GENOTOXICITY STUDY.pptx

TOXICOLOGY STUDY IS VERY ESSENTIAL FOR DRUG DISCOVERY. INTERNATIONAL COUNCIL FOR HARMONIZATION (ICH) HAS IMPLEMENTED SOME BASIC RULES AND REGULATION REGARDING THE TOXICITY STUDY ON ANIMAL DURING PRE-CLINICAL TRIAL, WHICH IS A PART OF DRUG DISCOVERY PROCESS. HERE SOME OF THE BASIC TEST ARE DISCUSSED ALONG WITH SOME BASIC CONCEPTS OF GENETICS. HOPE THIS WILL HELP THE STUDENTS TO UNDERSTAND THE TOPIC.

1. GENOTOXICITY STUDY Under the guidance of: DR. SHAMSHER SINGH BAJWA Professor and Head of Department of Pharmacology ISF College of Pharmacy, Punjab Presented By: PRATYUSH POREL M.Pharm , 2 nd Sem. Department of Pharmacology ISF College of Pharmacy, Punjab

2. CONTENTS SL NO. TOPIC NAME SLIDE NO. 01 INTRODUCTION: GENE 01 02 ALLELES, HOMOZYGOCITY & HETEROZYGOCITY 02 03 PHENOTYPIC & GENOTYPIC CHARACTERISTICS 03-04 04 ICH GUIDELINES ON GENOTOXICITY STUDY 05 05 CAUSES, MECHANISM AND STANDARD TESTS 06-09 06 AMES TEST (BACTERIAL REVERSE MUTATION TEST) 10-12 07 COMET ASSAY TEST 13 08 MAMMALIAN CHROMOSOMAL ABERRATION TEST 14-16 09 MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST 17-18 10 APPLICATIONS, LIMITATIONS AND FUTURE ASPECTS 19-20 11 REFERENCES 21-22

3. INTRODUCTION Genes are like a story and DNA is the language that the story is written in. - Sam Kean Gene 1 , unit of  hereditary  information that occupies a fixed position (locus) on a  chromosome . Genes achieve their effects by directing the synthesis of  proteins .

4. ALLELS AND HOMO AND HETEROZYGOSITY Allele 2 , any one of two or more  genes  that may occur alternatively at a given site (locus) on a  chromosome . Alleles may occur in pairs, or there may be multiple alleles affecting the expression ( phenotype ) of a particular  trait . The combination of alleles that an organism carries  constitutes  its  genotype . Homozygous: Organism has the paired alleles for any particular traits. Heterozygous: Organism has the different pair of alleles for any particular traits. Fig.02: Homozygosity and heterozygosity Fig.01: Alleles on chromosome Allele for phenotype A Allele for phenotype B Chromo- somes Alleles

5. PHENOTYPIC CHARACTERISTICS P henotype 3 , all the observable characteristics of an organism that result from the interaction of its genotype (total genetic inheritance) with the environment. Examples of observable characteristics include behavior, color, shape, and size. Fig.03: Different phenotypic characteristics

6. GENOTYPIC CHARACTERISTICS G enotype 3 , is the genetic makeup responsible for the all the observable characteristics of an organism (total genetic inheritance). Fig.04: Monohybrid cross in genetics Monohybrid cross: The cross between two monohybrid traits (TT and tt) is called a Monohybrid Cross. E.g., Height of the plant, position of the flower etc. Dihybrid cross: Dihybrid cross is the cross between two different genes that differ in two observed traits. E.g., Shape and color of the seed, shape and position of petals of the same flower etc. GREGOR JOHANN MENDEL

7. ICH GUIDELINES ON GENOTOXICITY STUDY

8. GENOTOXICITY Genotoxicity is a word used in genetics, that has the destructive effects on the genetic material of the cell (DNA, RNA), thus affecting the integrity of the cell. Genotoxins are one type of mutagens that causes genotoxicity followed by mutation. Genetic toxicology is the branch of toxicology that deals with those substances that can damage the genetic makeup even a chromosome. Genotoxic risk Somatic cells Germ cells Aging Cancer Sterility Genetic diseases Multifactorial diseases Cystic fibrosis Sickle cell anaemia Haemophilia Diabetes mellitus Cardio-vascular diseases Psychosis

9. CATEGORY OF GENOTOXINS Depending on their effects genotoxins can be classified as : Carcinogenic or cancer causing agents Mutagens or mutation causing agents Teratogens or birth defect causing agents MECHANISM FOR 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, alteration, flip-flop movements of nucleotides, mis-segregation or non-disjunction leading to damage of DNA and mutation. These types of chromosomal aberration may leads to abnormal cell growth, sometimes tumorigenic. Generally, ROS, UV rays, topoisomerase inhibitor and protein synthesis inhibitors are the causative agents.

