MOLECULAR CARCINOGENESIS3: 188- 197 (1990)
Molecular Mechanisms of Oxygen RadicaI Carcinogenesis
and Mutagenesis:The Role of DNA Base Damage
Lars H. Breimer
institute of Cancer Research, Chester Beatty Laboratories, London, England
INTRODUCTION
Oxygen is essential for life but can also harm cells. This
is because active forms of oxygen such as hydroxyl (OH')
and superoxide (0): radicals, hydrogen peroxide (HzO,),
and singlet oxygen (lo2)arise as byproducts and interme-
diates of aerobic metabolism and during oxygen stress
I1 -71. These can transiently or permanently damage nucleic
acids, lipids, proteins, and thiols. Free oxygen radicals have
been implicated in degenerative and inflammatory disor-
ders, aging, and cancer [ 1-1 01. They also function as the
effectors of ionizing and some aspects of ultraviolet (UV)
radiation, toxins, mutagens, and certain drugs [2,7,9- 141.
Activated phagocytes and other oxidant-generating systems
reportedly transform mammalian cells and act as promot-
ersof carcinogenesis [7,9,15-171. Indeed, high concentra-
tions of oxygen itself are mutagenic [ 181.Generally, before
causing damage reactive oxygen species are scavenged by
enzymes such as catalase and superoxide dismutase and
by antioxidants such as reduced glutathione and vitamins
C and E [1,2,7,19,20].
Reactive oxygen species induce all forms of DNA damage,
including base modification, base-free(apurinidapyrimidinic
(AP]) sites, strand breakage, and DNA-protein crosslinks,
but the specific spectrum of products depends on the reac-
tive species involved (1,3,4,7,10-141. For example, some
lesions are similar to those induced by ionizing radiation,
which exerts the bulk of its effects through free radicals,
in particular OH. [lo-141. The damage to DNA has been
suggested to contribute to the pathogenicity of these
agents, which is further supported by the existence of
several independent repair processes [ 10,21-231.
Damage to cellular DNA by oxidants has usually, partly
for technical reasons, been measured as strand breaks, a
phenomenon also observed in cells in culture exposed to
cigarette smoke, toxins such as paraquat, and known car-
cinogenic metals such as chromium and nickel 124-301.
These agents have generally been considered to act by
increasing the intracellular formation of 0;and H2Oz.
However, neither 0 ; nor H202 reacts directly with DNA
[ 10, 31 -341. Instead, OH' generated by local transition
metal catalysis [1,4,7,28,31-341 is the likely effective agent.
The literature on free oxygen radical pathogenicity has
grown explosively in the 1980s and has been the subject
of several detailed reviews [I-141. Here I review certain
recent data on the genotoxic effect of reactive oxygen spe-
cies, concentrating on the role of base damage.
0 1990 WILEY-LISS, INC.
CHEMICAL NATURE OF LESIONS
The majority of knowledge on the lesions induced by
reactive oxygen species has been derived from studies using
ionizing radiation [I 0- 141.However, leukocyte-derivedreac-
tive oxygen species induce far greater yields of base mod-
ifications than does ionizing radiation [35,361.Halliwell's
and Dizdaroglu's laboratories together have initiated a care-
ful and comprehensive study of the chemical modifications
of the nitrogenous bases in DNA by various reactive oxygen
species generated under biologically relevant conditions
1311. The products were analyzed by gas chromatography-
mass spectrometry with selective ion monitoring, a highly
sensitive technique by which the nature of the products is
determined from the molecular masses of ions produced
by their fragmentation. There is no need to radioactively
label the target DNA, which avoids the problem of auto-
irradiation 1371. It is also not dependent on synthesizing
the lesions to compare in chromatographic systems. They
used the commonly employed hypoxanthineixanthine oxi-
dase system to generate 0 ; and H202,which were fur-
ther catalyzed to OH' by FeiEDTA (Table l). In absolute yields
the main products were imidazole ring opened guanine
(FaPy-Gua) and 8-hydroxyguanine, followed by imidazole
ring opened adenine (FaPy-Ade)and cytosine glycol. How-
ever, the greatest relative increase in damaged base com-
pared with background levels by far was detected for
FaPy-Gua, followed by dihydroxycytosine, FaPy-Ade, and
cytosine glycol. When Fe3+ catalysis was allowed t o
proceed in the absence of EDTA (probably a more physio-
logical condition), the overall yield in damaged bases
was reduced by about 60%, and 8-hydroxyguanine, 8-
hydroxyadenine, and cytosine glycol were the main prod-
ucts, but FaFy-Gua and dihydroxycytosine were still the
most increased products.
If H202was used as a source of radicals with FeiEDTA
(ferric), there was at most a twofold difference between
the relative increases in the modified bases except for FaPy-
Gua, though in absolute levels the guanine derivatives still
predominated [32]. However, when nitrilotriacetic acid
(NTA), a powerful carcinogen and promoter [38],was used
Corresponding author: Institute of Cancer Research, Chester Beatty
Laboratories, Fulham Road, London SW3 6J8, UK.
