TOXICITY

„ Toxin: Any poisonous substance of microbial,

mineral, plant or animal origin.
„ Cytotoxicity VS Genotoxicity
„ Aims :
„ to gain specific information on substances that
could be an environmental or human hazard
(toxin)
„ toxicity is being examined to expand information
base on toxicological mechanisms
 Bahan Alam Perairan
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Antitumor/anticancer
Antioksidan
Antikolesterol
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 Cytotoxicity
Acute test
„ significant effect in short time (96 hours)

„ the effects seen are death

„ It is known as the "all or none" principle where the

organism responds or it doesn't respond.
„ the response measurement : Median Lethal
Concentration (LC50).
Organisms used for toxicity test
9 bacteria (most widely used)
9 algae
9 crustacea
9 fish
9 Others - higher plants, protozoa,
coelenterata, platyhelminthes,
rotifera, annelida, insecta and
mollusca
„ LC50 :The concentration of a toxin leading
to the death of 50% of the test population
over a given period (normally 96 hours).

„ chronic test
„ responses are observed weeks or months
„ the concentrations are usually much lower
„ subacute tests
„ intermediate between accute and chronic
„ similar assessmentpattern to the chronic test
Considerations about test organisms

9 same size
9 same developmental stage
9 healthy (they are not under any stress from
disease, lack of food or parasites)
9 It they are collected from field, they should be
from the same population
9 all specimens must be allowed to acclimate (or
get used to) the environmental testing conditions.
 Genotoxicity
„ Genotoxic compounds or mutagens : cause DNA
mutations to either the chromosome or the gene.
„ If these mutations cause cancer, then the compounds
are classified as carcinogens.
„ Usually the initial compound is not genotoxic: it
becomes genotoxic only after it has been
metabolized by some organism (bioassay species)
„ After conversion, the compound will either be
released to the environment or bioaccumulated
„ If the test species is not capable of metabolizing the
compound into its toxic form, the toxicity will not
be detected.
„ If other organisms in the ecosystem are able to
biologically transform the compound, then toxicity
will be exerted.
 Environmental Mutagenesis

„ An environmental mutagen, or genotoxin, is a

physical or chemical agent that is present in
the environment and can induce damage to the
genetic material or can cause an alteration in
the proper functioning of the genetic material
of an organism.
 Relationship Between Genetic Toxicology
and the Field of Toxicology
„ Genetic Toxicology deals with the study an
characterization of physical and chemical agents that
affect the hereditary material, and its mechanisms, of
living organisms.
„ The primary function of genetic toxicology is to
detect and understand the properties of agents that
induce deleterious effects in genetic elements at doses
below those that induce physiological toxicity.
„ Toxicology is a science
that deals with poisons,
with their effect and
with the problems
involved.
„ Genetic toxicology is a
sub-field of toxicology
that involved with
damage to the genetic
material.
„ Chromosomal damage is a change in the number or
structure of chromosomes.
„ Gene damage is a mutation of the gene itself
„ The DNA repair test assumes that if cells are
undergoing repair some damage must have occurred.
„ Another DNA repair test compares two strains of a
bacteria:
„ one is capable of DNA repair; t
„ he other is not.
„ If a chemical alters the DNA of the bacteria, then the
strain that is not capable of repair will die.
„ Bacteria commonly used in this test are Escherichia
coli, Bacillus subtilis, Proteus mirabilis, and
Salmonella typhimurium.
 Advantages of using bacteria in
genotoxicity assays
„ Their DNA is a single, circular molecule that can be
easily accessed by chemical penetration through the
cell wall
„ bacteria are genetically less complex than other
organisms,
„ it is easier to manipulate their DNA to produce more
sensitive organisms
„ bacteria are capable of testing complex mixtures, they
are typically 90% effective
„ These tests are relatively simple and may only require
a few hours
 The disadvantages
„ that it is difficult to extrapolate the mutagenicity of
bacteria to the dangers for humans or animals.
„ bacteria cannot detect the carcinogens that do not
cause DNA damage.
„ Some chemicals only cause chromosomal damage so
bacteria, which do not contain chromosomes, will not
be useful for testing these mutagens
„ there is no standard cut-off for whether or not a test is
positive or negative, Therefore results from different
laboratories will vary
„ different strains and different methods of storage, culture,
and enzyme addition.
„ Interpretation of data is also a problem. If
the organisms die due to acute toxicity,
then the mutagenic effects will not be
apparent and the sample should be
fractionated.
„ Positive tests may reflect problems with
the test conditions and not genotoxicity
„ Also a negative test result does not assure
that a chemical is not mutagenic
 Genotoxicity assay
„ Genetic mutations can occur in the forward or reverse
direction, to a new form or back to a previous form
„ AMES TEST : Bacterial Reverse Mutation
„ to evaluate a chemical's genotoxicity by measuring its ability
to induce reverse mutations
„ Salmonella typhimurium and Escherichia coli strains having
a unique mutation that has turned off histidine biosynthesis
in Salmonella or tryptophan biosynthesis in E. coli
„ the bacteria require exogenous histidine or tryptophan to
survive and will starve to death if grown without these
essential nutrients
„ The key to the assay is the bacteria can undergo a reverse
mutation turning the essential gene back on permitting the
cell to grow in the absence of either histidine or
 Ames Bacterial Reverse Mutation Assay
„ 5 strains are selected
strain TA 100
among the
following:
Salmonella
typhimurium (TA98,
TA100, TA1535,
TA1537, TA1538
and TA102)
„ Escherichia coli A: Vehicle Control B: Positive Control
(WP2 uvrA pKM101
and WP2 uvrA)
 Serratia marcescens
„ S. marcescens is a gram negative
bacillus (rod shaped) bacterium,
occurs naturally in soil, water
„ when grown at room temperature
(28˚C) produces a red pigment
called prodigiosin
„ When grown at temperatures
significantly above room
temperature
„ (37˚C) this red pigment is not
produced
 GreenScreen GC (genotoxicity &
cytotoxicity)
„ central component of the bioassay is a
yeast strain Saccharomyces cerevisiae,
„ genetically engineered to express green
fluorescent protein (GFP) when cells
experience DNA damage,
„ as a result of exposure to genotoxic
chemicals.
„ The promoter for a DNA repair gene
(RAD54) has been copied and placed in
front of a gene encoding GFP.
„ As a result, cells that sustain
chromosome damage become
increasingly fluorescent.
„ In addition, cytotoxicity is
simultaneously assessed by measuring
the reduction in cell proliferation.
 Biotechnological potential