10. STANDARD TESTS FOR GENOTOXICITY TEST CODES SPECIFICATIONS OF TESTS GUIDELINES TG 471 Bacterial reverse mutation (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 Skin linked recessive lethal test in Drosophila melanogaster TG 478 Genetic toxicology: Rodent dominant lethal test TG 479 In-vitro sister chromatid exchange assay in mammalian cells TG 480 Genetic toxicology: Saccharomyces cerevisiae, gene mutation assay

11. STANDARD TESTS FOR GENOTOXICITY cont... TEST CODES SPECIFICATIONS OF TESTS GUIDELINES TG 481 Genetic toxicology: Saccharomyces cerevisiae, mitotic recombinant assay TG 482 Genetic toxicology: DNA damage and repair, unscheduled DNA synthesis in mammalian cells in-vitro TG 483 Mammalian spermatogonial chromosome abbreviation 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 cells micronucleus test

12. TG 471: AMES TEST BACTERIAL REVERSE MUTATION TEST Prepare the culture of Histidine (-) Salmonella species. Petri dish 1 (CONTROL): Salmonella culture is added to the control plate with medium containing a minimal amount of histidine. Petri dish 2 (TEST): Salmonella culture is added to the test plate with medium containing a small amount of histidine along with the mutagen to be tested. Both dishes were incubated at 37 o C for 12 hours. In petri dish 1, histidine is completely exhausted only the natural revertant are grown in plate. In petri dish 2, until histidine is depleted all the His (-) cells are grown in presence of test mutagen. Higher no. of colonies represent greater mutagenicity. Control DR. BRUCE AMES

13. TG 471: AMES TEST cont … AMES TEST WORKFLOW Experimental tube Control tube Histidine selective media Incubation Incubation Control with natural revertants High no. of revertants indicates potential mutagen Suspected mutagen, agar, rat liver extract and salmonella strains Agar, rat liver extract and salmonella strains Fig.05: Negative and Positive Ames test

14. TG 471: AMES TEST cont … 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 (called as metabolic activator) is added to the test, to perform transformation. Then the bacterial sample is inoculated. But from the idea the negative test will not show any growth, but the growth can occurs due to spontaneous mutation, this is only the limitation . Fig.06: Ames test

15. TG 471: 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. Fig.07: Workflow of Comet assay test

16. TG 473: MAMMALIAN CHROMOSOMAL ABERRATION TEST Principle: The short-term in vitro mammalian cell chromosome aberration test is used to assess potential genotoxic hazard of test substances. Mammalian cells are cultured in vitro, exposed to a test substance, harvested, and the frequency of asymmetrical structural chromosome aberrations is measured. Human peripheral blood lymphocytes do not normally divide. The assessment of the effects of cyclophosphamide on lymphocytes, stimulated to divide in whole blood cultures in vitro, is described.

17. Procedure: Control and test group contain 5 animal each. Animals are exposed to the test substances once or two times in a day by an appropriate route. Animals are treated with metaphase arresting agents. Metaphase cells are observed for chromosomal aberration. TG 473: MAMMALIAN CHROMOSOMAL ABERRATION TEST Fig.08: Mammalian chromosomal aberration test

18. Fig.10: In vitro mammalian chromosome aberration test (TG 473) The cells are plated and treated with the metabolic activator (A), along with the test compound (B). This followed by exposure of cells to Colcemide / Nocodazole (C), which allows the arrest at metaphase. Various chromosomal type aberration (D) and chromatid type aberration are observed.

