Mutagenicity, Carcinogenicity, Genotoxicity Tests

It is a brief power point presentation about the types of Mutagenicity tests and about AMES Test.

1. Mutagenicity,carcinogenicityGenotoxicity Tests S. Archana (Reg no. 22701502) I MSc Biochemistry V.V.Vanniapermual college for women, Virudhunagar.

2. contents Introduction Types of mutagenicity tests Molecular level Gene level Chromosomal level Three important tests AMES Test HPRT Test Mouse Micronucleus Test

3. Introduction The purpose of mutagenicity testing is to identify substances that can cause genetic alterations in somatic and/or germ cells and use this information in regulatory decisions . Mutagenicity testing is the first step to screen the chemicals for their potential to be a pesticide, food additive, or drug . The most widely used mutation test is Ames test, developed by Ames, which is performed in different strains of Salmonella typhimurium and in Escherichia coli.

4. The most widely used mutation test is Ames test, developed by Ames, which is performed in different strains of Salmonella typhimurium and in Escherichia coli.

5. Types of mutagenicity tests It can be separated on the basis of 1)Molecular level 2)gene level 3) chromosomal level 1)Molecular level: Comet assay – The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells . Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix.

6. 2) Gene level test TK Gene Mutation test : The in vitro mammalian cell gene mutation test (OECD 490), also referred to as the mouse lymphoma assay , is used to detect a spectrum of genetic events denoting gene mutations induced by chemical substances in the cell lines that measure mutation at thymidine kinase (TK).

7. In vivo pig gene mutation assay: The Pig-a gene mutation assay has emerged as a valuable tool for quantifying in vivo and in vitro mutational events . The Pig-a locus is located at the X-chromosome, giving the advantage that one inactivated allele can give rise to a mutated phenotype, detectable by multicolour flow cytometry .

8. 3)Chromosomal level test In Vivo Micronucleus Assay: The micronucleus test (MNT) is used to determine if a compound is genotoxic by evaluating the presence of micronuclei. Micronuclei may contain chromosome fragments produced from DNA breakage ( clastogens ) or whole chromosomes produced by disruption of the mitotic apparatus ( aneugens ). SCE assay: It is a powerful technique to visually detect the physical exchange of DNA between sister chromatids . SCEs could result as a consequence of DNA damage repair by homologous recombination (HR) during DNA replication.

9. Micronucleus assay SCE

10. Important tests The three most commonly conducted assays are: 1)The Ames Salmonella typhimurium reverse mutation assay, 2)The Chinese hamster ovary (CHO) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in vitro cytogenetics assay, and 3)The mouse micronucleus test .

11. AMES TEST Ames test it is a biological assay to assess the mutagenic potential of chemical compounds. It utilizes bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. The test was developed by Bruce N. Ames in 1970s to determine if a chemical at hand is a mutagen. Objective To determine the mutagenic activity of chemicals by observing whether they cause mutations in sample bacteria.

12. 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. But culturing His- Salmonella is in a media containing certain chemicals, causes mutation in histidine encoding gene, such that they regain the ability to synthesize histidine   (His+ ). This is to say that when a mutagenic event occurs, base substitutions or frameshifts within the gene can cause a reversion to amino acid prototrophy . This is the reverse mutation. These reverted bacteria will then grow in histidine - or tryptophan-deficient media, respectively.

13. A sample’s mutagenic potential is assessed by exposing amino acid-requiring organisms to varying concentrations of chemical and selecting for the reversion event. Media lacking the specific amino acid are used for this selection which allow only those cells that have undergone the reversion to histidine / tryptophan prototrophy to survive and grow. If the test sample causes this reversion, it is a mutagen.

14. Method: I )  Isolate an auxotrophic strain of Salmonella  Typhimurium for histidine . ( ie . His- ve ) II)  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 . Note: small amount of histidine is required so bacteria starts growing. Once histidine is depleted only those bacteria mutated to gain the ability to synthesize histidine form colonies. III)  Also prepare a control suspension of His- ve  Salmonella  Typhimurium but without test chemicals. IV)   Incubate the suspensions at 37°C for 20 minutes V)  Prepare the two agar plate and spread the suspension on agar plate. VI)  Incubate the plates at 37°C for 48 hours. VII)  After48 hours count the number of colonies in each plate.

