolite, amosite, Arsenic and inorganic arsenic compounds (100C)
anthophyllite, chrysotile, crocidolite, and tremolite) anthophyllite, chrysotile, crocidolite, and tremolite) Asbestos (all forms, including actinolite, amosite,
(100C) (100C) anthophyllite, chrysotile, crocidolite, and tremolite) (100C)
Beryllium and beryllium compounds (100C) Beryllium and beryllium compounds (100C) Beryllium and beryllium compounds (100C)
Cadmium and cadmium compounds (100C) Cadmium and cadmium compounds (100C) Cadmium and cadmium compounds (100C)
Chromium(VI) compounds (100C) Chromium(VI) compounds (100C) Chromium(VI) compounds (100C)
Nickel compounds (100C) Nickel compounds (100C) Nickel compounds (100C)
Silica dust, crystalline, in the form of quartz or Silica dust, crystalline, in the form of quartz or Silica dust, crystalline, in the form of quartz or cristobalite
cristobalite (100C) cristobalite (100C) (100C)
Haematite mining with exposure to radon Haematite mining with exposure to radon Haematite mining with exposure to radon (underground)
(underground) (100D) (underground) (100D) (100D)
Plutonium-239 (100D) Plutonium-239 (100D) Plutonium-239 (100D)
Radon-222 and its decay products (100D) Radon-222 and its decay products (100D) Radon-222 and its decay products (100D)
X- and γ-radiation (100D) X- and γ-radiation (100D) X- and γ-radiation (100D)
Alcoholic beverages (100E) Coal, indoor emissions from household combustion Coal, indoor emissions from household combustion of (100E)
Coal, indoor emissions from household combustion of of (100E) Second-hand tobacco smoke (100E)
(100E) Second-hand tobacco smoke (100E) Tobacco smoking (100E)
Second-hand tobacco smoke (100E) Tobacco smoking (100E) Benzene (100F)
Tobacco smoking (100E) Coke production (100F) 1,3-Butadiene (100F)
Bis(chloromethyl)ether; Chloromethyl methyl ether Engine exhaust, diesel (100F) Coke production (100F)
(technical grade) (100F) 2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F) Vinyl chloride (100F)
Coal gasification (100F) Engine exhaust, diesel (100F*)
Coal-tar pitch (100F) 2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F*)
Coke production (100F) Trichloroethylene (100F*)
Soot (as found in occupational exposure of chimney
sweeps) (100F)
2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F)
Engine exhaust, diesel (100F)
Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in
animals in specific organ and tissue systemsa (continued)

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Mesothelium (100% overlap)

Asbestos (all forms, including actinolite, amosite, Asbestos (all forms, including actinolite, amosite, Asbestos (all forms, including actinolite, amosite,
anthophyllite, chrysotile, crocidolite, and tremolite) anthophyllite, chrysotile, crocidolite, and tremolite) anthophyllite, chrysotile, crocidolite, and tremolite) (100C)
(100C) (100C) Erionite (100C)
Erionite (100C) Erionite (100C)

Digestive tract (20% overlap)

Helicobacter pylori (infection with) (100B) Helicobacter pylori (infection with) (100B) Aristolochic acid, plants containing (100A)
X- and γ-radiation (100D) Betel quid without tobacco (100E) Helicobacter pylori (infection with) (100B)
Radioiodines, including iodine-131 (100D) Chromium(VI) compounds (100C)
Alcoholic beverages (100E) Betel quid without tobacco (100E)
Betel quid without tobacco (100E) Benzene (100F)
Salted fish, Chinese-style (100E) 1,3-Butadiene (100F)
Tobacco smoking (100E)
Tobacco, smokeless (100E)

Digestive organs (22% overlap)

Estrogen–progestogen oral contraceptives (combined) Arsenic and inorganic arsenic compounds (100C) Tamoxifen (100A)
(100A) Plutonium-239 (100D) Arsenic and inorganic arsenic compounds (100C)
Arsenic and inorganic arsenic compounds (100C) Thorium-232 (as Thorotrast) (100D) Thorium-232 (as Thorotrast) (100D)
Cadmium and cadmium compounds (100C) X- and γ-radiation (100D) Plutonium-239 (100D)
Thorium-232 (as Thorotrast) (100D) Aflatoxins (100F) X- and γ-radiation (100D)
Plutonium-239 (100D) Vinyl chloride (100F) Aflatoxins (100F)
X- and γ-radiation (100D) Trichloroethylene (100F*) 4-Aminobiphenyl (100F)
Alcoholic beverages (100E) Benzidine (100F)
Betel quid without tobacco (100E) 1,3-Butadiene (100F)
Tobacco smoking (100E) 2-Naphthylamine (100F)
Tobacco, smokeless (100E) 2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F)
Aflatoxins (100F) Vinyl chloride (100F)
Vinyl chloride (100F) Trichloroethylene (100F*)

Part 3 • Chapter 21. Analysis of tumour site concordance


Trichloroethylene (100F*) Polychlorinated biphenyls (100F)

239
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Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in

240
animals in specific organ and tissue systemsa (continued)

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Nervous system and eye (N/A)

UV-emitting tanning devices (100D)    


X- and γ-radiation (100D)
Solar radiation (100D)

Endocrine system (67% overlap)

Radioiodines, including iodine-131 (100D) Radioiodines, including iodine-131 (100D) Nickel compounds (100C)
X- and γ-radiation (100D) X- and γ-radiation (100D) Radioiodines, including iodine-131 (100D)
X- and γ-radiation (100D)

Kidney (33% overlap)

Arsenic and inorganic arsenic (100C) X- and γ-radiation (100D) Diethylstilbestrol (100A)
Cadmium and cadmium compounds (100C) Trichloroethylene (100F*) Estrogen-only menopausal therapy (100A)
X- and γ-radiation (100D) Phenacetin (100A)
Tobacco smoking (100E) X- and γ-radiation (100D)
Trichloroethylene (100F*) Trichloroethylene (100F*)

Urothelium (70% overlap)

Aristolochic acid, plants containing (100A) Aristolochic acid, plants containing (100A) Aristolochic acid, plants containing (100A)
Cyclophosphamide (100A) Cyclophosphamide (100A) Cyclophosphamide (100A)
Phenacetin (100A) Phenacetin (100A) Phenacetin (100A)
Arsenic and inorganic arsenic compounds (100C) Arsenic and inorganic arsenic compounds (100C) Arsenic and inorganic arsenic compounds (100C)
X- and γ-radiation (100D) 4-Aminobiphenyl (100F) 2-Naphthylamine (100F)
Tobacco smoking (100E) 2-Naphthylamine (100F) 4-Aminobiphenyl (100F)
Coal-tar pitch (100F) ortho-Toluidine (100F) ortho-Toluidine (100F)
Soot (as found in occupational exposure of chimney
sweeps) (100F)
4-Aminobiphenyl (100F)
Benzidine (100F)
2-Naphthylamine (100F)
ortho-Toluidine (100F)
Engine exhaust, diesel (100F*)
Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in
animals in specific organ and tissue systemsa (continued)

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Lymphoid and haematopoietic tissues (47% overlap)

Azathioprine (100A) Azathioprine (100A) Azathioprine (100A)


Chlorambucil (100A) Chlorambucil (100A) Chlorambucil (100A)
Cyclophosphamide (100A) Cyclophosphamide (100A) Cyclophosphamide (100A)
Thiotepa (100A) Thiotepa (100A) Estrogen-only menopausal therapy (100A)
Helicobacter pylori (infection with) (100B) X- and γ-radiation (100D) Thiotepa (100A)
Fission products including strontium-90 (100D) Benzene (100F) Silica dust, crystalline, in the form of quartz or cristobalite
Thorium-232 (as Thorotrast) (100D) 1,3-Butadiene (100F) (100C)
X- and γ-radiation (100D) 2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F) X- and γ-radiation (100D)
Radioiodines, including iodine-131(100D) Ethylene oxide (100F)
Radon-222 and its decay products (100D) Benzene (100F)
Tobacco smoking (100E) 1,3-Butadiene (100F)
Ethylene oxide (100F) 2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F)
Benzene (100F)
1,3-Butadiene (100F)
Formaldehyde (100F)
Trichloroethylene (100F*)
2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F)
Polychlorinated biphenyls (100F*)

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PART 3
CHAPTER 21
Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in

242
animals in specific organ and tissue systemsa (continued)

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Skin (35% overlap)

Azathioprine (100A) Methoxsalen in combination with UVA (100A) Methoxsalen in combination with UVA (100A)
Methoxsalen in combination with UVA (100A) Solar radiation (100D) Solar radiation (100D)
Arsenic and inorganic arsenic compounds (100C) UV-emitting tanning devices (100D) UV-emitting tanning devices (100D)
Solar radiation (100D) Coal-tar distillation (100F) Coal, indoor emissions from household combustion of (100E)
UV-emitting tanning devices (100D) Mineral oils, untreated or mildly treated (100F) Tobacco smoking (100E)
X- and γ-radiation (100D) Shale oils (100F) Benzene (100F)
Coal-tar distillation (100F) Soot (as found in occupational exposure of chimney Bis(chloromethyl)ether; Chloromethyl methyl ether
Mineral oils, untreated or mildly treated (100F) sweeps) (100F) (technical grade) (100F)
Shale oils (100F) Coal gasification (100F)
Soot (as found in occupational exposure of chimney Coal-tar distillation (100F)
sweeps) (100F) Coal-tar pitch (100F)
Polychlorinated biphenyls (100F*) Coke production (100F)
Mineral oils, untreated or mildly treated (100F)
Shale oils (100F)
Soot (as found in occupational exposure of chimney sweeps)
(100F)
2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F)
ortho-Toluidine (100F)
Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in
animals in specific organ and tissue systemsa (continued)

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Connective tissues (43% overlap)

Plutonium-239 (100D) Plutonium-239 (100D) Cadmium and cadmium compounds (100C)


Radium-224 and its decay products (100D) Radium-224 and its decay products (100D) Chromium(VI) compounds (100C)
Radium-226 and its decay products (100D) Radium-226 and its decay products (100D) Nickel compounds (100C)
Radium-228 and its decay products (100D) Radium-228 and its decay products (100D) Fission products including strontium-90 (100D)
X- and γ-radiation (100D) X- and γ-radiation (100D) Plutonium-239 (100D)
Radioiodines, including iodine-131 (100D) Vinyl chloride (100F) Radium-224 and its decay products (100D)
Vinyl chloride (100F) Radium-226 and its decay products (100D)
2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F) Radium-228 and its decay products (100D)
X- and γ-radiation (100D)
4-Aminobiphenyl (100F)
Bis(chloromethyl)ether; Chloromethyl methyl ether
(technical grade) (100F)
1,3-Butadiene (100F)
ortho-Toluidine (100F)
Vinyl chloride (100F)

Female breast, female reproductive organs, and female reproductive tract (31% overlap)

Diethylstilbestrol (100A) Diethylstilbestrol (100A) Cyclophosphamide (100A)


Estrogen-only menopausal therapy (100A) Estrogen-only menopausal therapy (100A) Diethylstilbestrol (100A)
Estrogen–progestogen oral contraceptives (combined) Estrogen–progestogen oral contraceptives Estrogen-only menopausal therapy (100A)
(100A) (combined) (100A) Estrogen–progestogen oral contraceptives (combined)
Tamoxifen (100A) X- and γ-radiation (100D) (100A)
Asbestos (all forms, including actinolite, amosite, X- and γ-radiation (100D)
anthophyllite, chrysotile, crocidolite, and tremolite) Benzene (100F)
(100C) Benzidine (100F)
X- and γ-radiation (100D) 1,3-Butadiene (100F)
Alcoholic beverages (100E) Vinyl chloride (100F)
Tobacco smoking (100E)
Ethylene oxide (100F)
Polychlorinated biphenyls (100F*)

Part 3 • Chapter 21. Analysis of tumour site concordance


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PART 3
CHAPTER 21
Table 21.8. Comparison of 60 Group 1 agents with sufficient or limited evidence of carcinogenicity in humans and sufficient evidence of carcinogenicity in

244
animals in specific organ and tissue systemsa (continued)

Humansb Humans and animalsb Animalsb


Agent (Monographs Volumec) Agent (Monographs Volume) Agent (Monographs Volume)

Male reproductive organs including prostate and testes (overlap N/A)

Diethylstilbestrol (100A)    
Arsenic and inorganic arsenic compounds (100C)
Cadmium and cadmium compounds (100C)
Thorium-232 (as Thorotrast) (100D)
X- and γ-radiation (100D)

Other groupings (0%)

2,3,7,8-Tetrachlorodibenzo-para-dioxin (100F)   X- and γ-radiation (100D) [exocrine glands NOS]


[all cancers combined] Benzene (100F) [exocrine glands NOS]
Fission products including strontium-90 (100D) 1,3-Butadiene (100F) [exocrine glands NOS]
[all solid cancers] Vinyl chloride (100F) [exocrine glands NOS]
Plutonium-239 (100D)

N/A, not applicable: denotes organ and tissue systems when overlap is not possible (positive data are available in either humans or animals, but not in both); UV, ultraviolet.
a Organ and tissue systems in the anatomically based tumour nomenclature system (see Supplemental Table 1. Animal and human tumour sites for 111 Group 1 agents identified up

to and including Volume 109 of the IARC Monographs). Data inputs for human and animal data with sufficient evidence of carcinogenicity are from Supplemental Table 2. Database of
animal and human tumour sites for 111 distinct Group 1 agents up to and including Volume 109 of the IARC Monographs. Agents that lack sufficient evidence in both humans and animals
are not shown, with the exception of limited additional data inputs for limited evidence of human sites from Volumes 100A–F, Volume 107, and Volume 109 (in italics) and included data
for ethylene oxide, estrogen–progestogen oral contraceptives, and diethylstilbestrol. Data for male reproductive organs are also included, although they are not part of the concordance
analyses. 2,3,7,8-Tetrachlorodibenzo-para-dioxin is included, but its designation of “all cancers combined” for human data precludes specific site analyses between species.
b Agents with sufficient evidence in humans, animals, and both humans and animals.

c Part A, B, C, D, E, or F in Volume 100 of the IARC Monographs in which the agent is included. Volume 100F* denotes chemical agents and related occupations identified as Group 1

agents after the publication of Volume 100.


d Number of agents with sufficient evidence in both humans and animals, as a percentage of the total number of agents that cause tumours in either humans or animals (or both) in the

specified organ and tissue system (see Table 21.7).


have passed the age of highest risk limited human data are considered, Table 21.8, they are not part of the
for testicular cancer, further study among agents that have similar concordance analyses in Table 21.7,
cannot clarify the association be- chemical and mechanistic charac- because of a lack of sufficient evi-
tween this exposure and this type of teristics. For example, if the limited dence in either humans or animals.
cancer (IARC, 2012e). Human data evidence of tumours of the upper TCDD is included in Table 21.8, but
for this agent will remain limited for aerodigestive tract for chromium(VI) its designation as an agent affecting
this end-point, although supported compounds in humans noted in “all cancers combined” in humans
by the induction of testicular tumours Table 21.8 were admitted as evi- precludes site-specific tumour con-
in rodents. dence of carcinogenicity in humans, cordance analyses. Nevertheless,
With ongoing studies, more evi- concordance between animals and the limited evidence of carcinogeni-
dence can be gathered that provides humans would be established within city of TCDD in humans in the res-
increasing certainty about potential this organ and tissue system. piratory system and lymphoid and
cancer risks to humans. Although Concordance may also be in- hematopoietic tissues is consistent
IARC had previously evaluated TCE creased if less stringent criteria are with the sufficient evidence of car-
in 1979, 1987, and 1995, this subs- applied than are used by IARC for cinogenicity in animals in these two
tance was not declared to be carci- determining sufficient evidence of organ and tissue systems. These ex-
nogenic to humans – causing kidney carcinogenicity in animals. In evalua- amples illustrate increased site con-
cancer – until 2012, after the emer- ting the available animal data on es- cordance by applying less stringent
gence of new data (IARC, 2014). trogen–progestogen oral contracep- criteria than those applied for the
Although it was noted that a posi- tives (IARC, 2012e), it was concluded concordance analysis presented in
tive association had been observed that “the data evaluated showed a Table 21.7.
between liver cancer and exposure consistent carcinogenic effect of Table 21.8 shows human data in-
to TCE, the lack of data was cited several estrogen–progestogen com- dicating biological plausibility for the
as the rationale for its designation binations across different animal mo- upper aerodigestive tract and lung
as demonstrating only limited evi- dels in several organs.” Similarly, the to be targets for agents for which
dence of carcinogenicity in humans synthesis statement in the evaluation the portal of entry is the lung (as
in the previous evaluations. In 2013, of diethylstilbestrol (IARC, 2012e) with dusts, particles, and particles
an updated pooled analysis of three notes: “The oral administration of that serve as a vehicle for a mix-
Nordic studies with 10–15 years of diethylstilbestrol induced tumours of ture of other carcinogens, such as
additional follow-up demonstrated the ovary, endometrium, and cervix, during tobacco smoking and coke