Abbreviations: OH , hydroxyl radical; 0 i, superoxide radical, Faqi-
Gua, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (imidazole ring
opened guanine); FaPy-Ade, 4.6-diamino-5-formamido~rimidrne (imid-
azole ring opened adenine); AP site, apurinidapyrimidinic, also known
as base-free site, NTA, nitrilotriacetlc acid.
 OXYGEN IN CARCINOGENESIS
as the chelator the yields were almost 10-fold higher [32],
supporting proposals that this representsthe carcinogenic
mechanism of this compound [39]. Further, they found that
Cu2+ in combination with HzOz was more efficient at
inducing base damage than was Fe3+,and this effect was
enhanced by the presence of ascorbic acid [401. Cytosine
glycol, thymine glycol, 8-hydroxyadenine, and especially
8-hydroxyguanine were the major products. They con-
cluded that the copper ions were bound to the DNA, where
they generated OH' that immediately attacked the DNA
bases. Thus there would appear to be differences in the
spectrum of damage induced by different free radical-
generating systems.
Their finding of a predominance of guanine and cyto-
sine modifications would predict that changes at these
bases should be overrepresented in mutagenesis induced
by agents acting through an increased flux of reactive oxy-
gen species, if OH' generated locally by transition metal
ion catalysis is the critical event. This is at variance with
genetic studies that indicate that changes at A.T base pairs
are of particular significance in oxidizing mutagenesis [41]
(see below). However, so far these chemical studies have
centered on fairly simple forms of base modifications and
not more complex breakdown products such as 5-hydroxy-
5-methylhydantoin in the case of thymine and urea, which
is a potential end-product of all bases in DNA [ I 0,141 but
the methodology to measure these lesions has now been
developed (Halliwell, personal communication). Moreover,
AP sites are known to be mutagenic [42-451, and the yield
of AP sites and any sequence specificity in their genera-
tion (i.e., "hotspots") needs to be determined. Studies using
ionizing radiation suggest that each base is equally prone
to generate an AP site and that this form of damage may
account for 10% of all damage [l1-1 31.
One problem in the study of the action of free radicals
on DNA IS that phenol, commonly used to purify DNA,
can partake in free radical reactions. Thus the level of
"background" lesions in DNA can vary considerably [31].
Indeed, it is in the nature of free radical reactions that an
agent may protect against radicals in certain circumstances
and to enhance damage in others [46]. Another problem
is that different free radical-generating systems appear to
cause different damage even if the same species of radical
is thought to be the effector. For example, Fe/EDTA-catalyzed
cleavage of RNA and DNA shows similar reactivity toward
single and double stranded forms, but Cu/phenanthroline
preferentially cuts duplex 5-form DNA [47]. However, stud-
ies with scavengers indicate that Fe/EDTA generates OH' out-
side DNA, whereas Cu/phenanthroline intercalates into the
nucleic acid [48]. It has been reported that nickel-catalyzed
hydroxyl radical DNA strand breakage is seen at C, T, or G
but not A, though C is preferred, whereas Fe-catalyzed
hydroxyl radical generation caused breakage at all bases,
but favored G and T [29], though these subtle differences
have yet to be independently confirmed. Singlet oxygen
appears to damage G specifically but, depending on the
catalyst, the reaction is specific for single- or double-
stranded DNA [49-511. Depending on the catalyst it may
also induce strand breakage. Thus the "finger prints"
789
of damage induced by various free radical-generating
systems in vitro and in vivo need to be established, and
some caution is needed when comparing results from dif-
ferent laboratories.
GENETIC EFFECTS OF DEFINED LESIONS
8-H yd roxypu rines
8-Hydroxyguanine and, by inference, 8-hydroxyadenine
are the most likely mutagenic base lesions induced by reac-
tive oxygen species [52-54].8-Hydroxyguanine has been
shown to directly miscode in model experiments and to
induce miscoding of neighboring bases [52]. Thus it can
potentially cause multiple amino acid substitutions. When
placed in a site-directed manner between T and C, the
8-hydroxyguanine residue directed the insertion of all four
bases with almost equal frequency, indicating that it lacks
specific base pairing. In addition to misreading of the
8-hydroxyguanine residue itself, the pyrimidines next to it
were also misread. Thus, depending on its position in rela-
tion to the reading frame, 8-hydroxyguanine could cause
paired amino acid changes. Such mutations would be
unlikely to revert and would therefore not appear as
single base damage events in indirect assays. Mispairing
due to 8-hydroxypurines at intronlexon boundaries (AG
and GT) would be expected to produce aberrant or in-
com plete messages.
8-Hydroxyguanine disappears from DNA in vivo though
the mechanism of repair is unknown [53]. Singlet oxygen
has been reported to induce G.C ---t T.A and G.C + C.G
transversions in the bacterial laczsystem, but the chemical
nature of the DNA lesion was not directly defined, though
it blocked DNA replication in vitro [50]. The frequency of
frameshifts, which occurred in runs of Gs or Cs, and dou-
ble mutations, which occurred at G.C base pairs, was spec-
tacularly increased. Although these changes may have been
due to 8-hydroxyguanine, other modifications were not
formally excluded. Recently it has been demonstrated that
8-hydroxyguanine can be specifically generated without
strand breakage in intact duplex DNA by the action of meth-
ylene blue and light in the presence of oxygen [51]. This
will allow a direct assessment of its role in carcinogenesis
as well as detailed characterization of the mechanism of
its repair.
