genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic
3. Introduction
Genotoxicity is a word in genetics defined as a
destructive effect on a cell genetic material affecting its
integrity
Genotoxins are mutagen they can cause mutation
Genotoxins include both radiation and chemical
genotoxins
Genotoxins can be divide in three type
1. Carcinogenic or cancer causing agent
2. Mutagen or mutation causing agent
3. Teratogen or birth defect causing agent
5. Agent that cause direct or indirect damage to the DNA
1. ROS
2. UV and ionizing radiation
3. Nucleoside analogues
4. Topoisomerase inhibitor
5. Protein synthesis inhibitor
Importance
Genotoxiciy assay have become an integral component of
regulatory reqirement
Compound which are positive in these test have the potential to
the human carcinogens and mutagen so it used in prediction
6.
7. Mechanism of genotoxicity
They damage to the genetic material is caused by the
interaction of the genotoxic substance with the DNA
structure and sequence
These genotoxic substance interact a specific location or
base sequence of the DNA structure causing lesion
breaking fusion deletion ,mis –segregation or non
disjunction leading to damage and mutation
8. TG471:AMES TEST
The Ames test is a widely employed method that uses
bacteria to test whether a given chemical can cause
mutations in the DNA of the test organism
The procedure was described in a series of papers in the
early 1970s by Bruce Ames and his group at the
University of California
9. Principle
Ames test uses several strains of bacteria (Salmonella,
E.coli) that carry a particular mutation.
Point mutations are made in the histidine (Salmonella
typhimurium) or the tryptophan (Escherichia coli)
operon, rendering the bacteria incapable of producing
the corresponding amino acid.
These mutations result in his- or trp- organisms that
cannot grow unless histidine or tryptophan is supplied.
10. Procedure
Isolate an auxotrophic strain of Salmonella Typhimurium
for histidine. (ie. His-ve)
Prepare a test suspension of his-ve Salmonella Typhimurium
in a plain buffer with test chemical (eg. 2-aminofluorene).
Also add a small amount of histidine.
Also prepare a control suspension of His-ve Salmonella
Typhimurium but without test chemicals.
Incubate the suspensions at 37°C for 20 minutes
Prepare the two agar plate and spread the suspension on
agar plate.
Incubate the plates at 37°C for 48 hours.
After48 hours count the number of colonies in each plate.
11.
12. Result interpretation
The mutagenicity of chemicals is proportional to number of
colonies observed.
If there is a large number of colonies on the test plate in
comparison to control, then such chemical are said to be
mutagens.
Uses
While ames test is used to identify the revert mutations which are
present in strains, it can also be used to detect the mutagenicity of
environmental samples such as drugs, dyes, reagents, cosmetics,
waste water, pesticides and other substances which are easily
solubilized in a liquid suspension
13. In Vitro Mammalian Cell Micronucleus Test
The in vitro micronucleus (mnvit) test is a genotoxicity
test for the detection of micronuclei (MN) in the
cytoplasm of interphase cells. Micronuclei may originate
from acentric chromosome fragments (i.e.Lacking a
centromere), or whole chromosomes that are unable to migrate
to the poles during the anaphase stage of cell division.
Therefore the mnvit test is an in vitromethod that
provides a comprehensive basis for investigating chromosome
damaging potential in vitro because both aneugens and
clastogens can be detected in cells that have undergone cell
division during or after exposure to the test chemical
14. Procedure
Detection of the frequency of micronucleus
Cell culture of human or other mammalian origin are
exposed to the test chemical formation of micronucleus in
interphase cells
Harvested and stained interphase cell are analyzed for the
presence of micronuclei treated with a cytokinesis
Assay detect the activity of clastogenic and aneugenic
chemical
16. MAMMALIAN ERYTHROCYTE MICRONUCLEUS
TEST
Animal are exposed to the test substance by an
appropriate route
If bone marrow > the animal are scarified bone marrow
extracted and preparation made and stained
If peripheral blood > the blood is collected at appropriate
time after treatment and smear preparation are made and
stained
Preparation are analyzed for the presence of micronuclei
17. Principle
For the detection of damage induced by the test substance
to the chromosome or the mitotic apparatus erythroblast
Identifies micronuclei containing lagging chromosome
fragment or whole chromosome
An increase in the frequency of micro nucleated
polynucleotide erythrocyte in treated animal is an
indication of induce chromosome damage because they
lack main nucleus
18. Procedure
Animal are exposed to the test substance by an appropriate
route
Bone marrow or blood is collected prepared stained
Preparation are analyzed for the presence of micronuclei
Each treated and control group must include at least 5
analyzable animal per sex
Administration of the treated consist of a single dose or two
daily doses
The limit dosed is 2000mg/kg weight/day for treatment up to
14 day and 1000 mg/kg body weight/day for treatment longer
than 14 days
19. In Vitro Mammalian Chormasomal Abberation Test
Principle
After exposure of cell culture treated with a metaphase
arresting substance colchicine with and without metabolic
activation
Harvested stained and metaphase cell are analyzed
microscopically for the presence of chromosomes
aberration
20. Cell line
Cell strain
Cell culture
test substance
3 concentration of test substance
Dupilation culture during each concentration
Finally treated with m-phase arresting substance
Harvest and stained
22. In Vitro Mammalian Bone Marrow Chromosomal
Aberration Test
Principle
For detection of structural chromosome aberration
induced test compound only in bone marrow cell of
animal
Animal exposed to the test substance metaphase arresting
agent sacrificed at appropriate times after treatment
23. Procedure
Animal are exposed to the test substance by an
appropriate route
Then animal are treated with a metaphase arresting
agent
Chromosome preparation are then made from the bone
marrow cell and stained
Each treated and control group must include at least 5
analyzable animal per sex
The limit dosed is 2000mg/kg weight/day for treatment
up to 14 day and 1000 mg/kg body weight/day for
treatment longer than 14 days
24. Prior to sacrifice animal are injected i.p with an
appropriate dose of a metaphase arresting agent ,sampled
Cell are harvested from bone marrow and analyzed from
chromosome aberration
Chromosome preparation : bone marrow in hypotonic
solution ,spread on slides and stained
Result
The mitotic index should be determined as a measure of
cytotoxicity in at least 1000 cell per animal
25. REFERNCE
Genotoxicity Assessment: Methods and Protocols by
Mahima Bajpayee, Alok Dhawan
IOSR journal of pharmacy and biological sciences
volume 1 issue 2
https://microbiologyinfo.com/ames-test/
https://ntp.niehs.nih.gov/iccvam/suppdocs/feddocs
/oecd/oecd-tg487-2014-508.pdf