CHAPTER 21
that human exposure to TCE was and mammary adenocarcinomas in production). Lymphohaematopoietic

PART 3
associated with a possibly increased female mice. Osteosarcomas and cancers are a consistent end-point
risk of liver cancer (Hansen et al., Leydig cell tumours were induced for antineoplastic alkylating agents
2013). Inclusion of the limited data for in rasH2 [transgenic] and Xpa/p53 that induce these cancers after their
TCE-induced liver cancer in humans [knockout] male mice, respectively. use in chemotherapy to eradicate
allows for the observation of overlap Subcutaneous implantation of die- other neoplasms (IARC, 2012e), for
between animals and humans for thylstilbestrol induced mammary tu- radioactive materials (IARC, 2012f),
this end-point. mours in female Wistar rats. Perinatal and for several chemical agents and
This example illustrates that exposure to diethylstilbestrol induces related compounds that are metabo-
the inclusion of agents with limited lymphoma, uterine sarcomas, ade- lized to or are in themselves agents
evidence of carcinogenicity in nocarcinomas, and pituitary, vaginal, that are reactive with DNA (IARC,
humans enhances the ability to and ovarian tumours in female mice. 2012c).
identify concordant relationships. Uterine adenocarcinomas and mam- Table 21.8 also illustrates some of
Comparison between Table 21.7, mary and vaginal tumours were also the potential relationships between
which mentions only sites with suf- induced in female rats. In hamsters, agents that may act in a similar
ficient evidence in humans, and diethylstilbestrol perinatal exposure fashion in humans. Tobacco smoke
Table 21.8, which also lists sites with induced kidney tumours.” and its related agents (smokeless
limited evidence in humans, illus- Although agents affecting male tobacco and second-hand tobacco
trates increased coherence, when reproductive organs are included in smoke) affect several similar organ

Part 3 • Chapter 21. Analysis of tumour site concordance 245


and tissue systems. For radioactive cific and varies widely. Nonetheless, reactive. Implantation of estrone or
materials, almost all organs and all agents that induce liver cancer in estradiol in castrated male hamsters
sites are affected by ionizing radia- rodents induce cancer at some other results in the induction of renal car-
tion; these agents affect multiple tar- site in humans. In some instances cinomas exclusively (Li et al., 1983).
get tissues because they are able to the apparent lack of overlap between Metabolic activation of estrogens by
reach the nucleus and cause a vari- the animal and human databases cytochrome P450 may also be relat-
ety of DNA lesions and other effects can still reflect mechanistic con- ed to a mechanism similar to that for
reflected by the key characteristics of cordance for similar agents. Dyes PAHs (Cavalieri and Rogan, 2014).
human carcinogens (see Chapter 10, such as magenta, 4-aminobiphenyl, Thus, diethylstilbestrol and estrogen
by Smith, and Chapter 22, by Krewski benzidine, and 2-naphthylamine all may have mechanistic similarities
et al.; see also Smith et al., 2016). cause liver cancer in rodents and that result in an apparent lack of or-
Radioactive materials also do not urothelial cancer in humans. TCDD gan and tissue system overlap, with
require metabolism in order to induce and PCBs are both associated with the hamster kidney being indicative
cancer. Several dyes are associated liver cancer in rodents and tumours of human risk.
with urothelial cancer in humans and of the lymphoid and haematopoietic
act through a similar mechanism tissues in humans. Discussion
(IARC, 2012c). Agents that disrupt Human exposures to diethyl-
Since the early 1970s, the IARC
the endocrine system and related stilbestrol, estrogen-only men-
Monographs Programme has been
organs (e.g. PCBs, diethylstilbestrol, opausal therapy, and combined
evaluating potential cancer risks to
estrogen-only menopausal therapy, estrogen–progestogen oral con-
humans (Saracci and Wild, 2015).
combined estrogen–progestogen traceptives are all associated with
Separate evaluations of the avail-
oral contraceptives, and tamoxifen) cancers of the female breast, female
able animal and human evidence are
induce cancer at similar sites, includ- reproductive organs, and female
made, and these are then combined
ing the female reproductive organs reproductive tract. Kidney cancer
to make an overall evaluation of the
and the breast. Metals appear to is induced in male hamsters upon
strength of evidence of carcinogen-
have many target sites in common, exposure to either diethylstilbestrol
icity to humans. At the time of this
including the upper aerodigestive or estrogens used in menopausal
analysis, 120 distinct agents have
tract, the respiratory system, the kid- therapy. Data from a control group
met the IARC criteria for determin-
ney, and the prostate. that received only estrogen, present-
ing causality and for designation of
As noted previously, the animal ed in the Monograph on combined
these agents as carcinogenic to hu-
database is predominantly popu- estrogen–progestogen oral contra- mans (Group 1). Of these, 111 dis-
lated by results from studies in ro- ceptives, indicate a similar result tinct Group 1 agents were included
dents. Respiratory tract tumours (IARC, 2012e). Although there ap- in the data set of tumours and tumour
are induced in rodents by many of pears to be concordance in rodents sites in animals and humans devel-
the same agents that cause such for the tumours induced by these oped by Grosse et al. (Annex 1).
tumours in humans. For the mes- agents, there does not appear to be The well-established weight-of-
othelium, where tumour formation overlap with humans: rodent kidney evidence criteria for the evaluation of
in humans or animals is rare and versus female breast and reproduc- the available human, animal, mech-
is specifically induced by a small tive organs. However, there may be anistic, and exposure data used by
number of agents, there is good mechanistic concordance between IARC are detailed in the Preamble to
agreement between the human and these two end-points, because both the IARC Monographs (IARC, 2006)
animal databases. Many agents me- diethylstilbestrol and estrogen may and provide clear guidance to the
tabolized in the liver to reactive com- damage DNA through oxidative Working Groups convened to review
pounds induce liver cancer in animal damage, formation of unstable ad- agents. If the criteria for sufficient ev-
models, with less apparent overlap ducts, and induction of apurinic sites. idence of carcinogenicity in both ani-
with the human data (see digestive In male Syrian hamsters the major mals and humans are satisfied, then
organs, Table 21.8). Susceptibility of metabolites of diethylstilbestrol are causality can be reasonably inferred,
the liver in rodents to cancer induc- catechols that easily oxidize to cat- and this can be strengthened by
tion is species-, sex-, and strain-spe- echol o-quinones, which are DNA- mechanistic considerations.

246
However, an immediate challenge er respiratory tract are considered model. Animal studies may also
in making comparisons for tumour together as the respiratory system. show tumours that are species- and/
site concordance between species Aggregation also allows more data or sex-specific.
was how to compare tumours in to be considered for analysis, which As part of the determination of
animals and in humans. A detailed increases the robustness of the en- weight of evidence, agents that in-
historical discussion of approaches suing conclusions. For the concor- duce tumours at multiple sites and
to the coding of human tumours was dance analyses, data at both the across multiple species are consid-
provided by Muir and Percy (1991), individual tumour site level and the ered to present a more robust can-
considering the topographical, mor- organ and tissue system level were cer hazard to humans. However,
phological, and histological char- the experimental animal database
examined.
acteristics of the lesion to be clas- used for the analysis consists pri-
Although the present analysis
sified. In the absence of a common marily of rodent data. It is notable
demonstrates generally good agree-
coding system for animal and human that of the 111 Group 1 agents ex-
ment between tumour sites in ani-
tumours, an anatomically based tu- amined here, three agents caused
mals and in humans after exposure
mour taxonomy system was devel- tumours in humans and in four ani-
to Group 1 carcinogens, concor-
oped during the course of the work mal species (mice, rats, hamsters,
dance was not demonstrated with
presented here.
every agent and tumour site. There and non-human primates): asbestos,
Although this system worked which causes lung tumours in all five
well for the purposes of the present are several factors and important
species; plutonium-239, which caus-
concordance analysis, there are limitations that may result in lack of
es skin tumours in these species;
some animal sites that do not have tumour concordance based on these
and 2-naphthylamine, which causes
a human counterpart, including the data. For many of the 111 agents,
urinary tract/uroendothelial tumours
Harderian gland and the Zymbal relevant and reliable data to support
in these species. These agents are
gland. Tumours at these unique sites a complete analysis of concordance
examples of carcinogens that cause
occurred rarely and were included are unavailable for either animals or
the same type of tumour in multiple
within the category of “other group- humans. For some agents, notably
species, thereby demonstrating a
ings” in the anatomically based tu- the human tumour viruses, relevant
high degree of interspecies tumour
mour nomenclature system used animal models are lacking, thereby
site concordance.
here. Other sites that are unique to precluding the possibility of obtaining
The present analyses exclude
animals but are, however, closely results on concordance. There may
the human tumour viruses evaluated
also be little motivation for conduct-

CHAPTER 21
related to a similar human site were in Volume 100B, because, with the

PART 3
aligned with the corresponding hu- ing animal tests for other agents,
possible exception of human T-cell
man tumour site; for example, the such as leather dust in occupational
lymphotropic virus type 1 (HTLV-
forestomach was considered as part environments or acetaldehyde asso- 1), the use of animals to assess the
of the stomach in the anatomically ciated with consumption of alcoholic potential cancer risks of human tu-
based taxonomy system. beverages. Mixtures such as those mour viruses is problematic (IARC,
This tool, developed for tumour in combined estrogen–progestogen 2012b). The best animal models to
comparisons across and within spe- menopausal therapy may also not study human viruses are non-hu-
cies, included 39 individual tumour have been evaluated in animals, man primates, which are difficult to
sites for which agents showed suf- particularly if the components of the use experimentally both because
ficient evidence of carcinogenicity in mixture had been previously evaluat- of the time and expense involved in
humans and/or animals, which were ed separately. Relevant animal tests conducting studies with long-lived
further aggregated into 14 organ and may still provide only limited or inad- species and because the incidence
tissue systems. This aggregation al- equate evidence of carcinogenicity of cancer is low in non-human pri-
lows comparisons to be made at a through limitations in study design mates. Although transgenic mouse
higher level of organization, reflect- or conduct, or if the mechanism of models have been developed for
ing anatomical and physiological action of the agent of interest was evaluating human cancer viruses,
similarities among certain tumour specific to humans and not easily such models are considered more
sites; for example, the lung and low- replicated in an experimental animal informative for understanding cancer

Part 3 • Chapter 21. Analysis of tumour site concordance 247


mechanisms than for human cancer Study limitations may also include UVC, UVB, and UVA). Although the
risk assessment (see Chapter 9, by inadequate power as a result of small skin was not explicitly mentioned as
Lambert and Banks). sample size. If human exposures to a human tumour site for UV radiation
The criteria for sufficient evidence the agent of interest are extremely in Volume 100D, the skin is implicitly
of carcinogenicity in animals as out- low, a particularly large, well-con- suggested as being a human tumour
lined in the Preamble to the IARC ducted study would be required to site for this agent. In the present
Monographs (IARC, 2006) gener- achieve reasonable sensitivity. analysis, the lack of explicit designa-
ally require independent replication Failure of human studies to identi- tion of the skin as a human tumour
in two different animal species, or fy tumour sites can occur when these site for UV radiation precluded its
studies do not consider all possible use. A similar situation occurred for
particularly strong results in a sin-
sites. Most case–control studies fo- areca nut, for which the oral cavity
gle species. The IARC Monographs
cus on only one or a limited number might have been considered as a
generally do not identify animal
human tumour site, although this site
tumour sites for agents with only of tumour sites. Human studies that
was not explicitly designated in the
limited evidence of carcinogenicity fail to identify a relevant tumour site
Monograph.
in animals. The criteria developed may have low sensitivity, possibly
An agent can be categorized by
by Grosse et al. (Annex 1) further because they do not focus on the
IARC as a Group 1 carcinogen in the
restrict the use of tumour data for most appropriate study population.
absence of sufficient evidence for
agents with sufficient evidence in As noted above for TCE, evidence
carcinogenicity in humans when it is
experimental animals (e.g. tumour on specific tumour sites may not
clear that the mechanisms by which
sites were not identified in the ab- yet have accrued at the time of an
the agent causes cancer in animals
sence of two or more animal studies evaluation. After the first evaluation
also operate in humans. Such “mech-
of adequate design and quality point- of tobacco smoking in Volume 38 of
anistic upgrades” have occurred with
ing at the same tumour site with a the IARC Monographs (IARC, 1986),
various levels of human evidence,
similar histological origin in the same cigarette smoking was subsequent-
including for aristolochic acid (lim-
ly shown – in Volume 83 – to cause
species). Although melphalan pro- ited evidence of carcinogenicity in
cancer at a much larger number of humans; IARC, 2012e), B[a]P (inad-
duced tumours of the forestomach,
tumour sites, including cancers of equate evidence in humans; IARC,
skin, and lung as well as lymphosar-
the nasal cavities and nasal sinus- 2012c), ethylene oxide (limited ev-
comas in mice and mammary gland
es, oesophagus, stomach, liver, kid- idence in humans; IARC, 2012c),
tumours and peritoneal sarcomas in
ney, and uterine cervix, and myeloid 4,4′-methylenebis(2-chloroaniline)
rats (IARC, 2012c), none of these tu-
leukaemia (IARC, 2004). Thus, the (MOCA) (inadequate evidence in
mour sites were replicated in a sec-
potential for underestimation of in- humans; IARC, 2012c); and neutron
ond animal species, and hence are
terspecies tumour site concordance radiation (inadequate evidence in hu-
not included in the data set of Grosse
may result from missing tumour sites mans; IARC, 2012f).
et al. (Annex 1).
for agents for which sufficient evi- For further discussion of mecha-
Human evidence is also subject
dence of carcinogenicity in humans nistic upgrades and key character-
to limitations. As noted above, the
already exists. istics of Group 1 agents developed
opportunity may no longer be avail-
How human study data are report- for this analysis, see Chapter 10, by
able to conduct further informative
ed in the Monographs may also af- Smith, Chapter 22, by Krewski et al.,
studies in humans of a substance fect the ability to conduct analyses to Smith et al. (2016), and Birkett et al.
like diethylstilbestrol. The absence establish tumour site concordance. A (2019). Ten key characteristics of
of sufficient evidence in humans may specific example of this constraint is human carcinogens described by
be due to a lack of evidence in ap- ionizing radiation. No specific human Smith et al. (2016) focus on wheth-
propriate epidemiological or clinical tumour sites were identified for ion- er the agent (1) is electrophilic or
studies, or to the inability of exist- izing radiation (all types), internalized can be metabolically activated to
ing studies to detect an association radionuclides that emit α-particles, electrophiles, (2) is genotoxic, (3) al-
between exposure to the agent of internalized radionuclides that emit ters DNA repair or causes genomic
interest (including exposures early or β-particles, and UV radiation (band- instability, (4) induces epigenetic
later in life) and a tumour outcome. width 100–400 nm, encompassing alterations, (5) induces oxidative

248
stress, (6) induces chronic inflam- ty to determine concordance may The concordance analyses reported
mation, (7) is immunosuppressive, change as additional Group 1 agents here are based either on 39 tumour
(8) modulates receptor-mediated are identified, or as additional ani- sites or on the broader classification
effects, (9) causes immortalization, mal or human evidence on current of 14 organ and tissue systems.
and/or (10) alters cell proliferation, Group 1 agents becomes available.
Effects of sex, strain, and
cell death, or nutrient supply. These New mechanistic data could affect
route of exposure
considerations will be relevant in IARC evaluations of agents currently
planned future analyses of coher- classified in Group 2A (probably car- Risks of cancer can differ between
ence between tumours in animals cinogenic to humans) and Group 2B male and female animals, among
and humans, taking into account (possibly carcinogenic to humans), different strains of the same animal
key characteristics of carcinogens. and hence affect the concordance species, and by route of exposure.
However, mechanistic upgrades limit estimates reported here. Birkett et al. Because of incomplete information
the ability to identify tumour site con- (2019) noted that additional informa- on these three factors in the data-
cordance when human tumour sites tion on the 10 mechanistic key char- base used in the present analysis,
are not identified. acteristics of human carcinogens it was not possible to evaluate how
Exposure assessment is one described by Smith et al. (2016) is concordance might vary by sex,
of the most difficult aspects of ep- available in the general scientific lit- strain, or exposure route.
idemiological investigations (Nieu­ erature, beyond what is summarized
Effects of dose
wenhuijsen, 2003). In some cases, in the IARC Monographs.
such as ecological studies that com- In addition to the restrictions used Because the primary objective of the
pare two population groups subject by Grosse et al. (Annex 1) for inclu- IARC Monographs Programme is to
to notably different exposure circum- sion of certain experimental animal identify agents with the potential to
stances, exposure may not be mea- data, other limitations of the data- cause cancer in humans in qualita-
sured at all. In other cases, however, base affect the ability to determine tive terms, rather than to quantify the
exposures may be very well deter- tumour site concordance, including level of risk at a given dose, informa-
mined, as with the use of personal incomplete information on tumour tion on dose dependence in cancer
dosimeters to measure exposures to histology, limited information on the risk is not systematically collected in
agents such as ambient air pollution effects of sex, strain, and route of the Monographs, although this is cur-
or ionizing radiation, or in the dose exposure, and limited information on rently under review by IARC (IARC
regimens of pharmaceutical drugs or dose-dependent effects. These and Advisory Group to Recommend on
medical radiation. In the future, en- other limitations are discussed brief- Quantitative Risk Characterization,

CHAPTER 21
hanced exposure assessment meth- ly below.