Ring-Saturated Pyrimidines
Partly for technical reasons, however, most studies have
concentrated on the fate of thymine in DNA, in particular
thymine glycol, which can be simply generated specific-
ally in DNA by oxidation with alkaline osmium tetroxide
[reviewed in lo]. It used to be considered the main product
of oxidizing injury, though its yield in free radical reactions
varies and has probably been overestimated [l1-13.531.
Indeed, Halliwell and coworkers found comparatively lit-
tle generation of thymine glycol under physiological con-
ditions (Table 1). Thymine glycol has been regarded as a
lethal rather than mutagenic lesion. A curious observation
has been that mutants deficient in the nth gene product,
endonuclease Ill which acts as a thymine glycol-DNA
glycosylase, are not more sensitive to HzOz and ionizing
790 BRNMER

Table 1. Yield of Base Products in DNA by Hydroxyl Radical Treatment*

Xanthine/xanthine oxidase [31]t Hydrogen peroxide l321t
Fe3'(11)-EDTA Fe3+(11)- no EDTA Fe3 +(ll)-EDTA Fe3+(II)-NTA
Yield Relative Yield Relative Yield Relative Yield Relative
Base product ( n m o h g ) increase (nmolirng) increase (nmolimg) increase (nmolimg) increase
FaF yG ua 8.8 900 0.14 15 1.1 60 5.9 300
8-Hydroxyguanine 4.3 9 4 1.4 3 13.4 30
FaPy-Ade 1.6 40 i::7 2 0.3 5 1.8 30
Cytosine glycol 1.5 20 0.32 4 0.1 2 1.3 25
Dihydroxycytosine 0.9 90 0.08 8 0.1 3 1.3 65
8-Hydroxyadenine 0.9 3 0.5 1.5 0.2 3 1.9 27
Thymine glycol 0.9 4 0.2 - 0.25 3 1.4 18
*Significant levels of 8-hydroxyguanine, &hydroxyadenine, and thymine glycol were present in the DNA before free radical treatment. Adapted
from Aruoma et al. [31,32].
tfree radical generator.
FaPy-Gua, imidazole ring opened guanine; FaPy-Ade, imidazole ring opened adenine, NTA, nitrilotriacetic acid.

radiation [55]. Two independent roups have now shown
that DNA containing thymine i y c o l is a substrate for
UvrABC exinuclease 156,571. This would explain those
results, and consequently further . coli studies extended
to multiple mutants are necessary. However, a uvrABCnth
double mutant may not be viable under oxic conditions.
The E. coli double mutan4 nfo xth is deficient in two
enzymes (endonuclease IV and exonuclease Ill) involved
in repair of DNA damaged by reactive oxygen species and
is sensitive to oxidant stress [45,581.
Essigmann's group has shown that thymine glycol placed
in a unique site resulted in targeted mutations a t a fre-
quencyof 0.3%, which was exclusivelyT -,C when single-
stranded bacteriophage was replicated in . coli (591.
Thymine glycol was not mutagenic in duplex DNA, and
the effect was independent of SOS and nth repair. This
is consistent with the property that endonuclease 111 acts
on single-stranded DNA at only about 2-3% the rate of
its action on double-stranded DNA [601, and UvrABC
requiring double-stranded substrate. However, mutations
were not a random event but occurred in positions where
the 3' residue was an adenine, in keeping with the require-
ments of a 3' purine to allow in vitro bypass [10,42,61],
and which by wobbling allows a thymine glyco1.G mispair
to form. Thus, if thymine glycol were to arise a t or near a
replication fork it could direct T + C transitions. In view
of the considerable yield of 5-6 bond-saturated cytosine
derivatives reported by Aruoma et al. [3 1,32,40,481, similar
studies on the role of these lesions are needed. Never-
theless, by analogy, they should not be expected to be
mutagenic. This may partly explain why mutations at G.C
base pairs are not as overrepresented in free oxygen
radical-induced mutations.
Ring-Fragmented Bases
The more complex ring-fragmented and ring-contracted
pyrimidine derivatives and imidazole ring-opened purines
are non-informative lesions that block DNA replication in
vitro [lo-141 and are considered to play little part in muta-
genesis. In view of Essigmann's work on the role of thy-
mine glycol, this concept should be reassessed. However,
in interpreting the action of mutagens and carcinogens in
vivo it should be remembered that the radical and hydro-
peroxide base derivatives that are intermediates in dam-
age can be restituted or modified by cellular processes
before being fixed [62,63].
