Genotoxicity studies

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

1. Submitted By- Shruti Rajeev Gautam ( M.Pharm 1𝑠𝑡 year ) Guide- Dr. Vivek Paithankar Vidya Bharati College of Pharmacy, Amravati. 2019-2020 Seminar on G enotoxicity Studies

2. Index Introduction Mechanism of genotoxicity Ames Test In Vitro & In Vivo Mammalian Cell Micronucleus Test Chromosomal Aberration Test Reference

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 Carcinogenic or cancer causing agent Mutagen or mutation causing agent Teratogen or birth defect causing agent

4. Genotoxic Risk Somatic Cell Germ Cell Cvs Cancer Multifactorial Disease Genetic Defect Diabetes Psychoses Cvs Cystic Sickle Hameomphilia Fibrosis Cell Anaemia

5. Agent that cause direct or indirect damage to the DNA ROS UV and ionizing radiation Nucleoside analogues Topoisomerase inhibitor 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. 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

7. 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

8. 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 .

9. 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 .

10. 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

11. 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

12. 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

13. Result Percentage of vehicle in the final culture medium should also be indicated

14. 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

15. 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

16. 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

17. 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

18. 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

19. Result % Of cell with structural chromosome aberration

20. 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

21. 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

22. 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

23. 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