PART 3
2013). Therefore, analyses of con-
odologies may serve to strengthen cordance considering dose–re-
Incomplete information on
the ability of epidemiological studies tumour histology sponse relationships seen in animals
to identify Group 1 agents (Cohen- and humans were not attempted at
Hubal et al., 2010; National Research Because of incomplete information
this time.
Council, 2012). Biomarkers of expo- on the histology of lesions in both an-
sure are expected to play an impor- imal and human studies, it was not Multisite/multiorgan
tant part in the future of exposure sci- possible to conduct concordance an- carcinogenicity
ence (Gurusankar et al., 2017). alyses for specific histological sub-
The data set assembled and eval- types of cancers at a given site (such Several agents, notably radiation
uated by Grosse et al. (Annex 1) was as adenocarcinoma or squamous and tobacco smoke, induce malig-
retrieved from the IARC Monographs. cell carcinoma of the lung). The con- nant lesions at multiple sites or in
Thus, these agents do not represent cordance analyses reported here multiple organ and tissue systems.
a “random sample” of all potential are necessarily restricted to tumours Volume 100F (IARC, 2012c) sum-
human carcinogens, and the data occurring in a given organ or tissue marizes the evidence that 1,3-buta-
set is populated by the available an- (such as lung cancer) or in a more diene induces haemangiosarcomas
imal and human evidence that was broadly defined organ and tissue sys- of the heart, malignant lymphomas,
the focus of the Monographs from tem (such as the upper aerodigestive bronchiolo-alveolar neoplasms,
which they were drawn. The abili- tract and the respiratory system). and squamous cell neoplasms of

Part 3 • Chapter 21. Analysis of tumour site concordance 249


the forestomach in male and fe- Predictive value of animal outlined in the Preamble to the IARC
male B6C3F1 mice, and acinar cell tests for carcinogenicity Monographs (IARC, 2006). Most
carcinomas of the mammary gland, Group 1 agents are identified on
Using a database comprising 150
granulosa cell neoplasms of the ova- the basis of sufficient evidence in
agents tested for toxicity in animals
ry, and hepatocellular neoplasms humans, and for the purpose of the
and humans, Olson et al. (2000)
in female mice. Assessing species overall evaluation, there is no imme-
estimated the positive predictive val-
concordance with multisite carcino- diate recourse to animal data. Of the
ue (PPV) and the negative predictive
gens is inherently more difficult than 111 Group 1 agents considered in
value (NPV) for human toxicity (ex-
with carcinogens that affect a single this chapter, 102 demonstrated suf-
cluding cancer). In this context, the
organ or tissue. Understanding the ficient evidence of carcinogenicity in
PPV is defined as the probability of
mechanistic and other attributes of humans; the remaining nine agents
observing human toxicity in clinical
such multisite carcinogens will be were placed in Group 1 because the
testing, given that toxicity has been
useful in translating results in experi- mechanisms by which tumours oc-
observed in animal tests. The PPV
mental animals to humans. curred in animals were considered to
for human toxicity was estimated to
be directly relevant to humans, or on
Measures of concordance be 71% for rodent and non-rodent
the basis of other relevant mechanis-
species combined, 63% for non-ro-
For simplicity of presentation, con- tic considerations. For example, neu-
dents alone, and 43% for rodents
cordance was evaluated here in tron radiation was placed in Group 1
alone. Although a statement of the
terms of the “overlap” between tu- despite inadequate evidence in hu-
PPV and the NPV of animal cancer
mour sites seen in animals and hu- mans, because the biophysics of ra-
tests for human carcinogenicity may
mans. Although more formal sta- diation damage is similar for different
be desirable, this cannot be done
tistical analyses of concordance as types of ionizing radiation.
on the basis of the IARC concor-
described in Supplemental Material Bearing in mind the contribution
dance database considered in this
II (online only; available from: http:// of animal data to the identifica-
chapter. This is because both the
publications.iarc.fr/578) were consid- tion of Group 1 agents in the IARC
PPV and the NPV depend on the
ered during the course of this work, Monographs, it is possible with the
prevalence of true positives in the
the consensus of the Working Group present IARC concordance data-
database (Altman and Bland, 1994).
was to represent concordance in base to make a statement about the
Because the IARC concordance da-
terms of the simpler, more directly likelihood of positive results in ani-
interpretable, indicators of “overlap” tabase comprises Group 1 agents
mals among the Group 1 agents that
in Table 21.7 and Fig. 21.10. that are known causes of cancer in
have been shown to cause cancer
humans, the PPV of animal cancer
Small sample size in humans. Excluding mechanistic
tests will artificially be calculated as
upgrades (nine agents) and Group 1
100%, whereas a lower PPV would
After the 111 Group 1 agents tabu- agents that lack appropriate animal
be obtained with a more represen-
lated by Grosse et al. (Annex 1) up data (20 agents), all Group 1 agents
tative database that includes agents
to and including Volume 109 of the with sufficient evidence of carcino-
that do not cause cancer in humans.
IARC Monographs were filtered to genicity in humans have also provid-
However, identifying agents that do
include only agents that provided ed sufficient or limited evidence of
not cause cancer in humans is not
sufficient evidence of carcinogenicity carcinogenicity in one or more ani-
in at least one tumour site in humans the focus of the IARC Monographs
mal species.
and at least one tumour site in ani- Programme; at present, only one
mals, 60 agents remained eligible for agent – caprolactam – is classified Conclusions
concordance analysis. Because the as probably not carcinogenic to hu-
sample size for some tumour sites mans (Group 4). The IARC Monographs Programme
is small (only two agents – asbestos In considering the relevance of is widely recognized as one of
and erionite – caused tumours of the animal data in the context of the the most authoritative sources of
mesothelium), caution is needed in IARC Monographs, it is important information on the identification of
interpreting the concordance results to keep in mind how animal data are agents that may be carcinogenic
presented in this chapter for these used in the identification of Group 1 to humans. The Monographs are
sites. agents, according to the criteria prepared with the involvement of

250
leading scientific experts world- the previous 99 Volumes of the IARC Monographs Programme (Wilbourn
wide, who apply the guidance pro- Monographs, providing a veritable et al., 1986) and commented upon by
vided in the Preamble to the IARC “encyclopaedia of carcinogens”. other authors (Tomatis et al., 1989;
Monographs (IARC, 2006) to eval- This information, supplemented with Huff, 1994; Maronpot et al., 2004).
uate the weight of evidence that an data on Group 1 agents identified in However, it is important to note that
agent may present a cancer risk to Volumes 101 to 109, formed the ba- the present database cannot be
humans. Up to and including Volume sis for the analyses included in this used to estimate the predictive value
109, more than 2000 scientists have chapter. After both PCB 126 and of animal cancer tests for humans,
contributed to the development of the dioxin-like PCBs were subsumed because it comprised by design only
IARC Monographs; nearly 200 sci- within the broader category of PCBs, Group  1 agents; the PPV and the
entists were involved in Volume 100 113 – 2 = 111 district Group 1 agents NPV of the animal data for humans
alone. Since its beginning in 1971– were included in the concordance would be 100% and 0%, respectively
1972 (Saracci and Wild, 2015), the analyses presented in this chap- (an artefact of a database that com-
IARC Monographs Programme has ter. The importance of human data prises human carcinogens only).
evaluated more than 1000 agents in the IARC carcinogen evaluation Despite the challenges in eval-
for their potential to cause cancer process is highlighted by the obser- uating concordance between tu-
in humans, with 120 of these agents vation that 102 of the 111 distinct mour sites in animals and humans,
assigned to Group 1, indicating that Group 1 agents identified at the time the IARC concordance database
the weight of evidence supports the this analysis was done demonstrated is a useful source of information for
conclusion that the agent is carcino- sufficient evidence of carcinogenicity comparing animal and human data
genic to humans. in humans. with respect to the tumours caused
A noteworthy aspect of the pro- Analysis of concordance between in different species by the 111 dis-
cess used by IARC to identify the tumour sites in animals and humans tinct Group 1 agents identified by
causes of cancer in humans is the was restricted to 60 Group 1 agents IARC up to and including Volume
reliance on leading experts in the demonstrating sufficient evidence for 109 of the IARC Monographs.
Working Groups that conduct the at least one tumour site in animals Future Monographs may benefit
evaluations documented in the and in humans. Substantial overlap from a more systematic summary
Monographs to interpret the data between animal and human tumours of the animal and human data on
according to the weight-of-evidence was seen in some organ and tissue agents evaluated within the IARC
guidelines provided in the Preamble systems but not in others. This anal- Monographs Programme, including

CHAPTER 21
to the IARC Monographs (IARC, ysis focused on tumours seen in the data on the types of tumours seen in

PART 3
2006). With the trend towards great- 14 organ and tissue systems in the animal and human studies, possibly
er reliance on systematic review anatomically based tumour classi- using the anatomically based tumour
(National Research Council, 2014) fication system rather than 39 indi- nomenclature system introduced
and structured weight-of-evidence vidual tumour sites, because of the in this chapter to facilitate compari-
approaches to the evaluation of sparseness of data at the individual sons between animals and humans.
toxic substances (Rhomberg et al., tumour site level. Data on route of exposure, sex, and
2013), the continued involvement The principle that agents that are animal strain would also support
of international experts in the IARC carcinogenic in experimental animals comparisons of animal and human
Monographs to interpret the often should be regarded as presenting a tumours at a finer level of biological
extensive human, animal, and mech- carcinogenic risk to humans was fur- resolution. Data on the exposure or
anistic data is a major strength of the ther confirmed in the course of this dose levels at which tumours are
IARC Monographs Programme. investigation. Excluding agents for seen in animals and humans would
Collectively, the IARC Mono- which animal data are lacking or oth- further support evaluation of the rel-
graphs provide a rich source of erwise uninformative, all agents that ative carcinogenic potency of agents
information on the causes of can- cause cancer in humans also cause evaluated in animals and humans.
cer in humans. In particular, Volume cancer in one more animal species, Information on tumour sites affected
100 presents a review and update a finding consistent with an earlier by agents evaluated within the IARC
of 107 Group 1 agents identified in evaluation of results from the IARC Monographs Programme should be

Part 3 • Chapter 21. Analysis of tumour site concordance 251


recorded in as much detail as possi- with sufficient evidence (82 agents) that cause tumours of the meso-
ble to facilitate future evaluations of or limited evidence (9 agents) of car- thelium, only two Group 1 agents
the concordance between tumours cinogenicity in animals. The most meeting the criteria for inclusion in
seen in animals and humans on a common tumours observed in both the concordance analysis caused
site-specific basis. humans and animals were those of tumours at this site. Although the
the respiratory system (including present analysis demonstrates good
Summary larynx, lung, and lower respirato- concordance between animals and
ry tract). In humans, such tumours humans for many, but not all, tumour
Since its inception in the early 1970s, were observed for 31 of the 111 dis- sites, limitations of the available data
the IARC Monographs Programme tinct Group 1 carcinogens identified may result in underestimation of
has developed 119 Monographs up to and including Volume 109 of concordance.
Volumes on more than 1000 agents the IARC Monographs, comprising
for which there exists some evi- mostly chemical agents and related Acknowledgements
dence of cancer risk to humans; of occupations (14 agents), arsenic,
these, 120 agents met the criteria Pascal Lajoie assembled the tumour
metals, fibres, and dusts (7 agents),
for classification as carcinogenic to site concordance database analysed
and personal habits and indoor com-
humans (Group  1). Volume 100 of in this chapter while working as a
bustions (5 agents). After tumours
the IARC Monographs, compiled in Visiting Scientist under the direction
in the respiratory system, those in
2008–2009 and published in 2012, of Yann Grosse at IARC during the
lymphoid and haematopoietic tis-
provided a review and update of the summers of 2011 and 2012. Mélissa
sues (26 agents), the urothelium (18
107 Group 1 agents identified as of Billard also contributed to the devel-
agents), and the upper aerodigestive
2009. These agents were divided opment of the concordance database
tract (16 agents) were most often
into six broad categories: pharma- while working as a Visiting Scientist
seen in humans, and tumours in di-
ceuticals; biological agents; arsenic, under the direction of Yann Grosse
gestive organs (19 agents), the skin
metals, fibres, and dusts; radiation; and Robert Baan at IARC during the
(18 agents), and connective tissues
personal habits and indoor com- summers of 2013 and 2014. Daniel
(17 agents) were most often seen
bustions; and chemical agents and Krewski is the Natural Sciences
in animals. Exposures to radiation
related occupations. The data set and Engineering Research Council
(particularly X- and γ-radiation) and
developed by Grosse et al. (Annex 1) of Canada Chair in Risk Science at
tobacco smoke were associated with
for human and animal tumours and the University of Ottawa. Julian Little
tumours at multiple sites in humans.
tumour sites associated with ex- is the Canada Research Chair in
Although the IARC Monographs do
posure to these agents, as well as Human Genome Epidemiology at the
not emphasize tumour site concor-
five additional Group 1 agents de- University of Ottawa. The authors
dance between animals and hu-
fined in subsequent Volumes of the would like to thank Nawal Farhat and
mans, substantial concordance was
Monographs, were used to analyse Mohamed Taher for their assistance
observed for several organ and tis-
the degree of concordance between in reviewing and checking the final
sue systems, even under the strin-
sites where tumours arise in humans draft of this chapter. This chapter is
gent criteria for sufficient evidence
and in experimental animals (mice, dedicated to the memory of Jan M.
of carcinogenicity used by IARC. Of
rats, hamsters, dogs, and non-hu- Zielinski, who succumbed to can-
the 60 agents for which at least one
man primates). An anatomically cer in 2016; before becoming ill, Dr
tumour site had been identified in
based tumour nomenclature system, Zielinski led the team that conducted
both humans and animals, 52 (87%)
representing 39 tumour sites and 14 the analyses reported here.
cause tumours in at least one of the
organ and tissue systems for which same organ and tissue systems in
agents presented sufficient evidence humans and animals. It should be
of carcinogenicity in humans and/or noted that some caution is needed
in experimental animals, was devel- in interpreting concordance at sites
oped and used as the basis for in- where the sample size is particu-
terspecies comparison. The present larly small: although perfect (100%)
analysis identified 91 Group 1 agents concordance was noted for agents

252
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role of human biological monitoring in health IARC (2012c). Chemical agents and related J Cancer Res. 80(5):419–23. http://dx.doi.
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CHAPTER 21
PART 3

Part 3 • Chapter 21. Analysis of tumour site concordance 255


part 3.
statistical analyses of concordance and key characteristics

chapter 22.