GENETIC CONSEQUENCES OF REACTIVE OXYGEN
SPECIES IN MAMMALIAN CELLS
Studies Using Chemical Agents
Dose-dependent mutagenesis has been observed at the
X-linked hprt locus and at a stably integrated E. coli gpt
gene when mammalian cells in culture have been exposed
to hyperoxia and KO2, but no molecular analysis was under-
taken [25]. Tyrrell's group has reported a substantial dif-
ference in the effect of reactive oxygen species, in particular
hydroxyl radicals, on a target gene depending on whether
the mutagenesis was carried out in vivo or in vitro (Tables
2 and 3) [34,64]. In brief, they analyzed alterations in the
supF gene in a shuttle vector. (Shuttle vectors can repli-
cate both in mammalian and bacterial hosts thereby sim-
plifying analysis.) Either the vector was treated with H202
with or without FeiEOTA t o catalyze a Fenton reaction to
generate OH' before transfection into the mammalian
cells, or cells carrying the vector were exposed to H20z,
the resulting mutations being analyzed and compared with
spontaneously arising mutations. They confirmed that
H202itself does not induce mutations, which is in agree-
ment with other work showlng that H202 does not react
directly with DNA.
They found that when HzOz and Fe/EDTA was used in
vitro (i.e., exposing the plasrnid to OH' prior t o trans-
fection), the main mutation was a combination of a base
pair change and a deletion, and there were less pure
base changes than in the spontaneously arising mutants.
However, when HzOzwas used in vivo (i.e., added t o the
cells containing the plasmid) the increase in combined
deletion and base change was less marked and took
place at the expense of pure deletions. In either case the
most common deletion was the loss of one base pair
(i.e., a simple frameshift), most deletions were less than
4 bp in size, and the frameshifts tended to occur in runs
 OXYGEN IN CARQNOGENESIS 191
Table 2 . General Classification of Mutations in a Table 4. General Classification o f Mutations
Shuttle Vector Arising Spontaneously or at the CHO aprt Locus Arising Spontaneously or
Following Hydrogen Peroxide Treatment* Following y Irradiation*
Treatment Mutation Spontaneous (%) y Irradiation (YO)
H202 H202 Altered mobility 10 23
Mutation Spontaneous (%) in vivo (YO)in vitro (YO) Base change 63 42
Altered mobility 17 11 15 Deletion 20 25
Deletion only 28 18 25 Multiple 1 9
Base change Duplication 5 1
only 52 53 36 Frarnwhifts 7 11
Deletion & *Derived from Miles and Meuth [681and Phear et al [71 I
base change 3 16 23
Insertion only <1 1 -

Deletion & to the nature of the frameshifts (see below) and illustrates
insertion 0 1 1 the difficulty of extrapolating between not only different
Frameshifts 12 50 60 free radical-generating systems but genetic targets.

*Derived from Moraes et al [34,641 Studies Using Ionizing Radiation

Table 3. Distribution of the Relative Frequency of
Base-Pair Changes Arising at the Right-Hand Base of
Certain Sequences in a Shuttle Vector After Mutagenesis*
Treatment
H202 H202
Sequence Spontaneous (%) in vivo (YO) in vitro (YO)
5'-TC-3'
51 66 28
3'-AG-5'
5'-CC-3'
44 29 36
3'-GG-5'
Other 5 5 36
*Derived from Moraes et al. [34,641.
of Gs or Cs. Spontaneously arising mutants exhibited an
increase in aeletions that were larger than and different
from those arising by H202treatment in that they gener-
ally occurred between two repeats, one of which was left
in the genome [65].
The specificity of the base pair changes was also remark-
ably different (Table 3). In the in vivo-induced mutants vir-
tually all arise at G.C base pairs. Indeed, not only do the
HzOzand the spontaneous base changes arise at the right
hand base pairs of Sf-TC-3':3'-AG-5' or 5'-CC-3':3'-
GG-5' but G.C + A.T transitions are by far the predomi-
nant form. The base changes in the in vitro-treated plasmid,
on the other hand, represented an increase of alterations
at A.T base pairs. This is consistent with other reports that
A.T base pairs are more prone to oxidizing mutations at
least in prokaryotic systems [41]. However, they are not in
agreement with the chemical studies of Halliwell's group,
which would indicate that changes at G.C base pairs are
more likely to occur.
The generation of frameshifts is consistent with free radi-
cals causing strand breaks with the loss of one base and
with increased risk of slippage in regions of repeated
purines [66]. But these results are clearly different from
the results obtained by ionizing radiation (which is gener-
ally considered to act mainly through OH') at the Chinese
hamster ovary (CHO) aprt locus, particularly with respect
A difficulty in interpreting the effect of exposing cells in
culture to chemical agents generating reactive oxygen spe-
cies are the quenching effects of media components, and
also plasma membrane damage. Thus, it would be desir-
able to develop a system of caged free radical release
whereby the component can enter in an inactive form and
be released through an innocuous signal such as a flash-
ing light [67]. Ionizing radiation represents a partial means
of achieving such intranuclear or, at least intracellular, re-
lease of reactive oxygen species. This is because ionizing
radiation does not act as a physical agent to damage DNA,
like a bullet cutting through a fiber, but instead exerts the
bulk of its effects through free radicals, in particular OH'.
