Genotoxicity studies

Presented by,
Tejo lahari sri iragavarapu
m.Pharmacy 1st year
pharmacology
Genotoxicity :
Genotoxicity is the property of chemical agents that damage the genetic information within a cell causing
mutations, which may lead to cancer.

Genotoxin:
A genotoxin is a chemical or agent that can cause DNA or chromosomal damage.

Guidelines for genotoxicity:

1. TG 471- bacterial reverse mutation test( Ames test)

2. TG 472- genetic toxicology: E. coli
3. TG 473- in vitro mammalian chromosome Aberration test
4. TG 474- in vivo mammalian erythroxyte micronucleus test
5. TG 475- in vivo mammalian bone marrow chromosome Aberration test
6. TG 476- in vitro mammalian cell gene mutation test
7. TG 487- in vitro mammalian cell micronucleus test
Ames test
(TG 471):

 It was developed in the 1970s by Bruce Ames, prof. of biochemistry

 It is a fast, inexpensive, and sensitive assay of the ability of chemical compound or mixture to induce
mutation in DNA
 It is based on reverse mutation or back mutation so-called bacterial reverse mutation assay
 He developed this method because previous methods were expensive and time- consuming

• Bacteria :
1.Salmonella typhimurium :TA1535; TA1537 or TA97a or TA97; TA98; and TA100 ) strains
2. E.coli :WP2 uvrA, WP2 uvrA(pKM101)

• Mutagens cause reverse mutations and bacterial cells there by get the property to grow on the medium without histidine and
form colonies.
Principle:

 Identifies substances that induce gene mutations by base substitution or frame shift

 Two species of bacteria i.e. S. typhi and E. coli with identified mutations in an amino acid i.e. His and
Trp as the receptor locus which involves substitution, addition or deletion of one or few DNA base pairs

 The principle of the Ames test is that it detects mutations that revert mutation present in test strains
and restores the functional capability of bacteria to synthesize an essential amino acid

• Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an
exogenous metabolic activation system.

• Treatment mixture is incubated and then mixed with an overlay agar plating onto minimal medium.

• after two or three days of incubation, revertant colonies are counted and compared.

methods
a.Plate incorporation method
b. preincubation method
Description of the method:
• Preparation:

1. bacteria: fresh cultures of bacteria should be grown up to the late exponential or early stationary
phase of growth (app. 109cells/ml) temp. of culture -37 degrees Celsius, Salmonella strain of
bacteria used.

2. medium: an appropriate minimal agar eg. Containing Vogel banner minimal med. E and glucose
overlay agar containing Histidine and biotin or tryptophan to allow for few cell divisions is used.

3. Metabolic activation: bacteria should be exposed to test sub. both in presence and absence of an
appropriate metabolic activation A commonly used system is cofactor supplemented post-mtd
fraction (S9) prepared liver of rodents

4. Test substance preparation: solid test substance dissolved or suspended in appropriate solvents or vehicles and
diluted if appropriate before treatment of bacteria  liquid test substance may be added directly to the test
system and /or diluted before treatment  fresh preparation should be employed •
1. Plate incorporation method:
steps:
Prepare culture of salmonella histidine auxotrophs (His-)

mix bacterial cells and test substance in dilute molten top agar with a small amount of
histidine in one set, and control with a complete medium plus a large amount of histidine

Pour molten mixture onto agar plates and incubate at 37 degrees for 2-3 days

until histidine is depleted all the His- cells will grow in the presence of test mutagen

when histidine is completely exhausted only the revertant will grow on the plate

The high no. of colonies represents the greater mutagenicity

2. Preincubation method:

The test substance/solution is preincubated with test strain ( approx. 108 cells) and sterile buffer or
metabolic activation system (0.5ml) for 20 min. or temp. 30-37degrees

before mixing with overlay agar poured onto the surface of a minimal agar plate

Usually, 0.5 or 0.1 ml of test substance, 0.1 ml of bacteria, and 0.5 ml of S9 mix or sterile buffer are
mixed with 2ml of overlay agar

tubes should be aerated during pre-incubation by using the shaker

All plates in the given assay were incubated at 37 degrees for 2-3 days

after the incubation period, no. of relevant colonies per plate is counted
Reporting:

 Test substance
 Solvent/vehicle
 Strains
 Test condition

• Result:

 Sing of toxicity
 Sign of precipitation
 Individual plate count
 Mean no. of revertant colonies per plate and standard deviation
 Dose-response relationship
 Statistical analysis
In vitro mammalian cell micronucleus test( TG 487):

Micronuclei(MN) are small nucleus that forms whenever a chromosome or its fragment is incorporated with
daughter nuclei during cell division

Principle:

 Detection of the frequency of micronuclei

 The in vitro micronucleus test is a genotoxicity test for the detection of MN in the cytoplasm of interphase
cells

 MN may originate from acentric chromosome fragments(i.e. lacking a centromere) or whole chromosomes
that are unable to migrate to poles during the anaphase stage of cell division

 Therefore the MN in vitro test is an vitro method 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 test chemical
Procedure:

 Detection of frequency of MN

 Cell cultures of human or other mammalian origin are exposed to test the chemical,
formation of MN in interphase cells

 Harvested and stained interphase cells are analysed for the presence of MN, treated
with a cytokinesis blocker

 Assay detects the activity of clastogenic and aneugenic chemicals

Reporting:
The report should include-

1. % of vehicles in the final culture medium should also be indicated

2. Detect frequency of MN
In vivo mammalian erythrocyte micronucleus test(TG 474):
Principle:

 For detection of damage induced by test substance to the chromosome or the mitotic apparatus
erythroblast

 Identifies MN containing lagging chromosome fragments or whole chromosome

 An increase in the frequency of micro nucleated polynucleotide erythrocytes in treated animals is

an indication of induced chromosome damage because they lack the main nucleus
Procedure:
Animals are exposed to test substances by an appropriate route

each treated and control group must include at least 5 analysable animals per sex

Administration of treated group single dose or two daily dose

The limit dose is 2000mg/kg body weight/day for treatment up to 14 days and 1000mg/kg body weight/day
for treatment longer than 14 days

if bone marrow > the animal is sacrificed bone marrow is extracted and preparation made and stained

If peripheral bold> the blood is collected at an appropriate time after treatment and smear preparation is
made and stained

Preparation is analyzed for the presence of MN

In vitro mammalian chromosomal Aberration test(TG 4730):
Principle:

 After exposure of cell cultures, treated with metaphase arresting substance colchicine with or
without metabolic activation
 Harvested, stained and metaphase cells are analyzed microscopically for the presence of
chromosome aberrations
 Cell lines: CHO, CHL V79, TK6

Structural aberration may be two types

1. Chromosome
2. Chromatid

 Observed only in metaphase of 1st or 2nd mitotic division after treatment

 Damage induced pre-S-phase chromosome aberration
 Damage-induced post-S- phase chromatid aberration
Procedure:

cell line

cell strain

cell culture

treated with a test substance (3 conc. Of test sub.) duplication of each culture during each conc. finally,

treated with m-phase arresting substance- culture treated with colcemid or colchicine 3 hr before to
harvesting process involves hypotonic treatment of cells and fixation and staining

• Result: % of cells with structural chromosome aberration counted

 In vivo mammalian bone marrow chromosome aberration test(TG 475):
Principle:

 for the detection of structural chromosome aberration induced by test compound only in bone
marrow cells animals (rodents)

 Animals are exposed to test substance metaphase arresting agents, sacrificed at appropriate
times after treatment

 Bone marrow cells are usually obtained from femurs or tibias immediately after sacrifice and
stained using establishing methods

 Blood: tail vein or other appropriate blood vessels, smear preparations are made and then
stained

 DNA-specific stain eg. Acridine orange

Procedure:

Animals exposed to the test substance by an appropriate route

each treated and control group must include at least 5 analysable animals per sex

Animals are injected i.p. with an appropriate dose of metaphase-arresting agents- the limit dose
is 2000mg/kg body weight/day up to 14 days and 1000mg/kg body weight/day for treatment
longer than 14 days

chromosome preparation is then made from bone marrow cells and stained, and metaphase cells
are analyzed for chromosome aberration Chromosome preparation: bone marrow in hypotonic
solution spread on slides and stained

• Analyasis: The mitotic index should be determined as a measure of cytotoxicity in at least 1000
cells per anima