Analysis of key characteristics


of human carcinogens
Daniel Krewski, Mustafa Al-Zoughool, Michael Bird, Nicholas Birkett, Mélissa Billard, Brittany Milton,
Jerry M. Rice, Vincent J. Cogliano, Mark A. Hill, Julian Little, and Jan M. Zielinski (deceased)
in collaboration with other participants (see the Contributors list) in the Workshop on Tumour Site Concordance
and Mechanisms of Carcinogenesis, which was convened by IARC in April and November 2012 in Lyon

CHAPTER 22
PART 3
Introduction evaluate and integrate the evidence limited evidence of carcinogenicity,
provided by human epidemiological inadequate evidence of carcinogen-
Since its establishment in the ear- studies, animal cancer bioassays, icity, or evidence suggesting lack of
ly 1970s, the IARC Monographs and information on possible biolog- carcinogenicity. The information on
Programme has evaluated more ical mechanisms of action, to clas- biological mechanisms of action may
than 1000 agents with evidence of sify agents into one of the following be evaluated as strong, moderate, or
human exposure and for which some categories: carcinogenic to humans weak, and is taken into consideration
suspicion exists of an increased (Group 1), probably carcinogenic to in the overall evaluation.
cancer risk to humans. The IARC humans (Group 2A), possibly carci- The role of mechanistic informa-
Monographs Programme has devel- nogenic to humans (Group 2B), not tion in evaluating carcinogenicity
oped detailed criteria against which classifiable as to its carcinogenicity to has increased substantially during
to evaluate the available scientific humans (Group 3), and probably not the history of the IARC Monographs
evidence on the carcinogenic poten- carcinogenic to humans (Group 4). Programme. In 1991, IARC convened
tial of such agents. These criteria, These evaluations involve classifying a Working Group on the Use of Data
which are described in the Preamble the data from both the human and on Mechanisms of Carcinogenesis
to the IARC Monographs (Cogliano the animal studies as providing suf- in Risk Identification, to explore how
et al., 2004; IARC, 2006), are used to ficient evidence of carcinogenicity, mechanistic data could be used to

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 257


identify agents with the potential to processes at the cellular and mo- electrophile can react with cellu-
cause cancer in humans. The con- lecular level and thought to be rele- lar macromolecules such as DNA,
sensus report of the Working Group vant to carcinogenesis. Information RNA, and proteins to form adducts.
documented several mechanisms on these 24 end-points was derived Some chemical carcinogens are di-
that were considered to be relevant from human in vivo, human in vitro, rect-acting electrophiles (e.g. form-
to human carcinogenesis at that animal in vivo, and animal in vitro aldehyde; sulfur mustard, and ethyl-
time, including genotoxicity, cell studies (see Al-Zoughool et al., 2019). ene oxide), whereas others require
proliferation, receptor mechanisms During the November 2012 meeting, biotransformation by enzymes in a
in mitogenesis, alterations in DNA the Workshop participants identified process termed metabolic activation
repair, intercellular communication, 10 broader key characteristics of car- (e.g. polycyclic aromatic hydrocar-
and immune defects and immuno- cinogens (see Chapter 10, by Smith, bons and benzene) (Miller, 1970).
suppression (Vainio et al., 1992). and Smith et al., 2016). Information Characteristic 2: Is genotoxic
Toxicokinetic and other variables on these characteristics was extract-
were also identified as factors affect- ed from the IARC Monographs and Genotoxicity is the ability to induce
ing multistage carcinogenesis. Since used to develop a database of key DNA damage or other chromosomal
1991, IARC (2006) and other orga- characteristics for Group 1 agents alterations, as measured by three
nizations – for example, the United (see Al-Zoughool et al., 2019). This associated toxicological end-points:
States National Toxicology Program chapter focuses on the key charac- (i) DNA damage: an alteration in
(National Toxicology Program, 2014) teristics of the Group 1 agents iden- the chemical structure or integrity
tified in the IARC Monographs up to of DNA, including a break in a DNA
and the United States Environmental
and including Volume 106, and pre- strand, and/or chemical modifica-
Protection Agency (EPA) (EPA,
tions such as covalent binding to
2005) – have stressed the increas- sents the results of an exploratory
the nucleotide bases (Hoeijmakers,
ing importance of mechanistic infor- analysis of this database.
2009); (ii) gene mutations: changes
mation in cancer risk assessment.
in the normal nucleotide sequence of
Related risk assessment practic- Methods
cellular DNA that may have a central
es concern mode of action (Meek
Key characteristics role in human carcinogenesis (Ding
et al., 2014) and pathways of toxic-
et al., 2008); (iii) clastogenic effects
ity (Krewski et al., 2014; Bourdon- As mentioned above, Chapter 10, reflect damage to chromosomes, in-
Lacombe et al., 2015; Cote et al., by Smith, and Smith et al. (2016) cluding DNA breakage, or the rear-
2016), as well as dosimetric consid- describe 10 key characteristics of rangement, gain, or loss of chromo-
erations (Gurusankar et al., 2017). human carcinogens, as listed in some fragments (Snyder, 2010).
This chapter examines the avail- Table 22.2. The toxicological end-
able data on mechanisms of action Characteristic 3: Alters DNA
points initially considered by the
of the Group 1 agents identified up repair or causes genomic
Workshop participants and used
instability
to and including Volume 106 of the as indicators of these characteris-
IARC Monographs (Table 22.1). The tics are also noted in Table 22.2. Alterations in DNA repair result in de-
present analysis is based on a re- A brief summary of each of these fects in processes that monitor and
view of human cancer mechanisms, characteristics and the associated correct DNA replication fidelity. Such
conducted by the participants in the toxicological end-points is provided defects can confer strong mutator
two-part Workshop on Tumour Site below. phenotypes that result in genomic
Concordance and Mechanisms of instability.
Characteristic 1: Is electrophilic
Carcinogenesis, which was con-
or can be metabolically Characteristic 4: Induces
vened by IARC in April and Novem-
activated to electrophiles epigenetic alterations
ber 2012 in Lyon. This approach
initially involved retrieval of informa- The first characteristic refers to Induced epigenetic alterations are
tion from the IARC Monographs on agents that act as electrophiles stable changes in gene expression
24 toxicological end-points identi- themselves or that can be metab- and chromatin organization that are
fied as likely indicators of biological olized to form electrophiles. An independent of mutation and that

258
Table 22.1. Number of Group 1 agents in Volumes 100–118 of the IARC Monographs, by type of agenta

Type of agent Volume Total

100 105 106 107 109 110 111 113 114 117 118

Pharmaceuticals 23 – – – – – – – – – – 23

Biological agents 11 – – – – – – – – – – 11

Arsenic, metals, 10 – – – – – 2b – – – – 12
fibres, and dusts

Radiation 18 – – – – – – – – – 1c 19

Personal habits and 12 – – – – – – – 1d – – 13


indoor combustions

Chemical agents and 33 1e 1f 2g 2h 1i – 1j – 1k 1l 43


related occupations

Total 107 1 1 2 2 1 2 1 1 1 2 121


a At the time that the present analysis was conducted, mechanistic information was available only for the 109 Group 1 agents
evaluated in the IARC Monographs up to and including Volume 106.
b Fluoro-edenite fibrous amphibole; occupational exposures associated with the Acheson process in the manufacture of silicon

carbide fibres.
c Ultraviolet radiation from welding.

d Processed meat.

e Diesel engine exhaust.

f Trichloroethylene.

g Polychlorinated biphenyls (PCBs); dioxin-like PCBs.

h Outdoor air pollution; particulate matter in outdoor air pollution.

i 1,2-Dichloropropane.

j Lindane.

k Pentachlorophenol (PCP).

l Welding fumes.

CHAPTER 22
can be inherited through cell divi- reactive oxygen and detoxification of leading to the recruitment and acti-

PART 3
sion. Epigenetic phenomena include the radical species within cells and vation of inflammatory cells. Strong
genomic imprinting, X-chromosome tissues. Reactive oxygen species links exist between inflammation
inactivation, global reconfiguration induce a cascade of events that can and the induction of oxidative stress
of the DNA methylome, changes in include DNA mutation and oxidative and genomic instability; this makes
chromatin compaction states and DNA damage. Both are key events in it difficult to separate out the rel-
histone modification patterns, and carcinogenesis (Klaunig et al., 2011). ative importance of each of these
altered expression of microRNAs mechanisms. These linkages might
Characteristic 6: Induces
(miRNAs). These phenomena occur be the basis of the relationship be-
chronic inflammation
during organ development and con- tween chronic inflammation and can-
tribute to the lineage-specific epi- Chronic inflammation can arise from cer (Multhoff and Radons, 2012).
genome that is maintained over the persistent infection (e.g. with human
Characteristic 7: Is
lifetime of an organism. papillomavirus or with Helicobacter
immunosuppressive
pylori) as well as from exogenous ir-
Characteristic 5: Induces
ritants (e.g. silica or asbestos fibres). Immunosuppression is an induced
oxidative stress
Persistent infection and chronic in- reduction in the capacity of the
Oxidative stress results from an flammation disrupt local tissue ho- immune system to respond effec-
imbalance between formation of meostasis and alter cell signalling, tively to foreign antigens, including

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 259


Table 22.2. Key characteristics and toxicological end-points demonstrated by agents known to cause cancer in
humans (adapted from Al-Zoughool et al., 2019)

Key characteristic Corresponding toxicological end-points

Is electrophilic or can be metabolically activated Metabolic activation


to electrophiles Protein adducts
ADME (differences in absorption, distribution, metabolism, and
elimination)

Is genotoxic DNA damage


Cytogenetic/clastogenic effects
Gene mutations

Alters DNA repair or causes genomic instability DNA repair alteration, leading to genomic instability

Induces epigenetic alterations Epigenetic alterations (DNA methylation, histone modification, and altered
expression of microRNAs)

Induces oxidative stress Oxidative stress

Induces chronic inflammation Chronic inflammation


Chronic irritation

Is immunosuppressive Immune effects

Modulates receptor-mediated effects Receptor-mediated effects


Hormonal effects

Causes immortalization Immortalization


Alterations in telomere length

Alters cell proliferation, cell death, or nutrient Cell-cycle effects


supply Bystander effects
Alterations in cell signalling pathways
Angiogenic effects
Cell death
Inhibition of gap-junctional intercellular communication

antigens on tumour cells. In contrast receptors, thereby inducing or mod- Characteristic 9: Causes
to other key characteristics, immuno- ifying a plethora of signal transduc- immortalization
suppression does not play a direct tion pathways that, among other re-
Immortalization refers to a cell evad-
part in transforming normal cells into sponses, stimulate cell proliferation. ing normal cellular senescence and
tumour cells, but enables them to es- Receptor-mediated effects can in- proliferating indefinitely. In culture,
cape immune surveillance. Among
duce hormonal effects whereby ex- normal cells have a fixed number of
other roles, the immune system also
ternal agents can interfere with the replication cycles before they enter
plays a major part in the inflammato-
synthesis, secretion, transport, bind- cellular senescence and stop repli-
ry response to injury. cating. Evasion of senescence is fre-
ing, action, or elimination of natural
Characteristic 8: Modulates quently associated with activation of
hormones in the body. These exter-
receptor-mediated effects telomerase (Willeit et al., 2010) and
nal factors can also demonstrate re-
plays a critical part in carcinogenesis
activity similar to endogenously pro-
Modulation of receptor-mediated ef- (Reddel, 2000). Carcinogenesis may
fects can occur when agents mimic duced hormones, which can lead to involve activation of a telomerase
the structure of endogenous ligands them mediating changes in homeo- that prevents loss of telomere length,
that bind to cells and activate cell stasis, reproduction, development, leading to immortalization of cells
surface receptors or intracellular or behaviour. (Willeit et al., 2010).

260
Characteristic 10: Alters cell (evaluated in Volume 106; Guha for 109 Group 1 agents identified in
proliferation, cell death, or et al., 2012; IARC, 2014). Had these the IARC Monographs up to and in-
nutrient supply two agents been evaluated within cluding Volume 106. The 86 Group 1
Volume 100, they would have been agents for which separate mech-
Cell proliferation is affected by alter-
included in Volume 100F; they have anistic summaries are provided in
ations in the rates of cell growth with-
therefore been listed with other the IARC Monographs up to and
in a tissue. It may be a direct effect or
chemical agents and related occupa- including Volume 106 are listed in
a secondary regenerative effect after
tions in Volume 100F*. Table 22.3, along with their relation-
induction of cell death by cytotoxic
Although additional Group 1 ship to the 111 distinct agents identi-
agents. Two associated toxicological
agents have since been identified fied up to and including Volume 109
end-points are (i) cell-cycle effects,
(Table 22.1), the present analysis is used by Krewski et al. (Chapter 21)
i.e. alterations in the functioning restricted to Group 1 agents identi- in a parallel analysis of overlap be-
of the complex series of factors fied in the IARC Monographs up to tween tumours and tumour sites in
that control the cell cycle and cell and including Volume 106, the most animals and humans.
division, which have been associat- recent volume for which mechanis-
ed with carcinogenesis (Diaz-Moralli tic information was available at the Database of mechanistic
et al., 2013), and (ii) alterations in cell time of the present analysis. Group 1
characteristics
signalling pathways, which relate to agents not included in the present A database of toxicological end-
the ability of the agent to interfere analysis are (i) polychlorinated bi- points was assembled for the 86
with cell signalling pathways, leading phenyls (PCBs) and dioxin-like PCBs
Group 1 agents identified up to and
to expression of a carcinogenic trait (reviewed in Volume 107; Lauby-
including Volume 106 of the IARC
or phenotype in the cell. Secretan et al., 2013; IARC, 2016b),
For cell death, necrosis triggers Monographs. The database in-
(ii) outdoor air pollution and (iii) par-
the invasion of cells such as mac- cludes information from in vivo and
ticulate matter in outdoor air pollu-
rophages into the affected area, in vitro studies in humans and ani-
tion (both evaluated in Volume 109;
and enhances the proliferation and mals. Information on the 24 toxico-
Loomis et al., 2013; IARC, 2016a),
spread of cancer cells. Defects in logical end-points was retrieved from
(iv) 1,2-dichloropropane (reviewed in
programmed cell death can cause Section 4 (“Other relevant data”) of
Volume 110; Benbrahim-Tallaa et al.,
cancer; evasion of apoptosis is a re- the IARC Monographs (Al-Zoughool
2014; IARC, 2017a), (v) fluoro-eden-
quirement for both neoplastic trans- et al., 2019).
ite fibrous amphibole and (vi) occu-
formation and sustained growth of Recognizing that, among other
pational exposures associated with

CHAPTER 22
cancer cells. limitations, new data may have be-
the Acheson process used in the