Selectable Loci
The effect of ionizing radiation on a number of mam-
malian loci is now well established [reviewed in 10.681.
Gross gene rearrangements have been generally observed,
but the proportion varies considerably (1 5-80%). which
may reflect differences between the actual targets, their
flanking regions, or the mammalian species or cell lines
employed. Comparisons of the changes at the aprt locus
of CHO cells developed by Meuth in spontaneously arising
and ionizing radiation-induced mutants have shown that
ionizing radiation caused an increase in gross rearrange-
ments [10,68-761. When mutants of unchangedgenestruc-
ture were analyzed a t the DNA sequence level, the majority
were found to be simple base substitutions, which hence
overall were the most common class of lesion induced at
this locus (Table 4). There was no statistically significant
difference in the pattern of distribution of type of base
change between spontaneous and ionizing radiation-
induced changes. These results are more similar to the
mutants of the supF system that arose spontaneously or
were generated by HzOzexposure in vivo but are in marked
contrast to those obtained when the plasmid was treated
with OH' in vitro prior to transfection. More radiation-
induced mutants had multiple local substitutions, proba-
bly reflecting localized release of energy [I 1-1 31.
Frameshiftswere induced at about twice the frequency
by ionizing radiation, but the mechanism involved in their
192 BRElMER
generation is unlikely to be the same. Spontaneousframe-
shifts occurred in runs of Cs or Gs, presumably because
of slippage of DNA polymerase during replication, but
radiation-induced frameshifts did not show such specific-
ity, suggesting imperfect repair of strand breaks. Thus the
frameshifts induced in vitro by OH' in the shuttle vector
did not resemble those induced in vivo by ionizing radia-
tion, which is considered to exert the bulk of its effects
through OH'. This implies either that the frameshifts
induced by ionizing radiation are created through some
other active species or intermediary, or that the data
obtained using the shuttle vector system are not a good
model for autosomal mammalian genes.
Small deletions were slightly more common in the
radiation-induced group of mutants but were found in
different sections of the gene. Spontaneous deletions
clustered in a region rich in direct and inverted repeats,
which can potentially form stable secondary structures and
occur between short direct repeats, suggesting that this
region might be particularly prone to distortion or slippage
of the template during replication or could, after damage,
serve as substrates for the recombinases involved in immu-
noglobulin switching [65,66,7 11. The ionizing radiation-
induced deletions were clustered in a different region, and
only half involved direct repeats [681. Thus as far as dele-
tions are concerned the only similarity between the shut-
tle vector data and aprrdata applies to spontaneous events.
In view of these differences the molecular changes in
the aprtgene induced by exposing cells to H202 (or its less
membrane-damaging derivative dibutyryl peroxide) need
to be determined and compared with the effect of ioniz-
ing radiation in the shuttle vector system.
It would appear that spontaneous mutations occur pri-
marily at G C base pairs but that several processes are
involved. Under resting conditions spontaneous mutations
presumably arise through a mixture of reactions, includ-
ing deamination of cytosine and 5-methylcytosine, meth-
ylation of pyrimidines, hydrolytic depurination (as well as
by repair enzyme action), and free radical attack. The envi-
ronment of the nucleus is likely to be hypoxic [reviewed in
71. Indeed, Breimer and Lindahl[60] found that when cells
were irradiated under conditions that minimized DNA
repair, the pattern of thymine lesions in their DNA was sim-
ilar to the spectrum seen under hypoxic conditions in vitro.
Treating cells with H202may cause a small increase in the
flux of OH' near DNA. 8-Hydroxypurines and formamido-
pyrimidines represent oxidation and reduction products of
the same purine radical resulting from addition of OH' to
the carbon-8 of the purine ring [321. The relative yields of
the two purine derivatives varies between different transi-
tion metal ion-chelator complexes, but in general the
8-hydroxypurines appear to be favored by chelators that
have been shown to be carcinogenic [32,38,39]. Thus treat-
ment of cells with H20z or other forms of oxidant stress
may represent a supply of oxidizing power to the nucleus
adequate to favor formation of 8-hydroxyguanine. (When
OH' are generated in vitro and allowed to interact
with pure DNA, the subtle conditions of the eukaryotic
nucleus may not be adequately represented.) One way of
investigating this hypothesis would be to repeat the in vitro
shuttle vector experiments using the methylene blue dye
method, which generates 8-hydroxyguanine specifically
[51I, and to compare the spectrum of mutations with those
obtained with hydroxyl radicals. Also, if different chelators
were used to give different yields of base damage in the
DNA (cf. Table 1) it may be possible to estimate the rela-
tive mutagenic importance of the various modifications.
(It is unlikely that the methylene blue dye method can be
used on cells in vivo.)
Transforming Genes
Activation of oncogenes is often associated with the
development of both experimentally induced tumors and
human cancers [771. The transformation of cells into can-
cerous growth is a complex multistep process, and onco-
gene activation is not necessarilya primary event [78-801.
Several groups have now reported that there are distinct
transforming genes involved in ionizing radiation-induced
tumors or transformation of cells in culture [81-831.
