M pharm pharmacology

1. Genotoxicity studies
Submitted By: Ayodhya Tanaji Paradhe Submitted To: Prof. Vaishnavi Ma’am
M Pharm 1st Year (Pharmacology) (Pharmacology Department)
Sudhakar Rao Naik Institute Of Pharmacy Pusad

2. Content
 Introduction
 OECD guidelines for genotoxicity
• Ames test
• In vitro mammalian cell micronucleus test
• In vivo mammalian erythrocyte micronucleus test
• In vitro mammalian chromosomal Aberration test
• In vivo mammalian bone marrow chromosome aberration test
 Importance
 Refences

3. Introduction
Genotoxicity:
 Genotoxicity is word used in genetic that describes the property of substance that has
destructive effect on the genetic material of the cell (DNA,RNA) thus affecting the
integrity of the cell
 Genotoxicity study can be defined as various in vitro and in vivo tests designed to
identify any substance which may induced damage to genetic material either directly or
indirectly by various machanisms
 Is branch of science deals with study of agents or substance that can damage the cell
DNA chromosome
 Genotoxin:
 Those agents which cause genotoxicity called genotoxins
 These are essentially mutagens that cause damage to DNA and chromosomal material
and bring about mutation
 Genotoxins can include chemical substance as well as radiation

4. Types:
1. Carcinogenetic or cancer causing agents
2. Mutagen or mutation causing agents
3. Teratogen or birth defect causing agents
History:
 Origin of genetic toxicology in 1900, genetic toxicity independent branch of science
started in 1927
 OECD genetic toxicology was first published in 1987
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. 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
Agents cause direct or indirect damage to DNA:
 ROS
 UV and ionizing radiation
 Nucleoside analogues
 Topoisomerase inhibitors
 Protein synthesis inhibitors
Mechanism of genotoxicity study:
 The damage to genetic material is caused by interaction of genotoxic substance with the
DNA structure and sequence
 These genotoxic sub. Interact at a specific location or base sequence of the DNA structure
causing lesions, breakage, fusion, deletion, mis-segregation or non-disfuction leading to
DNA damage and mutation

6. genotoxic risk
1. somatic cell 2. germ cell
CVS cancer genetic defect multifactorial disease
1. cystic fibrosis 1. DM
2. sickle cell anemia 2. psychoses
S3. haemophilia 3. CVS

7.  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 comp. 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
 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

8.  The revertant bacteria detected by their ability to grow in absence of amino acid required by
parent Ames strain
 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

9. The choice and condition of the metabolic activation system depend on class of
chemical being tested
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
• Procedure:
Two methods:
1. Plate incorporation method
2. preincubation method

10. 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

12. 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

14. • 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

15.  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

16. • 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

17. • Reporting:
The report should include-
 % of vehicles in the final culture medium should also be indicated
 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

18. • 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

19.  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

20. • 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

21.  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

22. 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 animal

23.  Importance:
• Genotoxicity assays have become an integral component of regulatory requirement
• Compounds that are +ve in this test have the potential to be human carcinogens and
mutagens so it used in the prediction
• Important to identify substances that can cause genetic alteration in somatic and germ
cells and this information is used in regulatory decision
• These tests should enable the identification of hazards concerning DNA damage and
fixation
• Important role in predicting if the compound has the potential to cause genotoxicity and
carcinogenicity

24.  References:
1. Genotoxicity assessment: methods and protocols by Mahima Bajpayee, Alok
Dhavan
2. Essential concepts in toxicology by prof. Gupta
3. OECD website, www.oced.org, genotoxicity test guidelines: 2016 OECD test
guidelines:471,473,474,475,487
4. http://microbiologyinfo.com/Ames-test/