Genotoxicity

study of genotoxicity

1. GENOTOXICITY SUB.by - narsingh kashyap m.f.Sc . , Fish Genetic s and breeding IFPGS, OMR Campus, CHENNAI

2. History & Background Origin of genetic toxicology in 1900 , Genetic toxicity independent branch of science started in 1927. OECD Genetic Toxicology TGs was first published in 1987. : OECD ,ICH, SCHEDULE Y (D&C India).

3. Major test guidelines OECD (The Organization for Economic Cooperation and Development) -1961 ICH ( International Council for Harmonisation ) – 1990 SCHEDULE Y of Drug and Cosmetic act -1940

4. Introduction Genotoxicity is a word used in genetics that describes the possession of substance that has destructive effect on the genetic material of the cell (DNA,RNA) , thus affecting the integrity of the cell. Genotoxins are mutagens that can cause genotoxicity leading to the damage of DNA or chromosomal materials thus causing mutation. Genetic toxicology is the is the branch of science that deal with study of agents or substance that can damage the cells DNA chromosome.

5. Importance of genotoxicity studies Genotoxicity studies can be defined as various in-vitro and in-vivo tests designed to identify any substance or compounds which may induce damage to genetic material either directly or indirectly by various mechanisms. These tests should enable the identification of hazard with respect to DNA damage and fixation

6. Genotoxins can be of the following category depending on its effects 1) Carcinogens or cancer causing agents 2)Mutagens or mutation causing agents 3) Teratogens or birth defect causing agents

7. Agents that can cause direct or indirect damage to the DNA Reactive oxygen species. UV and ionizing radiations. Nucleoside analogues . Topoisomerase inhibitors . Protein synthesis inhibitors .

8. Mechanism of genotoxicity The damage to the genetic material is caused by the interactions of the genotoxic substance with the DNA structure and sequence. These genotoxic substance interact at a specific location or base sequence of the DNA structure causing lesions, breakage, fusion, deletion, mis -segregation or non-disjunction leading to damage and mutation

9. Standard test battery for genotoxicity TG 471 Bacterial Reverse Mutation Test (Ames Test) TG 472 Genetic Toxicology: Escherichia coli, reverse assay TG 473 In-Vitro Mammalian Chromosome Aberration Test TG 474 Mammalian Erythrocyte Micronucleus Test TG 475 Mammalian Bone Marrow Chromosome Aberration Test TG 476 In-Vitro Mammalian Cell Gene Mutation Test TG 477 Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster TG 478 Genetic Toxicology: Rodent Dominant Lethal Test TG 479 Genetic Toxicology: In-Vitro Sister Chromatid Exchange Assay in Mammalian Cells TG 480 Genetic Toxicology: Saccharomyces cerevisiae , Gene Mutation Assay

10. TG 481 Genetic Toxicology: Saccharomyces cerevisiae , Mitotic Recombination Assay TG 482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In-Vitro TG 483 Mammalian Spermatogonial Chromosome Aberration Test TG 484 Genetic Toxicology: Mouse Spot Test TG 485 Genetic Toxicology: Mouse Heritable Translocation Assay TG 486 Unscheduled DNA Synthesis (UDS) Test with Mouse Liver Cells In-Vitro TG 487 In-Vitro Mammalian Cell Micronucleus Test

11. TEST

12. TG 471 : AMES TEST (Bacterial reverse mutation test)

13. Ames test was brought forward by Bruce Ames in 1970. • He is professor in university of California , berkely . In department of biochemistry. • He developed this method because previous methods were expensive and time consuming.