Genomic DNA was extracted from cells exposed to or
tumors induced by ionizing radiation and used to trans-
form NIH 313 cells in standard assays. The same transform-
ing gene appeared to be present in each of the donor cells
because digestion prior to transfection of the DNA with a
panel of restriction endonucleases gave distinct patterns
of inactivation. It is noteworthy that none of the studies
revealed evidence of rearrangement or amplification of the
commonly studied oncogenes including Ha-ras, Ki-ras,
N-ras, rnyc, raf, src, fes, abl, mos, erbA, erbB, myb, fos,
sis, neu, and trk [81-831. Little's group also reported a tem-
poral correlation between the appearance of transforma-
tion of cells at about three weeks after irradiation and the
ability of the extracted DNA to induce transformation [831.
These results are consistent with previous work showing
that transformation is not a direct consequenceof the expo-
sure to ionizing radiation but occurs as a rare event in the
progeny of the irradiated cells at some later time [78,79],
presumably owing to delayed activation or rearrangement
of certain genes. Identification and subsequent character-
ization at the level of DNA sequence of the gene (or genes)
responsible for these events is now a top priority.
Partly for historical reasons, as well as convenience, the
rasgene family is probably the most extensively studied of
oncogenes [84]. In contrast to the above findings, Pellicer's
group has reported evidence of fa5 activation in mouse
thymomas induced by ionizing radiation [85-871. When y
rays were used the changes were found to be G.C + A.T
transitions mainly in Ki-ras. However, when they used neu-
trons, a different spectrum of alterations was seen: there
were two Ki-ras (codon 12) and one N-ras (codon 61) G.C
+ T.A transversions, but also one Ki-ras G.C + A.T trans-
ition a t codon 146. Changes in H a - m a t codon 146 have
been created artificially and shown to have lower mutagenic
activity than codon 12 changes [88], and these nude mice
tumors also had a 30% longer latency [87]. G.C --t T.A
tranversion is consistent with a singlet oxygen mechanism
as deduced from prokaryote work 1501.
 OXYGEN IN CARQNOGENESIS
Neutron irradiation transforms cells in vitro [89,90] and
inducestumors in animals [91,92]. Neutrons result in denser
clusters of ionized molecules than y irradiation, which
should produce greater damage that would possibly be
more difficult to repair and intuitively ought to result in
more complex changes such as deletions and rearrange-
ments. However, the ra5 system will only tolerate base
changes (i.e., it is equivalent to reversion at a nonsense
codon), and G.C --t A.T transition is a common sponta-
neous change, owing to either depurination, or deamina-
tion of 5methylcytosine, or 06-methylation of guanine.
These results stand in apparent contradiction to those above
reporting as yet unidentified oncogene involvement in ion-
izing radiation transformation. One explanation is that in
lymphoid tissue a ras mutation gives a selective advantage
to a clone without being the primary event.
IS MODULATION OF THE ACTION OF
TRANSCRIPTION FACTORS THE BASIS OF TUMOR
PROMOTION BY REACTIVE OXYGEN SPECIES?
By contrast with the data on the mutagenic effects of
reactive oxygen species that has been accumulated, much
less is known about the promotional activity of these
agents.
The regulation of gene expression involves the nonco-
valent binding of protein transcription factors to specific
DNA recognition sequences [93]. Reactive oxygen species
may influence these processes at the level of DNA-protein
interaction, protein-protein interaction, and RNA-protein
interaction.
Modification of a base in a recognition sequence may
alter the binding affinity. The effect would depend on
whether the factor was a positive or negative regulator.
Inhibition of binding can be achieved in vitro by N7-methyl-
ation of guanines (so called methylation interference, a tool
commonly used to characterize transcription factors).
Reactive oxygen species could also covalently cross-link
a factor to a base in its binding site, causing a more last-
ing effect on gene expression. Hydroxyl radicals induce
covalent DNA-protein cross-links between thymine and
cytosine and various aliphatic and aromatic amino acids
in vitro, involving the carbon-6 atom or the 5-methyl group
of thymine and cytosine [94-961. Cross-linking a t the
5-methyl group was favored by anoxic conditions. Experi-
ments with nucleosomes showed that histones H2A, HZB,
H3, and H6 could be cross-linked to DNA. Little is known
about the repair of these lesions, but it would require the
excision of at least one base and probably a stretch of
nucleotides (see below).
Tsos group was abie to induce malignant transformation
and mutations in cells in culture by allowing them to incor-
porate 5-bromodeoxyuridine into their DNA and irradiating
with UV light 1971. This was interpreted that cancer could
be induced by direct action on DNA not involving other
macromolecules. However, a similar protocol has been used
to cross-link transcription factors to DNA in vitro [98, 991.