PART 3
Adequate cell nutrition is essen- come available since 2009, when
manufacture of silicon carbide fi-
tial to proliferating cancer cells, and the various parts of Volume 100 were
bres (both evaluated in Volume 111;
agents that promote or inhibit the Grosse et al., 2014; IARC, 2017b); compiled, PubMed searches were
growth of blood vessels (angiogene- (vii)  lindane (Volume 113; Loomis conducted to identify evidence on
sis) will affect tumour growth. et al., 2015), and (viii) processed any of the 24 toxicological end-points
meat (Volume 114; Bouvard et al., linked to these agents that was not
Group 1 agents included in recorded in the IARC Monographs
2015).
the analysis (Birkett et al., 2019). The mechanistic
In some cases, the discussion of
Volume 100 of the IARC Monographs mechanisms of action in Section 4 database distinguishes information
provided a review and update of the (“Other relevant data”) of the IARC derived from the Monographs from
107 Group 1 agents identified as Monographs is based on groups of that found in the PubMed search,
of 2009. Since the publication of agents that act via the same mech- thereby permitting an assessment
Volume 100, mechanistic informa- anism. For example, internalized of the extent to which Section 4
tion has become available on two radionuclides that emit α-particles (“Other relevant data”) of the IARC
additional Group 1 agents: diesel are discussed in the Monographs as Monographs captured all relevant
engine exhaust (reviewed in Volume a group with the same mechanism information on these end-points.
105; Benbrahim-Tallaa et al., 2012; of action. Birkett et al. (2019) re- The analyses in the present chapter
IARC, 2013) and trichloroethylene viewed the mechanistic information are restricted to information taken

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 261


Table 22.3. Relationship between 86 agents used in the analysis of key characteristics of human carcinogens and
111 agents used in the analysis of concordance between tumours and tumour sites in humans and animals

Volumea Agent 86 agents used in the analysis of key 111 agents used in the analysis of
number characteristics concordance between tumours and tumour
sites in humans and animals

100A 1 Aristolochic acid Aristolochic acid


Aristolochic acid, plants containing

100A 2 Azathioprine Azathioprine

100A 3 Busulfan Busulfan

100A 4 Chlorambucil Chlorambucil

100A 5 Chlornaphazine Chlornaphazine

100A 6 Cyclophosphamide Cyclophosphamide

100A 7 Ciclosporin Ciclosporin

100A 8 Diethylstilbestrol Diethylstilbestrol

100A 9 Estrogen-only menopausal therapy Estrogen-only menopausal therapy

100A 10 Estrogen–progestogen menopausal therapy Estrogen–progestogen menopausal therapy


(combined) (combined)

100A 11 Estrogen–progestogen oral contraceptives Estrogen–progestogen oral contraceptives


(combined) (combined)

100A 12 Etoposide in combination with cisplatin Etoposide


(Group 2A) and bleomycin (Group 2B) Etoposide in combination with cisplatin and
bleomycin

100A 13 Melphalan Melphalan

100A 14 PUVA (psoralen–UVA photochemotherapy) Methoxsalen in combination with UVA

100A 15 MOPP MOPP

100A 16 Phenacetin Phenacetin


Phenacetin, analgesic mixtures containing

100A 17 1-(2-Chloroethyl)-3-(4-methylcyclohexyl)- 1-(2-Chloroethyl)-3-(4-methylcyclohexyl)-


1-nitrosourea (Methyl-CCNU) 1-nitrosourea (Methyl-CCNU)

100A 18 Tamoxifen Tamoxifen

100A 19 Thiotepa Thiotepa

100A 20 Treosulfan Treosulfan

100B 21 Opisthorchis viverrini and Clonorchis sinensis Clonorchis sinensis (infection with)
Opisthorchis viverrini (infection with)

100B 22 Epstein–Barr virus Epstein–Barr virus

100B 23 Helicobacter pylori Helicobacter pylori (infection with)

100B 24 Hepatitis B virus Hepatitis B virus

100B 25 Hepatitis C virus Hepatitis C virus

100B 26 Human immunodeficiency virus type 1 Human immunodeficiency virus type 1

100B 27 Human papillomavirus Human papillomavirus

262
Table 22.3. Relationship between 86 agents used in the analysis of key characteristics of human carcinogens
and 111 agents used in the analysis of concordance between tumours and tumour sites in humans and animals
(continued)

Volumea Agent 86 agents used in the analysis of key 111 agents used in the analysis of
number characteristics concordance between tumours and tumour
sites in humans and animals

100B 28 Human T-cell lymphotropic virus type 1 Human T-cell lymphotropic virus type 1

100B 29 Kaposi sarcoma-associated herpesvirus Kaposi sarcoma-associated herpesvirus

100B 30 Schistosoma haematobium Schistosoma haematobium (infection with)

100C 31 Arsenic and inorganic arsenic compounds Arsenic and inorganic arsenic compounds

100C 32 Asbestos (all forms, including actinolite, amosite, Asbestos (all forms, including actinolite, amosite,
anthophyllite, chrysotile, crocidolite, and anthophyllite, chrysotile, crocidolite, and
tremolite) tremolite)

100C 33 Beryllium and beryllium compounds Beryllium and beryllium compounds

100C 34 Cadmium and cadmium compounds Cadmium and cadmium compounds

100C 35 Chromium(VI) compounds Chromium(VI) compounds

100C 36 Erionite Erionite

100C 37 Leather dust Leather dust

100C 38 Nickel and nickel compounds Nickel compounds

100C 39 Silica dust, crystalline, in the form of quartz or Silica dust, crystalline, in the form of quartz or
cristobalite cristobalite

100C 40 Wood dust Wood dust

100D 41 Solar and UV radiation UV radiation (bandwidth 100–400 nm,


encompassing UVC, UVB, and UVA)
UV-emitting tanning devices
Solar radiation

CHAPTER 22
PART 3
100D 42 X- and γ-radiation X- and γ-radiation
Ionizing radiation (all types)

100D 43 Neutron radiation Neutron radiation


100D 44 Internalized radionuclides that emit α-particles Haematite mining with exposure to radon
(underground)
Plutonium-239
Internalized radionuclides that emit α-particles
Thorium-232 (as Thorotrast)
Radium-224 and its decay products
Radium-226 and its decay products
Radium-228 and its decay products
Radon-222 and its decay products

100D 45 Internalized radionuclides that emit β-particles Fission products including Sr-90
Radioiodines, including iodine-131
Phosphorus-32, as phosphate
Internalized radionuclides that emit β-particles

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 263


Table 22.3. Relationship between 86 agents used in the analysis of key characteristics of human carcinogens
and 111 agents used in the analysis of concordance between tumours and tumour sites in humans and animals
(continued)

Volumea Agent 86 agents used in the analysis of key 111 agents used in the analysis of
number characteristics concordance between tumours and tumour
sites in humans and animals

100E 46 Consumption of alcoholic beverages Acetaldehyde associated with consumption of


alcoholic beverages
Alcoholic beverages
Ethanol in alcoholic beverages

100E 47 Betel quid and areca nut Areca nut


Betel quid with tobacco
Betel quid without tobacco

100E 48 Coal, indoor emissions from household Coal, indoor emissions from household
combustion of combustion of

100E 49 N′-Nitrosonornicotine (NNN) and 4-(Methyl- N′-Nitrosonornicotine (NNN) and 4-(Methyl-


nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

100E 50 Salted fish, Chinese-style Salted fish, Chinese-style

100E 51 Second-hand tobacco smoke Second-hand tobacco smoke

100E 52 Tobacco smoking Tobacco smoking

100E 53 Tobacco, smokeless Tobacco, smokeless

100F 54 Acid mists, strong inorganic Acid mists, strong inorganic

100F 55 Aflatoxins Aflatoxins

100F 56 Aluminium production Aluminium production

100F 57 4-Aminobiphenyl 4-Aminobiphenyl

100F 58 Auramine production Auramine production

100F 59 Benzene Benzene

100F 60 Benzidine Benzidine

100F 61 Benzidine, dyes metabolized to Benzidine, dyes metabolized to

100F 62 Benzo[a]pyrene Benzo[a]pyrene

100F 63 Bis(chloromethyl)ether; Chloromethyl methyl Bis(chloromethyl)ether; Chloromethyl methyl


ether (technical grade) ether (technical grade)

100F 64 1,3-Butadiene 1,3-Butadiene

100F 65 Coal gasification Coal gasification

100F 66 Coal-tar distillation Coal-tar distillation

100F 67 Coal-tar pitch Coal-tar pitch

100F 68 Coke production Coke production

100F 69 Ethylene oxide Ethylene oxide

100F 70 Formaldehyde Formaldehyde

100F 71 Iron and steel founding, occupational exposure Iron and steel founding, occupational exposure
during during

264
Table 22.3. Relationship between 86 agents used in the analysis of key characteristics of human carcinogens
and 111 agents used in the analysis of concordance between tumours and tumour sites in humans and animals
(continued)

Volumea Agent 86 agents used in the analysis of key 111 agents used in the analysis of
number characteristics concordance between tumours and tumour
sites in humans and animals

100F 72 Isopropyl alcohol manufacture using strong acids Isopropyl alcohol manufacture using strong acids

100F 73 Magenta production Magenta production

100F 74 4,4′-Methylenebis(2-chloroaniline) (MOCA) 4,4′-Methylenebis(2-chloroaniline) (MOCA)

100F 75 Mineral oils, untreated or mildly treated Mineral oils, untreated or mildly treated

100F 76 2-Naphthylamine 2-Naphthylamine

100F 77 ortho-Toluidine ortho-Toluidine

100F 78 Painter, occupational exposure as a Painter, occupational exposure as a

100F 79 2,3,7,8-Tetrachlorodibenzo-para-dioxin, 2,3,4,7,8-Pentachlorodibenzofuran


2,3,4,7,8-Pentachlorodibenzofuran, 2,3,7,8-Tetrachlorodibenzo-para-dioxin
3,3′,4,4′,5-Pentachlorobiphenyl 3,3′,4,4′,5-Pentachlorobiphenyl

100F 80 Rubber manufacturing industry, occupational Rubber manufacturing industry, occupational


exposures in the exposures in the

100F 81 Shale oils Shale oils

100F 82 Soot (as found in occupational exposure of Soot (as found in occupational exposure of
chimney sweeps) chimney sweeps)

100F 83 Sulfur mustard Sulfur mustard

100F 84 Vinyl chloride Vinyl chloride

105 85 Diesel and gasoline engine exhausts Engine exhaust, diesel

106 86 Trichloroethylene Trichloroethylene

CHAPTER 22
107 Polychlorinated biphenylsb

PART 3
109 Outdoor air pollutionb

109 Particulate matter in outdoor air pollutionb

UV, ultraviolet.
a IARC Monographs Volumes 100A (IARC, 2012e), 100B (IARC, 2012b), 100C (IARC, 2012a), 100D (IARC, 2012f), 100E (IARC,

2012d), 100F (IARC, 2012c), 105 (IARC, 2013), 106 (IARC, 2014), 107 (IARC, 2016b), and 109 (IARC, 2016a).
b Because the mechanistic sections for Monographs Volumes 107–109 were not available for review at the time that the present

analysis was conducted, Group 1 agents in these volumes were not included in the present analysis.

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 265


Fig. 22.1. Key characteristics of 86 Group 1 agents. The number of agents is shown above each characteristic.

directly from the IARC Monographs: animal in vivo and animal in vitro sources of information noted above.
Birkett et al. (2019) present the re- sources, it is possible to aggregate To evaluate the extent to which the
sults of a sensitivity analysis incorpo- this information according to human Group 1 agents demonstrated more
rating the additional information ob- and animal sources (by combining than one key characteristic, the num-
tained through the PubMed search. across in vivo and in vitro sources) ber of agents demonstrating multiple
After the collection of informa- or according to in vivo and in vitro characteristics was also tabulated.
tion on the toxicological end-points sources (by combining across hu- A heat map showing the number
identified by the Workshop partici- man and animal sources). Of primary
(0, 1, 2, 3, or 4) of sources of infor-
pants during the April 2012 meeting, interest here is aggregation across
mation (human in vivo, human in vi-
the database of key characteristics all four sources combined, to obtain
tro, animal in vivo, and animal in vitro
was then created by mapping the an overall indicator of whether any of
studies) supporting a given charac-
24 toxicological end-points to the the key characteristics is associated
teristic for a specified agent was pre-
10 characteristics as indicated in with each of the 86 Group 1 agents
pared, to evaluate the consistency
Table 22.2. As noted by Al-Zoughool of interest.
of information provided by different
et al. (2019), two of the toxicological
Statistical analysis sources. A heat map showing the
end-points – susceptibility and
changes in gene expression – did Descriptive statistical methods were overlap between human and animal
not link to any of the key character- used to explore the key characteris- sources of information (after combin-
istics, and thus were not included in tics associated with the 86 Group 1 ing in vivo and in vitro sources in both
the development of the database of agents, beginning with a tabulation cases) on the key characteristics was
key characteristics. Because the da- of the number of agents demonstrat- also prepared, to evaluate the extent
tabase includes information derived ing any of the 10 characteristics, to which there was overlap between
from human in vivo, human in vitro, both overall and stratified by the four these two sources.

266
Fig. 22.2. Sources of information on key characteristics of 86 Group 1 agents (sources are human in vivo, human in vitro, animal in vivo, and animal in vitro
studies).

Human In Vivo
Human In Vitro
Animal In Vivo
Animal In Vitro

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens


267
PART 3
CHAPTER 22
Overall mechanistic data were epigenetic alterations”, and “induc- for each agent. As in Fig. 22.1, the
also tabulated by type of agent es chronic inflammation”. Human in single most prominent characteristic
(pharmaceuticals; biological agents; vitro studies provide the most infor- was genotoxicity: many agents (HIV-
arsenic, metals, fibres and dusts; ra- mation on an additional three key 1 is a prominent exception) showed
diation; personal habits and indoor characteristics: “alters DNA repair or a positive response for genotoxicity
combustions; and chemical agents causes genomic instability”, “induces in at least one of the four sources
and related occupations), to identify oxidative stress”, and “alters cell pro- of information, and for many agents
possible differences in mechanistic liferation, cell death, or nutrient sup- more than one source provided ev-
patterns by agent type. ply”, and equivalent information to idence of genotoxicity. For some
animal in vivo studies on “modulates agents (e.g. all radiation sources,
Results
receptor-mediated effects”. some pharmaceutical agents, and
The prominence of human stud- some chemical agents), genotoxicity
The key characteristics of the 86
ies as sources of information on was demonstrated in all four test sys-
Group 1 agents considered here are
the key characteristics of human tems, confirming that genotoxicity is
summarized in Fig. 22.1. The most
carcinogens may be attributed to central to the carcinogenic pathways
prevalent mechanistic characteristic
the increasing use of molecular and of these agents.
was “is genotoxic”, followed by “al-
genetic markers in human studies. Fig. 22.4 also shows that most
ters cell proliferation, cell death, or
Epidemiological studies conducted agents exhibit multiple key charac-
nutrient supply”, “induces oxidative
in the occupational or general envi- teristics, with evidence drawn from
stress”, “is electrophilic or can be
ronment often analyse biomarkers more than one source of mechanis-
metabolically activated to electro-
of DNA adduct formation, clasto- tic information. Radiation sources
philes”, and “induces chronic inflam-
genic effects, and gene mutations, and tobacco smoke are associated
mation”. Nearly all agents demon-
all of which reflect DNA damage. with many of the key characteristics,
strate genotoxicity as one of their
Therefore, human in vivo studies
mechanistic properties; a prominent suggesting that these agents act by
are a major source of information on
exception is human immunodeficien- multiple pathways.
genotoxicity.
cy virus type 1 (HIV-1). Evidence of Several Group 1 agents, including
Fig. 22.3 shows the number of several occupational exposures, are
genotoxicity was provided by expres-
agents demonstrating multiple char- complex mixtures of chemicals and
sion of the following toxicological
acteristics as evidenced from stud- other substances. Coal-tar pitch,
end-points: DNA damage, gene mu-
tations, and cytogenetic/clastogenic ies in animals and in humans. The occupational exposure to soot, and
effects (including chromosomal ab- 86 Group 1 agents considered here coke production have similar charac-
errations, micronucleus formation, exhibit an average of approximately teristics, probably due to the strong
and aneuploidy). four key characteristics; the modal presence in relevant workplaces of
Fig. 22.2 shows the key charac- value is two characteristics, exhibit- polycyclic aromatic hydrocarbons,
teristics exhibited by the 86 agents ed by 20 agents. All agents demon- although other factors such as the
classified according to the source of strate at least one key characteristic, nature of inorganic substances and
data (human in vivo, human in vitro, with two agents demonstrating nine their chemical composition could
animal in vivo, and animal in vitro characteristics and 14 agents show- also have a role. Other occupation-
studies) on these characteristics. ing six. No agent exhibited all 10 key ally relevant agents (e.g. exposures
Information on all the mechanistic characteristics. during iron and steel founding and
characteristics was available to dif- Fig. 22.4 presents a heat map aluminium production) demonstrate
ferent degrees from all four sourc- indicating the strength of evidence only a single key characteristic, al-
es. Information on genotoxicity was of the different characteristics for though this may reflect the difficulty
available from each of the four sourc- the 86 individual Group 1 agents. of testing for other characteristics
es for the majority of the agents. The intensity of the colour reflects in these occupational exposure
Human in vivo studies contribute the the number of sources of informa- situations.
most evidence on four of the 10 key tion (human in vivo, human in vitro, The degree of overlap between
characteristics for these 86 agents, animal in vivo, and animal in vitro human and animal sources of infor-
including “is genotoxic”, “induces studies) on each key characteristic mation on the 10 key characteristics

268
Fig. 22.3. Number of Group 1 agents demonstrating one or more key characteristics.

of human carcinogens is shown in icity. Ten agents – diethylstilbestrol, oxidative stress”), but Fig. 22.5 clar-
the heat map in Fig. 22.5. This heat Kaposi sarcoma-associated herpes- ifies that there are both human and

CHAPTER 22
map, prepared by combining the in virus, arsenic and inorganic arsenic animal data for only five of these.