14. Plate incorporation method STEPS OF AMES TEST: Prepare the culture of Salmonella histidine auxotroph's (His-). Mix the bacterial cells and test substance in dilute molten top agar with a small amount of histidine in one set, and control with complete medium plus large amount of histidine . Pour the molten mixture on the top of minimal agar plates and incubate at 37°C for 2-3 days. Until histidine is depleted all the His- cells will grow in the presence of test mutagen. When the histidine is completely exhausted only the revertants will grow on the plate. •High number of colonies represent the greater mutagenicity

15. There are some chemicals that are non mutagenic , but they become mutagenic when they come in contact in body metabolism. Bacteria does not have metabolizing capacity, therefore, the liver extract is added to this test, to promote transformation • And then the bacterial sample is inoculated. But from the idea the negative test will not show any growth, but the growth can occur due to spontaneous mutation, this is the only limitation of this test. C0NTI,,,

16. Pre-incubation method Pre-incubated with the test strain . 0.05-0.1ml (approx. 108 cells) & sterile buffer or the metabolic activation system (s9 0.5 ml) usually for 20 min @30-37°c [aeration+ shaker – 48 to 72hrs] • Mix overlay agar (2ml) and pouring onto the surface of a minimal agar plate • Report number of revertant colonies per plate • ( with +ve & -ve coloies nos) • Standard deviation

17. Comet assay test Principle- The basis for this assay is that loops of DNA containing a break lose their super coiling and become free to extend toward the anode when exposed to current during electrophoresis at high pH. • The results appear as structures resembling comets observed by fluorescence microscopy

18. TG 474: Mammalian Erythrocyte Micronucleus Test Animals are exposed to the test substance by an appropriate route If bone marrow > the animals are sacrificed, bonemarrow extracted, and preparations made and stained. If peripheral blood > the blood is collected at appropriate times after treatment and smear preparations are made and stained. Preparations are analysed for the presence of micronuclei

19. Fish as a model for the Aquatic genotoxicity Aquatic animal specially fishes are most susceptible to genotoxic effect caused by the pollutants usually agricultural wastes, chemicals heavy metal etc. The selection of fishes as a model in the eco- genotoxicalogical studies could be made since fish is a very sensitive bio- indicator of water quality and can highlight the potential danger of new chemical introduced in the aquatic environment and also respond in a manner similar to higher vertebrates. They have graater ability to metabolize xenbiotics and accumulate polllutants .

20. Contii ,,, They capable of inhabiting practically all zones of the aquatic habitat and have great Commercial and recreational value. They play different roles in the tropic web such as undergoing bio accumulation of environment pollutants and biotransformation of xenobiotics through cytochrome 450- dependent oxidative metabolism like mammals, besides they respond to mutagens at low concentration. In Addition as compared to mammalian cells, they have been shown to be more sensitive for the induction of DNA damage, there for they can used as sentinel organism for bio-monitoring studies.

21. Genotoxicity due to the heavy metals Aquatic ecosystem receive a number of toxic substances, among which heavy metals released from domestic ,industrial and other man-made activities are the significant importance ,due to their toxicity their bioaccumulation potential and their ability to induce damage in DNA. The studies carried out on various fishes have shown that these metals alter the physiological activities and biochemical parameters both in tissues and blood. The mercury toxicity for clarius batrachus resulted in marked decrease in hemoglobin and Erythrocyte count.

22. Genotoxicity due to the Microbial toxin Ricardo et.al (2010) evaluated the toxicity and genotoxicity in Astyanax bimaculatus ,as induced by an extract of cyanobacterial microcystins ,using two administration routes and different end points ,such as micronucleas and Apoptosis-necrosis testing and comet assaying. The genotoxicity caused by the microcystins LR and LA from a bloom collected in a eutrophic lake LC50 (72 h) was determined as 242.81 µg L-1 and LD50 (72 h) as 49.19 µg kg-1 bw . There observed a significant increase of DNA damage in peripheral erythrocyte ake LC50 was determine as 242.81

23. REFERENCE 1. Essential concepts in toxicology by prof.Dr.Gupta . 2. Shaily Umang Shah . Importance of Genotoxicity & S2A guidelines for genotoxicity testing for pharmaceuticals .IOSRJPBS.2012 1(2) 43-54 . 3. OECD website , www.oecd.org , genotoxicity test guidelines :2016 OECD Test guidelines; 471,473,474,475,483,487 .