Reactive oxygen species could also modify the transcrip-
tion factor itself, for example, by altering its protein-protein
interacting domain, which could lead to inappropriate gene
193
regulation. The effects of reactive oxygen species on pro-
teins are poorly understood but include direct degradation
and stimulation of proteolysis but also subtler modification,
including loss of phosphatefrom phosphoserinesand modi-
fication of glycosylation side chains [ I 00-1 021. At least
some transcription factors are dependent on posttransla-
tional modifications [93]. It has recently been proposed
that the oncogenic activation of the myb proto-oncogene
is a result of an inability to phosphorylate a regulatory site
because this site is deleted in the oncogenically activated
Myb protein [I 031, and free radical-induced dephosphor-
ylation could have similar consequences. An oxygen radi-
cal mechanism has been proposed to regulate some mRNA
translation by a sulfhydryl switch of reversible modifica-
tion of a regulatory protein, indicating a functional role
for reactive oxygen species in metabolism [1041. A similar
mechanism might be involved in some forms of regula-
tion of transcription.
The effect of alteration to protein-nucleic acid interaction
can only be expected to be transient. Only if it was preceded
by or occurred concomitantly with a genetic alteration or
premutagenic DNA lesion could a selective advantage be
expected to develop. However, these mechanisms represent
a possible aspect in multistep models of carcinogenesis.
POSSIBLE FUTURE DIRECTIONS
To prevent and treat cancer the molecular action of car-
cinogens must be elucidated [105]. Our understanding of
the role at the molecular level of reactive oxygen species
in carcinogenesis is hampered by the fragmented nature
of the studies. Consequently,complementary investigations
are urgently needed to harmonize the information. An obvi-
ous place of departure is the autosomal CHO aprtand the
supF shuttle vector systems, which have provided a large
body of data of spontaneously arising mutants, but in the
case of the CHO system, H 2 0 z studies are needed to com-
pare with the supF data and vice versa for ionizing radiation.
Because of the apparently contradictory results of the spe-
cificity of the base changes induced when the supF shuttle
vector was treated with H202in vivo or in vitro (Table 3). a
comparativelysimple study of the changes in DNA sequence
of mutations induced by H202should be performed at the
autosomal locus aprt of CHO cells, which is still the most
powerful mammalian locus for such detailed analysis. This
could be complemented by a combined study of the effect
of K02, which generates 0 ; on the shuttle vector and
aprt locus, and certainly a study of the effect of ionizing
radiation on the shuttle vector system. Such studies would
demonstrate the validity of the shuttle vector approach,
and the lesions found would indicate the mechanism
involved. Thus exposure of CHO cells to Hz02would be
expected, when compared with spontaneously arising
mutants, to increase the number of mutants showing a
base pair change at the righthand base of 5-TC-3 at
the expense of mutations at 5-CC-3. Further infor-
mation might be obtained from repeating the shuttle
vector protocols using ionizing radiation. If, as is generally
assumed, ionizing radiation exerts the bulk of its effects
through O H , the results of exposure in vitro should
194
be similar to those reported for Fe/EDTA/H202.The results
of irradiation in vivo would be particularly valuable be-
cause the pattern of mutations can be expected to be
different if the aprt data are generally applicable. They
may also help answer the question of the microenviron-
ment of the nucleus. Thus oxic conditions should favor the
generation of 8-hydroxypurines and ring-saturated pyri-
midines, whereas anoxic conditions would favor the open-
ing of the imidazole rings of purines and protein-DNA
cross-links.
Another simple study applicable to the shuttle vector
protocol would be to induce 8-hydroxyguanine lesions in
vitro [51I and analyze the mutations induced by passage
through the mammalian cells. As these lesions are con-
sidered miscoding rather than blocking DNA replication
and if FaPy-Gua (or, less likely, cytosine glycols) are involved
in generating frameshifts, a preponderance of base pair
changes, either singly or adjacent pairs, without deletions
may be seen.
One limitation of CHO aprtsystem is that it appears to
permit deletions only in one direction and therefore under-
estimates the frequency of deletions. The supF shuttle vec-
tor system used by Tyrrell's group can, owing to the size
of the supF reporter gene and essential flanking plasmid
sequences, only cope with deletions of about 100 bp in
size. For completeness mutations should be collected in
parallel a t another locus such as hprtor dhfr, though they
do not allow such detailed analysis (101. However, Little
[lo61 has pointed out that all the autosomal loci commonly
used underestimate large scale events, including chromo-
some loss, because (partly for practical reasons) they are
hemizygous for the locus and neighboring regions (in the
case of hprt, which IS located on the X chromosome, this
is an inherent problem [lo]).
The mutagenic effects at the level of DNA sequence
change of known carcinogenic compounds such as chro-
mium and nickel should also bedetermined in these mam-
malian systems, because of the reported sequence specificity
of the DNA damage induced by these agents in vitro. These
studies may also be extended to compounds such as acet-
aldehyde and formaldehyde, which are implicated in the
carcinogenesisof alcoholic liver disease through a free radi-
cal mechanism, though little if anything is known of its
action [107-1 101.
The pattern of mutations in cell lines with altered oxy-
gen metabolizing systems should also be assessed. Though
such bacterial mutants are available, mammalian mutants
that also allow detailed analysis of the genetic effects are
not yet established. For example, mutants with altered lev-
els of superoxide dismutase would change the flux of Ot,
and similarly, catalase mutants would affect the intracel-
Mar levels of HzOz.As the OH' is highly reactive and does
not diffuse significant distances it must be formed locally
from HzOz and O;, which can be generated at signifi-
cant distances from the target molecule and diffuse there.