19. TG 474: MAMMALIAN ERYTHROCYTE MICRONUCLEUS TEST Principle: Animals are exposed to the test substance by an appropriate route If the bone marrow > the animals are sacrificed, bone marrow extracted and preparations are 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 Procedure: Animals are exposed to the test substance by an appropriate route. Bone marrow / blood cells are collected, prepared and stained. Preparations are analysed for the presence of micronuclei Each treated and control group must include at least 5 analysable animals per sex. Administration of the treatments consists of a single dose or two daily doses. The limit dose is 200mg/kg/body weight /day for treatment up to 14 days, and 1000 mg/kg/body weight/day for treatment longer than 14 days.

20. APPLICATIONS Assessment of genotoxicity in occupational exposed individual. A study with 838 drugs, analyzed by the different assays, pointed out that 56.3% of them were genotoxic. So, it is used for testing of new pharmaceutical product for its safety. Testing of various products used by humans cosmetics, food products. Nowadays, most of the toxicological studies are based on traditional methods, they are not always the best choice for predicting human health effects. Cell culture methodologies have substantially improved resulting in a number of promising cell models composed of relevant morphological and biochemical signaling processes that may reproduce the in vivo environment. LIMITATIONS

21. FUTURE ASPECTS The genotoxicity tests are an important for the safety assessment of chemicals and drugs according to the guidelines set by international regulatory authorities. The novel in vitro model system along with other emerging technologies such as high-throughput assay system, imaging technologies, computational approaches will help in developing completely new integrated approaches for genotoxicity testing and assessment, which will be more advanced and promising than the traditional methods. In the current scenario, more research, development, and data integration are required to meet the demands of regulatory testing. Only then, there is a possible likelihood for reduction and complete replacement of traditional testing method acceptable to regulatory authorities.

22. Britannica, T. Editors of Encyclopedia. "gene." Encyclopedia Britannica, January 25, 2024. https://www.britannica.com/science/gene. Britannica, The Editors of Encyclopedia. "allele". Encyclopedia Britannica, 2 Jun. 2023, https://www.britannica.com/science/allele. Accessed 5 February 2024. Britannica, The Editors of Encyclopedia. "genotype". Encyclopedia Britannica, 11 Jan. 2024, https://www.britannica.com/science/genotype. Accessed 7 February 2024. Edward E. Gahres , Tongue rolling and tongue folding: and other Hereditary Movements of the Tongue,  Journal of Heredity , Volume 43, Issue 5, September 1952, Pages 221–225,  https://doi.org/10.1093/oxfordjournals.jhered.a106313 Zhang XJ, He PP, Liang YH, Yang S, Yuan WT, Xu SJ, Huang W. A gene for freckles maps to chromosome 4q32–q34. Journal of investigative dermatology. 2004 Feb 1;122(2):286-90. Ordu KS, Agi CE. Inheritance pattern of hairline shape amongst Nigerian population. Int J Curr Microbio App Sci. 2014;3:61-5. REFERENCES

23. REFERENCES 7. Barton NH. Genetic hitchhiking. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences. 2000 Nov 29;355(1403):1553-62. 8. Britannica, The Editors of Encyclopedia. "genotype". Encyclopedia Britannica, 11 Jan. 2024, https://www.britannica.com/science/genotype. Accessed 7 February 2024. 9. Mallick A, Gautam A. Mendelian Crosses. 10. Kamath GH, Rao KS. Genotoxicity guidelines recommended by International Conference of Harmonization (ICH). Genotoxicity Assessment: Methods and Protocols. 2013:431-58. 11. Mortelmans K, Zeiger E. The Ames Salmonella/microsome mutagenicity assay. Mutation research/fundamental and molecular mechanisms of mutagenesis. 2000 Nov 20;455(1-2):29-60. 12. Speit G, Vasquez M, Hartmann A. The comet assay as an indicator test for germ cell genotoxicity. Mutation Research/Reviews in Mutation Research. 2009 Jan 1;681(1):3-12. 13. Araldi RP, de Melo TC, Mendes TB, de Sá Júnior PL, Nozima BH, Ito ET, de Carvalho RF, de Souza EB, de Cassia Stocco R. Using the comet and micronucleus assays for genotoxicity studies: A review. Biomedicine & Pharmacotherapy. 2015 May 1;72:74-82.