PART 3
vivo and in vitro sources of informa- compounds, cadmium and cadmium For chlornaphazine, Fig. 22.4 shows
tion on the key characteristics for compounds, asbestos, crystalline two sources of information, for “is
humans and for animals, indicates silica, solar and ultraviolet radiation, electrophilic or can be metabolical-
whether information on the key char- sulfur mustard, diesel and gaso- ly activated to electrophiles” and “is
acteristics for a given agent is de- line engine exhausts, and trichloro- genotoxic”, whereas the correspond-
rived from both human and animal ethylene – demonstrate overlap be- ing data in Fig. 22.5 show overlap
sources (reflecting concordance tween human and animal sources of between human and animal sources
between humans and animals), from information for at least five of the key only for “is electrophilic or can be
human sources alone, from animal characteristics. metabolically activated to electro-
sources alone, or from neither of Comparisons between the results philes”, with human but not animal
these. These results indicate overlap in Fig. 22.4 and Fig. 22.5 can pro- data on “is genotoxic”.
between human and animal sourc- vide additional insights into the key Fig. 22.6 shows the key character-
es of information for several agents. characteristics of the Group 1 agents istics of the six categories of Group 1
The concordance is particularly considered here. For example, in the agents considered in Volume 100:
strong for genotoxicity: information case of diethylstilbestrol, Fig. 22.4 pharmaceuticals; biological agents;
from both human and animal sourc- indicates that there is information arsenic, metals, fibres, and dusts;
es is available for 63 of the 85 agents from 1, 2, or 3 sources on nine key radiation; personal habits and in-
demonstrating evidence of genotox- characteristics (all except “induces door combustions; and chemical

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 269


Fig. 22.4. Heat map showing the strength of evidence for key characteristics of 86 Group 1 agents according to
the number of information sources (sources are human in vivo, human in vitro, animal in vivo, and animal in vitro
studies).

270
Fig. 22.5. Heat map showing the degree of concordance between human and animal sources of information on key
characteristics of 86 Group 1 agents (after combining in vivo and in vitro sources of information for humans and for
animals).

CHAPTER 22
PART 3

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 271


Fig. 22.6. Key characteristics of 86 Group 1 agents by type of agent (expressed as a percentage of the number of
agents of each type demonstrating each of the 10 mechanistic characteristics): (a) pharmaceuticals; (b) biological
agents; (c) arsenic, metals, fibres, and dusts; (d) radiation; (e) personal habits and indoor combustions; and
(f) chemical agents and related occupations.

272
Fig. 22.6. Key characteristics of 86 Group 1 agents by type of agent (expressed as a percentage of the number of
agents of each type demonstrating each of the 10 mechanistic characteristics): (a) pharmaceuticals; (b) biological
agents; (c) arsenic, metals, fibres, and dusts; (d) radiation; (e) personal habits and indoor combustions; and
(f) chemical agents and related occupations (continued).

CHAPTER 22
PART 3

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 273


Fig. 22.6. Key characteristics of 86 Group 1 agents by type of agent (expressed as a percentage of the number of
agents of each type demonstrating each of the 10 mechanistic characteristics): (a) pharmaceuticals; (b) biological
agents; (c) arsenic, metals, fibres, and dusts; (d) radiation; (e) personal habits and indoor combustions; and
(f) chemical agents and related occupations (continued).

274
agents and related occupations. al., 2019 and Birkett et al., 2019). Information drawn from the IARC
Genotoxicity was the most preva- The profiles of key characteristics Monographs showed that the over-
lent key characteristic demonstrat- of these agents show several inter- whelming majority of the agents ex-
ed by agents in the categories of esting patterns. First, all but seven amined here induce one or more of
pharmaceuticals; arsenic, metals, agents exhibited multiple character- these end-points. Even biological
fibres, and dusts; personal habits istics, an observation consistent with agents such as viruses that act pri-
and indoor combustions; and chem- previous findings on the complexity marily through non-genotoxic mech-
ical agents and related occupations, and heterogeneity of carcinogenic anisms induce cytogenetic effects
and genotoxicity was exhibited by pathways (Hanahan and Weinberg, and gene mutations as secondary
all agents in the category of radia- 2011; Floor et al., 2012; Baker, 2014; events through chronic inflammation
tion. Immortalization, genotoxicity, Pickup et al., 2014; Roessler et al., and oxidative stress.
and altered cell proliferation, cell 2014). Agents in the categories of Another important observation
death, or nutrient supply are promi- biological agents; arsenic, metals, fi- is that information on the key char-
nent characteristics of the biological bres, and dusts; personal habits and acteristics of the 86 Group 1 agents
agents. None of the biological agents indoor combustions; and radiation considered here is often derived from
demonstrated modulation of recep- demonstrated a wide spectrum of bi- multiple sources (human in vivo, hu-
tor-mediated effects, and none of the ological activity. Radiation has been man in vitro, animal in vivo, and ani-
agents in the category of personal linked to many hallmarks of cancer mal in vitro studies); for many agents,
habits and indoor combustions ap- (Boss et al., 2014): this mechanistic evidence is available from more than
peared to act through modulation of profile, with multiple pathways in- one of these sources. Concordance
receptor-mediated effects, through volved for most radiation agents, is between animal and human sources
immunosuppression or through im- consistent with the broad spectrum of information was seen for sever-
mortalisation. There are five agents of tumours associated with exposure al agents, particularly with respect
in the category of radiation, all of to ionizing radiation (Chapter 21, by to genotoxicity, an observation that
which demonstrate the following Krewski et al.). Viral oncogenesis lends additional support to the rele-
key characteristics: is genotoxic; al- is also multifaceted, and the multi- vance of animal data for human can-
ters DNA repair or causes genomic step nature of viral oncogenesis is cer risk assessment.
instability; induces oxidative stress; thought to be influenced by host ge- Some caution must be used in
causes immortalization; and alters netic variability (Mesri et al., 2014). interpreting the distribution of key
cell proliferation, cell death, or nu- Genotoxicity was the most prev- characteristics across the Group 1

CHAPTER 22
trient supply. The profiles of key alent mechanistic characteristic, agents considered here. It is possi-

PART 3
characteristics for pharmaceutical demonstrated by 85 of the 86 agents ble that the near universality of ge-
agents and for chemical agents and considered, possibly reflecting the notoxicity as a carcinogenic mech-
related occupations are remarkably fact that the process of carcino- anism may be related to the way in
similar, possibly reflecting the fact genesis necessarily involves geno- which the IARC Monographs were
that despite their different expo- mic changes. This finding is consis- compiled, with emphasis on the re-
sure circumstances, some of these tent with an earlier evaluation of 180 porting of genotoxicity data. This
chemical entities may act via similar Group 1, Group 2A, and Group 2B would have been partially mitigat-
mechanisms. agents conducted by Bartsch and ed by the inclusion of mechanistic
Malaveille (1989), who reported that information from outside the IARC
Discussion 80–90% of the agents in these three Monographs in the preparation of
categories demonstrated genotoxic the mechanistic database evaluated
The present analysis of key charac- characteristics. In the present ana- separately by Birkett et al. (2019). It
teristics of 86 agents classified as lyses, genotoxicity was considered should also be noted that the IARC
carcinogenic to humans (Group 1) by to include the following end-points: Monographs have been published
the IARC Monographs Programme DNA damage, cytogenetic effects over a long time span, extending
was based on mechanistic infor- (including chromosomal aberra- from the early 1970s to the present
mation retrieved from the IARC tions, micronucleus formation, and (Saracci and Wild, 2015). Studies of
Monographs (see Al-Zoughool et aneuploidy), and gene mutations. agents in earlier Monographs would

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 275


have focused on changes such as information sources, of which the The epigenetic characteristics of
DNA damage that could have been most notable was the identification the 74 Group 1 agents considered
detected by the techniques available of evidence for six additional agents in Volumes 100A–E were assessed
at that time. These agents may not that demonstrate modulation of re- by Herceg et al. (2013). As in the
have been evaluated exhaustively ceptor-mediated effects, beyond present analysis, those authors used
for pathways that have been iden- the seven agents noted in Fig. 22.1. DNA methylation, histone modifica-
tified more recently, such as those Nonetheless, the overall findings are tion, and altered expression of miR-
involving the multifactorial nature largely comparable with those pre- NAs as indicators of epigenetic alter-
of carcinogenesis, or for the multi- sented without the additional data ations. They considered information
plicity of pathways operating dur- search (for further details, see Birkett from both the IARC Monographs
ing the process of agent-induced et al., 2019). and the general scientific literature,
carcinogenesis. As the IARC Monographs Pro- and identified 22 of the 74 Group 1
A related limitation of the present gramme has evolved from its incep- agents (30%) as demonstrating epi-
analysis is that it did not distinguish tion in the early 1970s until the pres- genetic effects. The present analy-
direct genotoxicity of the agent or its ent time, the guidelines for carcin- sis, which examined Group 1 agents
metabolites from genotoxicity that ogen identification as set out in the in Volumes 100A–F as well as
occurs as a result of other respons- Preamble to the IARC Monographs Volumes 105 and 106, identified 33
es, because this information was (IARC, 2006) have been updated of the 86 Group 1 agents (38%) as
not generally provided in the IARC from time to time, with increasing mediating epigenetic change.
Monographs from which the mech- emphasis on the use of mechanis- In an earlier evaluation, Hernán-
anistic data on the Group 1 agents tic information in the overall eval- dez et al. (2009) reported that 45 of
were abstracted. It is recommended the 371 agents (12%) in Groups 1,
uation in the most recent updates.
that the distinction between primary 2A, and 2B at the time of their analy-
Nonetheless, the identification of
and secondary genotoxic effects be sis were not genotoxic. In their study,
Group 1 agents continues to rest
noted in future Monographs, when- an agent was considered non-ge-
heavily on the availability of suffi-
ever possible. notoxic if it showed negative results
cient evidence of carcinogenicity in
Another limitation of the present in the Salmonella mutagenicity as-
epidemiological or clinical studies.
results is that they are based on say (the Ames test) as well as in
Of the 111 distinct Group 1 agents
the information on mechanisms in the mouse lymphoma assay, the in
identified up to and including Volume
Section 4 (“Other relevant data”) of vitro chromosomal aberration test,
109, no less than 102 demonstrated
the IARC Monographs, which fo- the in vitro micronucleus test, the
sufficient evidence of carcinogeni-
cused primarily on “established” and in vivo micronucleus test, and the
city in humans, and the remaining
“likely” mechanisms. A full systemat- in vivo chromosomal aberration test
nine agents were placed in Group 1
ic review of the entire literature on bi- in bone marrow in rodents. These
after reference to mechanistic data
ological mechanisms for all Group 1 results support the role of non-ge-
or other considerations (as “mech- notoxic pathways in carcinogenesis,
agents was not undertaken, so the
anistic upgrades” according to the an observation that is reinforced by
database may not reflect all mech-
anistic characteristics of the differ- evaluation criteria outlined in the the prevalence of multiple charac-
ent agents. As a sensitivity analysis Preamble to the IARC Monographs; teristics of human carcinogens not
to examine the extent to which the see Table 21.4, in Chapter 21, by associated with genotoxicity in the
Monographs captured most of the Krewski et al.). Despite the inherent present analysis.
relevant information in this regard, reliance on human epidemiological To ensure that all relevant evi-
Birkett et al. (2019) conducted a data in identifying agents that may dence on the 10 key characteristics
supplementary PubMed search to increase human cancer risk, Section of human carcinogens is taken into
identify additional information on 4 (“Other relevant data”) of the IARC account in future Monographs eval-
key characteristics not cited in the Monographs increasingly provides uations of agents that may cause
Monographs, or published since detailed descriptions of the mecha- cancer in humans, a carefully de-
2009. Although this sensitivity anal- nisms by which agents under review signed systematic review of the sci-
ysis was not based on an exhaus- may act, including agents not as- entific literature would be required
tive search, it did identify additional signed to Group 1. in conjunction with each evaluation.

276
However, to conduct a series of com- The 10 key characteristics are and reproducible manner. The EPA
prehensive systematic reviews of the features of carcinogens rather than is also currently supporting the de-
key characteristics of all 86 agents mechanisms. The analysis presented velopment of software tools specifi-
considered in the present analysis here does not address the sequence cally designed for systematic review
would require a considerable ef- of events involved in carcinogenesis. of toxicological and epidemiological
fort, and was not attempted as part For example, if the carcinogenic data (ICF, 2017).
of the present project. The expert mechanism of action is being in- The strong evidence linking geno-
opinion of future IARC Working vestigated for a genotoxic agent toxicity to carcinogenesis is consis-
Groups charged with evaluating the that requires metabolic activation, tent with epidemiological data and
mechanistic data on new agents the mechanism needs to consider experimental research. Genotoxic
selected for evaluation by the IARC the entire metabolic pathway. If the effects include the formation of DNA
Monographs would be of consider- agent is not metabolized to produce adducts or induction of single- and
able value in this regard, but would an electrophile, DNA damage will double-strand DNA breaks. Several
ideally be supported by a concomi- not occur. In such a case, biological lines of evidence from epidemio-
tant systematic review of the relevant effects that occur after induction of logical studies and in experimental
scientific literature on the key char- DNA damage also would not be ob-
animals and model systems have
acteristics to ensure that the analysis served. This sequential relationship
shown that DNA adducts are strongly
would be as complete as possible. is also apparent for characteristics
associated with cancer (Kriek et al.,
Another issue that arises when such as chronic inflammation, which
1998; Phillips et al., 2015). Some
discussing key characteristics of hu- acts through the production of oxida-
genotoxic effects can lead to gene
man carcinogens is whether indirect tive stress, release of cytokines, and
mutation, an important event in the
effects should be considered. Many stimulation of cell proliferation, which
pathway towards carcinogenesis,
agents have a direct carcinogenic ultimately produces DNA damage.
especially if it involves oncogenes or
effect, but in other cases the carci- The results of the present analysis
tumour suppressor genes. Chromo-
nogenic characteristic is the result of can provide a basis for future efforts
somal aberrations are another type
a secondary event along the mecha- to categorize mechanistic data for
of genetic alteration that occurs fre-
nistic pathway. For example, cell pro- carcinogens through a systemat-
quently in many tumours, especial-
liferation can arise either as a result of ic review process. A full systematic
review of all agents and all potential ly solid tumours. Most tumour cells
a direct action of the agent on the cell
carcinogenic mechanisms is an in- display aneuploidy, and for some tu-
or indirectly, as a result of cytotoxici-
timidating prospect. However, such mours, characteristic chromosomal
ty that stimulates cell proliferation to