Further, if cells are treated with oxidant stress under con-
ditions that limit the repair processes [60] and the DNA or
chromatin be extracted and analyzed, a "finger print" of
the types of base lesions induced in DNA in vivo can be
BRNMER
established and compared with the various patterns of dam-
age induced in vitro by different systems.
The biological role of 5-hydroxymethylpyrimidines in
mammalian DNA needs to be resolved. Is the hydroxyl
group substitution at the methyl group of thymine (and
5-methylcytosine) merely generating an inefficient model
of the true substrate of this intriguing enzyme? 5-Hydroxy-
methyluracil (a-hydroxythymine) is not only a product of
OH' attack on the methyl group of thymine, but it is the
natural DNA base residue of certain bacteriophage genomes
[reviewed in 10,12,14,23]. Eukaryatic but not prokaryotic
cells possess DNA glycosylase activities directed against
5-hydroxymethylthymine and -cytosine [ 10,231. However,
the activity of the enzyme on DNA containing 5-hydroxy-
methyluracil residues is exceedingly low [231. These two
observations suggest that the true substrate may be an
alternative modification of the 5-methyl group that only
arises in eukaryotic cells. One possibility is that the enzyme
incises the glycosyl bond of thymines that have been cross-
linked by their 5-methyl to an amino acid residue, such as
tyrosine, which may be part of a polypeptide. Dizdaroglu's
group 1951 have shown that the formation of such cross-
links depends on OH' attack in an anoxic or hypoxic envi-
ronment, which is likely to exist in the eukaryotic nucleus
[7,60].To test this hypothesis, techniques t o synthesize
relevant analogues and incorporate them into (or selec-
tively modify the bases in) synthetic oligonucleotides
must be developed.
It is mandatory to relate data on mechanisms obtained
by studying prokaryotes and eukaryotic (often transformed)
cells in culture to the whole animal [ l l 11. One approach
is to use a shuttle vector established in a transgenic animal
[112,1 131. Physiologically relevant aspects of carcino-
genesis can then be studied because the mutagenic action
of any carcinogen in any tissue could potentially be as-
sessed at the level of DNA sequence changes. As yet there
are no data on agents considered to act through free
oxygen radicals.
Identification of the role and identity of transforming
genes implicated in free radical transformation, including
differences owing to various types of radicals such as those
generated from neutrons as opposed to y rays, would
appear to be a fruitful avenue of research.
The effect of reactive oxygen species on transcription
factor DNA interaction can comparatively simply be assessed
in vitro by exposing pure transcription factors to free
radical-generating systems and assess the effect by band
shift assays, methylation interference, and DNase protec-
tion assays [93]. Such work can be extended to in vitro
transcription protocols.
CONCLUSION
Cells can cope with reactive oxygen stress in four broad
ways: (a) by scavenging the reactive species before they
damage critical molecules such as DNA; (b) by enzymati-
cally converting the species into inert or less harmful forms;
(c) by restituting modifications before they are fixed, that
is, reverting reactive intermediates to their original form;
and (d) by repairing established modifications.
 OXYGEN /N CAR<3NOGENESfS
Just as it is simpler to measure blood pressure than flow
rate so it is simpler to study the repair of a defined and
stable end-product lesion rather than the fate of short-
lived intermediates. However, it would intuitively seem to
be more expedient to the organism to prevent damage
rather than to repair it; that is, possession of such systems
ought to represent a tremendous selective advantage, espe-
cially, as would seem likely, these systems would interfere
with tumor promotion as well as mutagenesis. During the
past five years a number of inducible systems of preven-
tion and repair have been described [ 1 14- 1 191.These over-
lap in their specificitiesof the stress signal. The involvement
of reactiveoxygen species in carcinogenesis is beyond doubt
but further work is needed to define the important inter-
mediates and lesions, not least the action of the defense
systems. The work to decipher the role of free oxygen radi-
cals in tumor promotion is likely to be complicated. How-
ever, such studies should help in devising improved protocols
for treatment and prevention.
ACKNOWLEDGMENTS
This work was supported by grants from the Medical
Research Council of UK and the Cancer Research Cam-
paign of UK.
I thank Drs. 5. Keyse, P Lawley, K. Willison, and R. Wood,
and Profs. P Brookes and B. Halliwell for encouragement
and advice, and Prof. B. Hahwell for communicating results
prior to publication.
Received Apri) 9, 1990; revised, May 21, 1990; accepted May 23,
1990.
NOTE ADDED IN PROOF
Significant mutations (50-300-fold increase) have been
reported to be induced in Ames Salmonella tester strains
by the visible lighthethylene blue protocol [120]. Base modi-
fications were the main DNA lesions, not strand breakage
or AP sites. The modified bases appeared to be removed
through DNA glycosylase action, but were also poor sub-
strates for UvrABC exinuclease.
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