15. 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. Very few numbers of colonies can be seen on control plate also. This may be due to spontaneous point mutation on hisidine encoding gene. Uses 1)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. 2)AMES test are used in pharmacy research before a compound given to a animal.

16. 3)Smoker Urine test for mutagenicity is possible by the use of AMES test. 4)Primary DNAdamage test can be done through AMES test. 5)This test is highly sensible for testing the mutagnicity of water and solid soil sample from the nuclear. Merits: 1)Simple, rapid and robust bacterial assay. 2)Ease and low cost of the test make it invaluable for screening substances in our environment for possible carcinogenicity. 3)Ames test can detects suitable mutants in large population of bacteria with high sensitivity.

17. limitation 1)Some substances that cause cancer in laboratory animals ( dioxin , for example) do not give a positive Ames test (and vice-versa) 2)Ames assay consists of Salmonella typhimurium  strains and so it is not a perfect model for human. 3)Some sample shows false postive test. That is scattered large population microbes integates only spontaneous mutation not mutation due to the compound taken as test.Aggregated population of microbes only positive for test compound.

18. HPRT GENE TEST In Vitro Mammalian Cell Gene Mutation Test (HPRT Gene) The in vitro mammalian cell gene mutation test is used to detect mutations of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in Chinese hamster ovary (CHO) or lung (V79) fibroblasts. Screening and Regulatory Support 1)Cultures are incubated with several concentrations of the test compound for three to four hours in the presence and absence of metabolic activation (S9).

19. 2) Cells are subcultured for seven to eight days after treatment to allow expression of the mutant phenotype, and then plated in media with and without 6-thioguanine (TG) to select for mutants and determine cloning efficiency. 3) A positive outcome is characterized by a statistically significant, dose-dependent increase in mutant frequency that exceeds historical negative control limits.

20. When To Perform Screening Reduced volume and/or abbreviated formats available REACH requirement As part of Annex VIII testing Mutagenicity To confirm presumed mutagenic activity arising from limited/small genetic damage (e.g., positive Ames or large colony MLA) To assess mutagenicity when the Ames assay may not be appropriate (e.g., antibiotics, nanomaterials )

21. The Mouse Micronucleus Test Screening and Regulatory Support Male and/or female rats or mice are treated with the test compound at three dose levels, usually two or three times at 24-hour intervals. Approximately 24 hours after the last dose, bone marrow or peripheral blood is collected to determine the frequency of micronucleated polychromatic erythrocytes (MN-PCEs) or micronucleated reticulocytes (MN-RETs), respectively. A positive outcome is characterized by a statistically significant, dose-dependent increase in MN-PCEs or MN-RETs that exceeds historical control limits. Can be combined with standard toxicology tests, the comet assay and the Pig-a assay. Administration routes include: oral, intravenous, infusion and inhalation

22. When to Perform Screening Abbreviated formats available or can be added to non-GLP tolerability studies IND-enabling As part of the ICH S2(R1) standard battery (Option 1 or 2) REACH requirement To follow up a positive result in any of the Annex VII or VIII genotoxicity tests

23. REFERENCE: https://www.criver.com/ https://microbiologyinfo.com/ https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/mutagen-testing https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352214/ https://www.slideshare.net/manoahram1/ames-test# https://www.slideshare.net/krupasagar12/ames-test-109209493 https://www.nature.com/articles/s41596-020-0398-1 https://www.mbbiosciences.com/genotoxicity https://www.sciencedirect.com/science/article/pii/B9780128007648000057 https://onlinelibrary.wiley.com/doi/10.1002/em.20627 https://www.mdpi.com/1422-0067/21/4/1534/htm https://currentprotocols.onlinelibrary.wiley.com/doi/abs/10.1002/0471143030.cb2207s25