CHAPTER 22
a review would provide a more com- abnormalities have been identified
replace cells, through alterations in

PART 3
prehensive examination of mech- (e.g. the Philadelphia chromosome
cell signalling without cytotoxicity, or
via inhibition of cell proliferation that anisms, because it would include in chronic myeloid leukaemia).
then results in selection of an altered studies that failed to find effects. It The complexity of the pathways
clone of cells with a high prolifera- might also support a process that involved in carcinogenesis and the
tion rate. Although the downstream involves a sequence of mechanistic fact that the cellular response to car-
effect is the same (increased cell steps and mechanistic characteris- cinogen exposure is modulated by
proliferation), the pathway leading to tics relevant to the development of host cell physiology, genetics, and
that result can be different. A similar cancer in humans. other variables have prompted the
issue arises with genotoxicity: many The importance of systematic re- development and application of sen-
agents are not directly genotoxic but view in assembling all relevant evi- sitive assays that measure toxicity
cause DNA damage by stimulating dence on a particular issue has been pathways and perturbations in the
a chain of molecular changes (e.g. emphasized in the recent review of molecular functioning of the cell. The
chronic inflammation). The current the EPA’s Integrated Risk Information newly proposed toxicological test-
database does not contain the in- System (IRIS) (National Research ing paradigm (Krewski et al., 2014)
formation needed to address these Council, 2014) and is currently being focuses on high-throughput screen-
issues and cannot be used to draw implemented within the IRIS pro- ing to detect changes in the molec-
conclusions about the detailed gramme as a way of summarizing ular pathways of the cell in response
mechanism of action of an agent. all relevant data in a comprehensive to chemical exposure. This new

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 277


paradigm would be useful in com- four characteristics per agent, no work of Krewski et al. (Chapter 21)
prehensive cancer risk assessment attempt was made to conduct a mul- that examined concordance be-
and would be able to detect distinct tivariate analysis of these character- tween animals and humans for 39
key mechanistic pathways operating istics to determine whether similar tumour sites and 14 organ and tissue
after carcinogen exposure. In a sim- agents tended to express similar systems, based on the database on
ilar initiative, the Kyoto Encyclopedia characteristics. Recalling that phar- tumours and tumour sites in humans
of Genes and Genomes (KEGG) maceuticals as a class demonstrat- and experimental animals developed
website has compiled a comprehen- ed a mechanistic profile similar to by Grosse et al. (Annex 1). The pro-
sive list of pathways associated with that of chemical agents and related files of key characteristics of agents
specific diseases (see the KEGG occupations, it is possible that the associated with specific tumour sites
pathway database at http://www.ge­ chemotherapeutic agents and some could be examined to obtain addi-
nome.jp/kegg/pathway.html). KEGG of the chemical agents act via the tional insights into the mechanisms
also identified major in vitro assays same carcinogenic mechanisms. by which specific tumours occur. It
that can be used to detect targets of Cyclophosphamide and benzene would be of particular interest to an-
these pathways. This attempt to un- (once used as a chemotherapeutic alyse whether certain tumour sites
derstand the biological mechanisms agent) may have some commonality demonstrate signature profiles.
of carcinogenesis is consistent with in this respect, as might treosulfan Extending the mechanistic data-
current practice of using in vitro as- and butadiene through the formation base to include additional informa-
says to detect changes in critical of the same diepoxide. Further study tion such as structural alerts rele-
signalling and other molecular path- of these two groups, in terms of both vant to carcinogenesis could also
ways in cancer development, as pro- mechanism of action and tumour site be informative. Although the present
posed by Krewski et al. (2014). concordance, may provide insight version of the mechanisms database
into tumours that result from long- includes the International Union of
Further analyses term exposure to chemotherapeutic Pure and Applied Chemistry (IUPAC)
The extensive database on key cha- agents. International Chemical Identifier
racteristics of human carcinogens Searching for patterns within (InChI) for key chemical coding (Stein
developed here offers considerable homogeneous classes of agents et al., 2003; Heller et al., 2015), this
potential for further analysis. More would also be of future research in- information has not been taken into
in-depth analyses are under way terest. For example, one could ex- account in the analyses completed to
to explore the level of agreement amine mechanistic patterns within date. One possible source of auxilia-
between mechanistic data derived subgroups of pharmaceuticals, in- ry information on toxicological end-
from human sources on the one cluding antineoplastic agents, hor- points that may be related to the 10
hand and from animal sources on monal products, immunosuppres- key characteristics is EPA’s Toxicity
the other, as well as from in vivo and sants, and analgesic mixtures. In a Forecaster (ToxCast) programme
in vitro sources, issues that have similar vein, Shin et al. (2015) have (Judson et al., 2014; Knudsen et al.,
received only limited attention here. recently used bioactivity profiles for 2015), which now includes in vitro,
An analysis of the key characteris- 38 agents derived from high-through- in vitro, and in silico data on diverse
tics demonstrated by Group 1 agents put in vitro assays to investigate pat- toxicological end-points for more
on a site-specific basis is also plan- terns of toxicity associated with dif- than 10 000 chemical substances,
ned; if agents that cause tumours ferent scenarios of use. some of which overlap with the set
at a specific site (e.g. the lung or Exposure to a single agent may of Group 1 agents considered in
the liver) are shown to demonstrate result in the development of more this chapter. The ToxCast database
similar characteristics, this could than one type of tumour, perhaps also includes information on several
provide new insights into site-speci- through different pathways that in- hundred toxicological assays, which
fic carcinogenesis. volve different mechanistic charac- could enrich the database of key
Although the present anal- teristics. It would be of interest to characteristics used in the present
ysis found that most Group 1 examine the key characteristics for analysis.
agents demonstrated multiple key agents associated with specific tu- With the elaboration of the 10
characteristics, with an average of mour types. This would extend the key characteristics articulated by

278
Smith (Chapter 10) and Smith et al. There could be value in revisiting relevant mechanistic information.
(2016), mechanistic evaluations of the present retrospective analysis Although this approach could expe-
new agents undertaken within the of the 86 Group 1 agents identified dite identification of relevant articles,
IARC Monographs Programme in the IARC Monographs up to and expert opinion and application of
are beginning to make use of these including Volume 106, with respect weight-of-evidence criteria would still
characteristics, including the use of to the conduct of a series of compre- have value in reducing the errors in
formal methods of systematic review hensive systematic reviews on the 10 the assignment of key characteristics
to identify relevant mechanistic infor- key characteristics of these agents, to specific agents.
mation. This has been successfully followed by an in-depth evaluation
attempted in recent evaluations of of the findings of the systematic re- Conclusions
some organochlorine insecticides views by experts in relevant disci-
and some chlorophenoxy herbicides plines. The development of criteria In considering the results present-
(Loomis et al., 2015; Volume 113) for evaluating the weight of evidence ed in this chapter, it is important to
and of red meat and processed meat for the key characteristics, similar to emphasize that these mechanistic
(Bouvard et al., 2015; Volume 114). that included in the Preamble to the analyses are a first step in under-
It is expected that the search IARC Monographs for human and standing the biological mechanisms
strategies used in future mechanistic animal data (IARC, 2006), might be by which cancer may occur in hu-
evaluations will be refined as expe- mans. Although considerable effort
contemplated at that time. Group 1
rience with the key characteristics was expended in developing the
agents identified beyond Volume 106
accumulates. In an earlier evaluation database of key characteristics and
for which mechanistic information
of evidence of epigenetic alterations their analyses in this chapter, these
had become available could also be
for 28 Group 1 agents, Chappell results should be viewed as prelim-
included in such an analysis.
et al. (2016) searched for evidence of inary, to be refined through more
Baker et al. (2016) have recently
DNA methylation, histone modifica- exhaustive systematic reviews of
applied supervised machine learn-
tion, and expression of non-coding the relevant scientific literature and/
ing techniques to classify PubMed
miRNAs, as was done in the pres- or through discussion with a broad
literature according to the hallmarks
ent analysis, but with the addition of
of cancer (Hanahan and Weinberg, panel of experts on the mechanisms
several more detailed search terms,
2000, 2011). In a case study of basal of carcinogenesis. The 10 key char-
specifically long non-coding RNA
cell carcinoma and melanoma, only acteristics were endorsed by the
(lncRNA), small RNA, chromatin,
46,727 of 121,488 abstracts from participants in the IARC Workshop
and promotor methylation. Chappell

CHAPTER 22
their original systematic literature on Tumour Site Concordance and
et al. (2016) noted that the great ma-

PART 3
search were classified as relevant, Mechanisms of Carcinogenesis,
jority (89%) of the studies on lncR-
reflecting the potential time savings which provided oversight for this
NAs included in their review report-
that may be achieved through auto- project; additional experience with
ed alterations in miRNAs, leading
matic classification. An approach to the exploration of these characteris-
to results largely consistent with the
extracting information on the 10 key tics in cancer research will serve to
search terms used here: 43% (12 of
28) of the agents evaluated by these characteristics of human carcinogens define their utility more fully. Equally
authors demonstrated evidence of would be to apply these machine important is to consider the nature
epigenetic alterations, similar to the learning techniques and biomedical of the evidence needed to establish
38% (33 of 86) of agents included in text mining methods to identify, in an that specific mechanistic charac-
the present analysis. Continued ex- automated fashion, articles associ- teristics are associated with human
perience with the evaluation of the ating these key characteristics with carcinogens. The current database
10 key characteristics of human car- specific Group 1 agents. Because has relied on the demonstration of
cinogens can be expected to further of the sheer size of a full systemat- certain toxicological end-points as
refine the criteria used for their iden- ic review of mechanistic information evidence of these mechanistic char-
tification, including both the toxico- on all Group 1 agents, the use of acteristics; further consideration of
logical events associated with these automated search algorithms of this these and other possible markers
key characteristics and the assays type could offer considerable effi- of the key characteristics of human
used as evidence of these events. ciency gains in identifying potentially carcinogens is warranted.

Part 3 • Chapter 22. Analysis of key characteristics of human carcinogens 279


Mechanistic considerations are extracted from the Monographs to Further in-depth analyses of this rich
becoming increasingly prominent assemble a database on the basis database of characteristics of human
in the IARC Monographs, thereby of 10 key characteristics of human carcinogens are expected to provide
enriching the body of evidence on carcinogens. After some grouping of additional insights into the mecha-
which future analyses of this type similar agents, the characteristic pro- nisms of human carcinogenesis.
may be based. The authors are plan- files were examined for 86 Group 1
ning to update the mechanistic da- agents for which mechanistic infor- Acknowledgements
tabase to include Monographs pub- mation was available in the IARC
Monographs up to and including The authors are grateful to Kate Z.
lished subsequent to Volume 106, a
Volume 106, based on information Guyton for many helpful comments,
task that will be greatly facilitated by
derived from human in vivo, human which resulted in both improvements
the documentation of key character-
in vitro, animal in vivo, and animal in the presentation of this chapter
istics of agents evaluated in recent
in vitro studies. The most prevalent and additional insights into the find-
Monographs.
key characteristic was “is genotoxic”, ings in this chapter, and to the editors
Summary followed by “alters cell proliferation, for their thorough review of several
cell death, or nutrient supply” and “in- preliminary drafts. Mélissa Billard
Since its inception in the early 1970s, duces oxidative stress”. Most agents was a Visiting Scientist at IARC
the IARC Monographs Programme exhibited several of the 10 key char- during the summers of 2013 and
has evaluated more than 1000 acteristics, with an average of four 2014, working under the direction of
agents with respect to their carci- characteristics per agent, a finding Yann Grosse and Robert Baan on
nogenic hazard; of these, up to and consistent with the notion that can- assembling the mechanistic data-
including Volume 119 of the IARC cer development in humans involves base analysed in this chapter. Julian
Monographs, 120 agents met the multiple pathways. Information on Little is the Canada Research Chair
criteria for classification as carcino- the key characteristics was often in Human Genome Epidemiology
genic to humans (Group  1). Volume available from multiple sources, with at the University of Ottawa. Daniel
100 of the IARC Monographs provid- many agents demonstrating concor- Krewski is the Natural Sciences and
ed a review and update of Group 1 dance between human and animal Engineering Research Council of
carcinogens. These agents were sources, particularly with respect Canada Chair in Risk Science at the
divided into six broad categories: to genotoxicity. Although a detailed University of Ottawa. This chapter is
pharmaceuticals; biological agents; comparison of the characteristics of dedicated to the memory of Jan M.
arsenic, metals, fibres, and dusts; different types of agent was not at- Zielinski, who succumbed to can-
radiation; personal habits and indoor tempted here, the overall characteris- cer in 2016; before becoming ill, Dr
combustions; and chemical agents tic profiles for pharmaceutical agents Zielinski led the analytical team that
and related occupations. Data on bi- and for chemical agents and relat- conducted the analyses reported
ological mechanisms of action were ed occupations appeared similar. here.

280
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282
annex 1.

Development of a data set on


tumours and tumour sites in
humans and in experimental
animals for Group 1 agents
identified up to and including
Volume 109 of the
IARC Monographs
Yann Grosse, Pascale Lajoie, Mélissa Billard, Daniel Krewski, Jerry M. Rice,
Vincent J. Cogliano, Michael Bird, and Jan M. Zielinski (deceased)

Introduction conducted by international Working For Volume 100 of the IARC


Groups of expert scientists accord- Monographs, a review was under-
Since its establishment in the ear- ing to a well-established and rigor- taken of relevant information on all
ly 1970s, the IARC Monographs ous protocol that is described in the the agents classified in Group 1 (car-
Programme has conducted hazard Preamble to the IARC Monographs cinogenic to humans). For conve-
ANNEX 1

evaluations of agents that may in- (IARC, 2006). The reviews and eval- nience, Volume 100 was organized
crease the risk of cancer in humans. uations are published as the IARC in six parts (100A–100F), covering
The reviews of the relevant literature Monographs on the Evaluation of pharmaceuticals (IARC, 2012e);
and the ensuing evaluations are Carcinogenic Risks to Humans. biological agents (IARC, 2012b);