Green Fluorescent Protein (GFP)

Aequorea victoria is probably the most

influential bioluminescent marine organism
 GFP
„ Wild-type GFP is a 27-kDa monomer consisting of 238 amino
acids
„ It has the unique characteristic of emitting green light when
excited with UV (360–400 nm) or blue (440–480 nm) light.
„ GFP is the only well-characterized example of a protein that
displays strong, visible fluorescence without any additional
substrates or co-factors
„ It maintains this fluorescence even after prolonged incubation
in strong denaturing agents such as 6 M guanidine HCl, 8 M
urea or 1% sodium dodecyl sulfate (SDS)
„ GFP has a broad range of pH stability, from pH 5.5–12.0 and
is thermostable, surviving temperatures up to 65°C.
GFP as Reporter Gene
GFP-based High-throughout drug
Screening

GFP is cloned under control of the oxidative stress promoter

SoxS. Therefore, when the cells are exposed to stress, they
will turn on the SoxS promoter and express GFP
Thus more stress will result in more GFP expression
 Table . Some of samples and their biological activities
No Group Bioactivity Scientific name
1 Coastal plant Antioxidant, natural color, cytotoxic Pandanus sp
2 Coastal plant Antimicrobes, antioxidant, biodesinfectant Pemphis sp
3 Coastal plant Antimicrobe & Antioxidant, cytotoxic Ipomoea sp
4 Sea star Antioxidant Astropecten sp
5 Sea worm aprodisiax Eunice sp
6 Sea cucumber Antimicrobes, cytotoxic Sticopus sp
7 Sea slug Antioxidant, aprodisiax Discodoris sp
8 Mangrove Antimicrobe, sunscreen, cytotoxic Xylocarpus sp
9 Fish antifouling Carcharhinus sp
10 Algae Antimicrobe Entheromorpha sp
11 Coastal plant Antimicrobe Clerodendrum
12 Algae Antioxidant Gelidiella sp
13 Coastal plant Antimicrobe, antioxidant, cytotoxic Solanum sp
14 Sponge cytotoxic Stylotella sp
15 Sponge cytotoxic Stylissia sp
16 Sponge cytotoxic Axinella sp
17 Sponge Cytotoxic Acanthella sp
18 Algae Cytotoxic unidentified