Annex 1. Development of a data set on tumours and tumour sites in humans and in experimental
animals for Group 1 agents identified up to and including Volume 109 of the IARC Monographs
283
arsenic, metals, fibres, and dusts what level of concordance may exist become less reliable or impossible.
(IARC, 2012a); radiation (IARC, between humans and experimen- Therefore, although the data set
2012f); personal habits and indoor tal animals with respect to tumours described in this Annex (Table A1;
combustions (IARC, 2012d); and and tumour sites. To this end, the online only; available from: http://
chemical agents and related occu- pertinent information in Volume 100 publications.iarc.fr/578) provides in-
pations (IARC, 2012c).The reviews was captured in a comprehensive ta- formation on all the Group 1 agents,
and analyses were discussed dur- ble (Table A1; online only; available the actual database of human carcin-
ing a two-part Workshop on Tumour from: http://publications.iarc.fr/578) ogens eligible for the concordance
Site Concordance and Mechanisms that could then serve as a basis to analysis is appreciably smaller (see
of Carcinogenesis, which was con- develop a database on tumour sites Chapter 21, by Krewski et al.).
vened by IARC on 16–18 April 2012 in animals and humans. The creation
and 28–30 November 2012 in Lyon. of such a database – designed to be Methods
The data set described in this amenable to biostatistical analysis
In making an evaluation of the evi-
Annex also includes information on (see Chapter 21, by Krewski et al.)
dence of carcinogenicity to humans,
five additional human carcinogens – was motivated by the interest in a
an IARC Monographs Working
that were added to Group 1 after statistical assessment of the degree
Group is generally asked to identi-
completion of Volume 100, i.e. diesel of concordance between animal and
fy organ sites in humans for which
engine exhaust (Volume 105; IARC, human tumour sites. This important
there is sufficient evidence of carci-
2013), trichloroethylene (Volume scientific question bears upon the
nogenicity of the agent under study.
106; IARC, 2014), polychlorinated bi- extent to which the animal cancer
However, the Working Group is not
phenyls (Volume 107; IARC, 2016b), data collected here may be extrap-
required to identify organ sites for
and outdoor air pollution and partic- olated to humans. It is anticipated
carcinogenicity in experimental ani­
ulate matter in outdoor air pollution that the database will also find other
mals at the time of the evaluation,
(Volume 109; IARC, 2016a). For applications, including in the devel-
but is required more simply to as-
ease of reference, these five agents opment of human tumour profiles to
sess the overall weight of the ev-
are included in an expanded group assist in the identification of addition- idence in experimental animals.
of chemical agents and related oc- al Group 1 agents. Consequently, for the purpose of this
cupations denoted by Volume 100F*. It should be noted that for agents IARC Scientific Publication, the spe-
Although additional Group 1 agents classified in Group 2A (probably car- cies-specific tumour sites in experi-
have been identified in subsequent cinogenic to humans) or Group 2B mental animals needed to be identi-
volumes of the Monographs, the (possibly carcinogenic to humans) fied for each Group 1 agent before
current data set extends only up to the information on cancer in humans proceeding to explore concordance
and including Volume 109, the last may often be lacking or may not be between animal and human cancers.
Monograph for which final results strong enough for a proper interspe- During the six meetings for
were available at the time this Annex cies comparison to be made. For this Volumes 100A–F, the respective
was completed. reason, the concordance analysis Working Groups identified studies in
The reviews and updates in (see Chapter 21, by Krewski et al.) is experimental animals that provided
Volumes 100A–F specifically fo- focused on agents in Group 1. In ad- results on species-specific tumour
cused on identification of tumours, dition, it was decided that sufficient sites. This was based on criteria
both in humans and in experimental evidence of carcinogenicity in hu- adapted from the Preamble to the
animals, resulting from exposure to mans and sufficient evidence of car- IARC Monographs. It was consid-
each of the Group 1 agents. In ad- cinogenicity in experimental animals ered that there is sufficient evidence
dition, the organs where the tumours were required for an agent to be in- for identifying a species-specific tu-
were reported to arise were docu- cluded in the statistical concordance mour site in experimental animals
mented where possible. The avail- analysis; with less than sufficient under any one of the following three
ability of this information on the more evidence of carcinogenicity, in hu- conditions:
than 100 human carcinogens in mans or in animals, the definition of • An increased incidence of mali-
Group 1 prompted an investigation of a tumour site in either species would gnant neoplasms or an appropriate

284
combination of benign and mali- Workshop participants to confirm the data set (see Table A1; online only;
gnant neoplasms originating from entries. Ultimately, more than 2000 available from: http://publications.
the same organ (or tissue) is iden- studies were reviewed, and more iarc.fr/578) but is not included in the
tified in two or more independent than 1000 of these contributed to the statistical analysis of concordance
studies in one species carried out at identification of species-specific tu- (see Chapter 21, by Krewski et al.).
different times or in different labora- mour sites in experimental animals. All the information on tumours
tories or under different protocols. Studies were not considered if any and tumour sites in humans and in
• An increased incidence of malig- one of the following exclusion de- experimental animals from IARC
nant neoplasms or an appropriate scriptors was applicable: Monographs Volumes 100–109 is
combination of benign and malig- • initiation–promotion studies; given in Table A1 (online only; avail-
nant neoplasms originating from • co-carcinogenicity studies; able from: http://publications.iarc.
the same organ (or tissue) is iden- • studies in genetically modified fr/578).
tified in both sexes of one species animals;
in one well-conducted study, ideally • studies with precancerous lesions Observations
performed under good laboratory as the outcome;
practice (GLP). For some Group 1 agents, there
• studies on the carcinogenicity of
• A single study in one species and were only a few studies that contrib-
metabolites and derivatives;
sex might be considered to provide uted to the identification of a tumour
• studies with non-laboratory animals
sufficient evidence for a specific site in experimental animals, and fre-
(livestock; companion animals);
organ site when malignant neo- quently the studies did not enable the
• studies with analogous agents
plasms occur to an unusual degree definition of an organ site, as a result
(similar chemical structure or simi-
with regard to incidence, type of tu- of inadequate reporting. There were
lar virus type).
mour, or age at onset. many instances where the reported
Confirmation of the tumours and tumour incidences were uninforma-
Results
tumour sites identified in Volume 100 tive, possibly as a result of the small
was performed by one member each Table A2 illustrates the format of number of animals tested. In other
from the IARC secretariat and from the data set on tumours and tumour cases, studies reported an increased
the project team at the University of sites, with one agent from each of incidence of tumours but without
Ottawa, Canada, who systematically Volumes 100A–F. From epidemio- mention of malignancy or proper de-
consulted the original publications logical studies, human tumour sites scription of histopathological details.
describing the studies cited in the with sufficient evidence and those Also, some reports did not specify
Volume 100 reviews. It was decided with limited evidence are mentioned. the purity of the administered agent.
by the Workshop participants that ex- For experimental animals, tumour In these cases the experts in the
traction of the following information sites are recorded only for agents that Monographs Working Groups and
was required for each study: spe- demonstrate sufficient evidence of the two team members (one mem-
cies, strain, sex, route of exposure, carcinogenicity, as indicated above. ber each from the IARC secretari-
and tumour site including histology. Strain, sex, and route of exposure at and from the project team at the
Further information would be record- reported for each animal study are University of Ottawa, Canada) had to
ed as “study details”, for example also captured. Comments are pro- consider the possibility of confound-
dose, number of test animals, num- vided as appropriate. For example, ing, because the existence of other
ber of control animals, age at start no human tumour site is specified for agents in the administered sample
of exposure, duration of exposure, aristolochic acid, because this agent could have contributed to the out-
duration of follow-up, and statistical was placed in Group 1 on the basis of come. In some studies, animals were
analyses. The two team members the classification of plants containing followed up for only short periods of
independently captured the informa- aristolochic acid as a Group 1 agent, time after treatment, especially in
ANNEX 1

tion, and any disagreements were re- supported by mechanistic data on studies investigating acute adverse
solved in a group discussion. Tables genotoxicity (IARC, 2012e). Together effects, which precluded observation
summarizing this information were with other “mechanistic upgrades”, of carcinogenic outcomes that may
created to enable peer review by the this agent is listed in the complete take longer to develop.

Annex 1. Development of a data set on tumours and tumour sites in humans and in experimental 285
animals for Group 1 agents identified up to and including Volume 109 of the IARC Monographs
Table A2. Template for presentation of data on tumours and tumour sites in humans and in experimental animals from the IARC Monographs

286
Volume Agent Sites with Sites Agent Species Histology Study, sex, strain, Comments
100 part sufficient evidence with tested in Site exposure route
Agent in humans limited experimental
number evidence animals
in
humans

A Aristolochic Aristolochic Rat Squamous cell Mengs et al. (1982) (Volume The experts consider
1 acid acid Forestomach carcinoma 82; Volume 100A), MF, concordance when an
Wistar, g.; Mengs (1983) agent in humans such
(Volume 82; Volume 100A), as Plants containing
M, Wistar, g.; Schmeiser aristolochic acid is tested
et al. (1990) (Volume 100A), in animals by one of its
M, Wistar, d.w.; Hwang et al. main components such
(2006) (Volume 100A), M, as Aristolochic acid.
Sprague-Dawley, g.

A Aristolochic Aristolochic Rat Transitional Mengs et al. (1982) (Volume The experts consider
1 acid acid Renal pelvis cell carcinoma 82; Volume 100A), M, concordance when an
Wistar, g. agent in humans such
as Plants containing
aristolochic acid is tested
in animals by one of its
main components such
as Aristolochic acid.

B Clonorchis Cholangiocarcinoma No data on animal


24 sinensis experiments
(infection listed because of
with) limited evidence of
carcinogenicity.
Table A2. Template for presentation of data on tumours and tumour sites in humans and in experimental animals from the IARC Monographs (continued)

Volume Agent Sites with Sites Agent Species Histology Study, sex, strain, Comments
100 part sufficient evidence with tested in Site exposure route
Agent in humans limited experimental
number evidence animals
in
humans

C Arsenic and Lung, urinary Kidney, Dimethylarsinic Mouse Bronchiolo- DMA(V): Tokar
35 inorganic bladder, skin liver, acid [DMA(V)], Lung alveolar et al. (2012a), M, CD1, d.w.;
arsenic prostate Monomethyl- carcinoma Sodium arsenite: Waalkes
compounds arsonous acid et al. (2003), F, C3H/HeNCr,
[MMA(III)], in utero; Waalkes et al.
Sodium (2006a, b), M, CD1, in utero;
arsenite Tokar et al. (2011), MF,
CD1, in utero + p.o.; Tokar
et al. (2012a), M, CD1, in
utero; MMA(III): Tokar et al.
(2012b), M, CD1, in utero

C Arsenic and Lung, urinary Kidney, Sodium Mouse Hepatocellular Sodium arsenite: Waalkes
35 inorganic bladder, skin liver, arsenite, Liver carcinoma et al. (2003), M, C3H/HeNCr,
arsenic prostate Monomethyl- in utero; Waalkes et al.
compounds arsonous acid (2004a), M, C3H/HeNCr, in
[MMA(III)] utero; Waalkes et al. (2006a,
b), M, CD1, in utero; Tokar
et al. (2011), MF, CD1, in
utero + p.o.; Tokar et al.
(2012a), M, CD1, in utero;
MMAIII: Tokar et al. (2012b),
M, CD1, in utero

C Arsenic and Lung, urinary Kidney, Dimethylarsinic Rat Transitional Wei et al. (1999, 2002), M,
35 inorganic bladder, skin liver, acid [DMA(V)] Urinary cell carcinoma F344/DuCrj, p.o.; Arnold
arsenic prostate bladder et al. (2006), F, F344, p.o.
compounds

D Fission Solid cancers, Sr-90 Mouse Osteosarcoma Nilsson (1970, 1971), M,


45 products leukaemia Bone CBA, i.p.; Nilsson et al.
including (1980), F, CBA, i.p.

animals for Group 1 agents identified up to and including Volume 109 of the IARC Monographs
Annex 1. Development of a data set on tumours and tumour sites in humans and in experimental
Sr-90

287
ANNEX 1
Table A2. Template for presentation of data on tumours and tumour sites in humans and in experimental animals from the IARC Monographs (continued)

288
Volume Agent Sites with Sites Agent Species Histology Study, sex, strain, Comments
100 part sufficient evidence with tested in Site exposure route
Agent in humans limited experimental
number evidence animals
in
humans

D Fission Solid cancers, Sr-90 Dog Osteosarcoma Gillett et al. (1992), MF,
45 products leukaemia Bone beagle, i.v.; White et al.
including (1993), MF, beagle, p.o.;
Sr-90 Gillett et al. (1987), MF,
beagle, inh.

E Acetaldehyde Oesophagus and Sufficient evidence in


63 associated upper aerodigestive experimental animals but
with tract combined no organ sites identified
consumption due to the absence of
of alcoholic two (or more) studies
beverages of adequate design and
quality pointing at the
same organ site (with a
similar histological origin)
in the same species.

F Acid mists, Larynx Lung No animal data available.


75 strong
inorganic

b.pouch, buccal pouch; d.w., drinking water; F, positive female; g., gavage; i.col., intracolonic; i.f., intrafetal; i.m., intramuscular; i.mam., intramammary; inh., inhalation; i.p., intraperitoneal;
i.pulmo., intrapulmonary; i.t., intratracheal; i.v., intravenous; M, positive male; MF, positive male and female; NR, not reported; per., perinatal; p.o., feeding; s.c., subcutaneous; skin, skin
application.
Conclusions addressing the important scientific of Yann Grosse at IARC during the
question, i.e. to which extent these summers of 2011 and 2012. Mélissa
The data set developed here to de- animal cancer data are comparable Billard also contributed to the devel-
fine tumour sites for carcinogeni- with human cancer data. The value opment of the data set while working
city in humans and in experimental of this data set is demonstrated by as a Visiting Scientist at IARC during
animals summarizes all available the initial concordance analyses that the summers of 2013 and 2014 un-
data on Group 1 agents identified have been conducted with the data- der the direction of Robert Baan and
in Volumes 100–109 of the IARC base derived from it (see Chapter 21, Yann Grosse. Daniel Krewski is the
Monographs. At the time of com- by Krewski et al.). Natural Sciences and Engineering
pletion of Volume 109, a total of 111 Research Council of Canada Chair
Group 1 agents had been identified, Acknowledgements in Risk Science at the University of
and these are included in the list Ottawa.
presented in Table A1 (online only; Pascale Lajoie assembled the data
available from: http://publications. set on tumours and tumour sites
iarc.fr/578). This comprehensive set presented here while working as a
of data constitutes a unique basis for Visiting Scientist under the direction

ANNEX 1

Annex 1. Development of a data set on tumours and tumour sites in humans and in experimental 289
animals for Group 1 agents identified up to and including Volume 109 of the IARC Monographs
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Disclosures of interests

Each participant was asked to Mélissa Billard’s travel expenses Association regarding the listing of
disclose pertinent research, employ- to attend the Workshop were paid by glass wool fibres in the 12th Report
ment, and financial interests. Current the R. Samuel McLaughlin Centre for on Carcinogens.
financial interests and research and Population Health Risk Assessment,
employment interests during the past where she was a master’s student. Agnes B. Kane received re-
4 years or anticipated in the future are search support from the Gulf of
identified here. Minor pertinent inter- Michael Bird is retired from Exxon Mexico Research Initiative (funded
ests are not listed and include stock Mobil Biomedical Sciences Inc. and by BP) to investigate remediation
valued at no more than US$ 1000 receives a pension and holds sig- of deep water oil spills, and from
overall, grants that provide no more nificant stock from the company. He Unilever to Brown University to de-
than 5% of the research budget of has an affiliate appointment with the velop predictive toxicity testing for
the participant’s organization and R. Samuel McLaughlin Centre for engineered nanomaterials.
that do not support the participant’s Population Health Risk Assessment,
research or position, and consulting which also paid for his travel expens- Robert J. Kavlock’s research
or speaking on matters not before es to attend the Workshop. unit receives research support
a court or government agency that from L’Oréal for the United States
does not exceed 2% of total pro- Bernard D. Goldstein is a Environmental Protection Agency’s
fessional time or compensation. All member of the Dow Chemical Toxicity Forecaster (ToxCast).
grants that support the participant’s Sustainability External Advisory
research or position and all consult- Committee. Daniel Krewski holds a Natural
ing or speaking on behalf of an inter- Sciences and Engineering Research
ested party on matters before a court Charles William Jameson Council (NSERC) of Canada
or government agency are listed as serves as a consultant for the North Industrial Research Chair in Risk
significant pertinent interests. American Insulation Manufacturers Science at the University of Ottawa.

Disclosures of interests 291


This Chair is funded under a com- work with both public and private on health risks from benzene ex-
petitive university–industry part- sector clients in Canada, the USA, posure, funded by the American
nership programme administered and Europe. Petroleum Institute. He is also a
by NSERC. He also serves as the partner in and holds stock of a small
Scientific Director for the R. Samuel Pascale Lajoie’s travel expenses vineyard and winery corporation.
McLaughlin Centre for Population to attend the Workshop were paid by
Health Risk Assessment and re- the R. Samuel McLaughlin Centre for Martyn T. Smith received pay-
ceives salary support and has ac-
Population Health Risk Assessment, ment from various United States law
cess to research funding from the
where she was a master’s student. firms for consulting and testimony
R. Samuel McLaughlin Foundation.
in litigation involving benzene and
He has received an unrestricted
Ronald L. Melnick served as a other chemicals. He also received
grant from the ExxonMobil Research
Foundation, administered through consultant and expert witness for payment for travel and honorari-
the NSERC Chair. He serves as Bush Lewis, PLLC on health effects um from the American Chemistry
Chief Risk Scientist and CEO for of 1,3-butadiene. Council (ACC) and for an invited
Risk Sciences International (RSI), lecture from the International Life
a Canadian company established Jerry M. Rice served as a chair Sciences Institute (ILSI)-Health and
in partnership with the University of of the external Scientific Advisory Environmental Sciences Institute
Ottawa. RSI undertakes consulting Panel for the Shanghai Health Study (HESI).

292
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ISBN 978-92-832-2215-6

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