Micronucleus Assay

Includes both invivo and invitro procedures of micronucleus assay

1. Micronucleus Assay PRESENTED BY G.R.HARIKA M.S .(Pharm.) 1 st semester Dept . of Regulatory Toxicology GUIDED B Y Dr. G.B. JENA Associate Professor Dept . of Pharmacology and Toxicology National Institute of Pharmaceutical Education and Research, S.A.S Nagar, Mohali

2. Flow of presentation: Introduction Mechanism Characteristics Types Models Conclusion

3. Introduction: A micronucleus assay is a test used for toxicological testing of potential genotoxic compounds It is commonly used because of its simplicity, reliability and reproducibility This assay is one of the most successful assays for genotoxic carcinogens, i.e., carcinogens that act by causing genetic damage

4. It has widespread acceptance and regulatory approval and is the OECD guideline for the testing of chemicals It is performed in erythrocytes generally, but currently its use has been extended to other tissues like liver, lung, skin etc Micronucleus assay is also exploring to wide areas and future advancements make this assay more promising in analyzing the chromosome damage caused by the genotoxic chemicals Jena, et.al ( 2002), Indian journal of pharmacology, 34, 2 86-99

5. Micronuclei formation http:// meddev.uio.no/elaring/lcms/ernaeringslaere/nutr-cancer-biology/nutrition-cancer

6. Mechanisms through which micronuclei can form: The mitotic loss of acentric chromosome fragments (forming structural aberrations ) Mechanical consequences of chromosomal breakage and exchange, such as from lagging chromosomes, an inactive centromere or tangled chromosomes (forming structural aberrations ) Mitotic loss of whole chromosomes (forming numerical aberrations ) Apoptosis Schmid , (1975 ),Mutation Research/Environmental Mutagenesis , 31,1 9-15.

7. Micronuclei is induced by: A Clastogen is a mutagenic agent giving rise to or inducing disruption or breakages of chromosomes leading to sections of the chromosome being deleted, added, or rearranged An Aneugens is a substance which makes daughter cells to have abnormal no of chromosomes

8. Micronuclei http://meddev.uio.no/elaring/lcms/ernaeringslaere/nutr-cancer-biology/nutrition-cancer

9. Micronuclei Characteristics : Micronuclei are morphologically identical to the main nuclei, but are smaller than it It is not linked or connected to the main nuclei It may touch but do not overlap the main nuclei It is non-retractile and they can therefore be readily distinguished from staining particles Fenech ,( 2000 ), Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 455, 1 81-95

10. Types of micronuclei: Micronuclei are of different types based on 3 criteria , Based on the presence of kinetochore Based on the presence of centromere, they are of two types- Centromeric micronucleus Acentromeric micronucleus

11. Based on the number of nuclei present in the cell (i.e., cell divisions that occur), they are of following types, Mononucleate Binucleate Trinucleate Quadrinucleate Multinucleate Fenech ,( 1997 ),Mutation Research/ GeneticToxicology and Environmental Mutagenesis, 392, 1 11-18.

12. Models of micronucleus assay : In-vitro : OECD guideline No . 487 In-vivo : OECD guideline No . 474

13. Principle : In-vitro Cell cultures are exposed to the test substances both with and without metabolic activation. After exposure to a test substance, and addition of cytochalasin B for blocking cytokinesis cell cultures are grown for a sufficient period to allow chromosomal damage to lead to the formation of micronuclei in bi- or multinucleated interphase cells. Harvested and stained interphase cells are then analyzed microscopically for the presence of micronuclei. Micronuclei are scored in those cells that complete nuclear division following exposure to the test item.

14. General considerations : Most commonly used cell lines are CHL/IU , CHO, SHE and V79 Mouse lymphoma L5178Y cells are also used though they have interactions with the cytochalasinB C ell types with low and stable frequency of micronuclei are mostly used because frequency of micronuclei formation may influence the studies Kirsch- Volders, et.al (2003 ),Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 540, 2 153-163 .

15. Media: C ells are cultured in Phytohaemaglutinin (PHA) medium at 37°C Cultures should not be contaminated with mycoplasama Ames , et.al (1975), Mutation Research/Environmental Mutagenesis and Related Subjects, 31, 6 347-363 .

16. Metabolic activation: Cells should be treated with the test substance both in the presence and absence of an appropriate metabolic activation system 1-10% v/v conc. of post mitochondrial fraction (S9) is the most commonly used metabolic activating system prepared from liver of rodents Initially it is treated with Aroclor1254 or in combination with phenobarbitone and napthoflavone Elliott, et.al ( 1992), Mutagenesis, 7, 3 175-177

17. Use of cytochalasinB: CytochalasinB helps in formation of binucleated cells by inhibiting the formation of micro filament and cytokinesis It is used in the concentration of 3-6 µg/ml to get 50% binucleated cells; along with the test substance and it should be given at least 6hours before the first mitosis Evaluation of micronuclei formation can be known by reduction of cell proliferation.

18. C ytokinesis-block proliferation index: CBPI indicates the number of cell cycles per cell during the period of exposure to cytochalasinB . Cytotoxicity = 100-100 {( CBPI T - 1) / (CBPI C -1)} where; T = test ; C = control No . mononucleate cells + 2 x No. binucleate cells + 3 x No. multinucleate cells CBPI = ---------------------------------------------------------------------------- Total no. of cells CBPI = 1; indicates 100% cytotoxicity

19. C ontrols: Positive controls : used to identify clastogens , aneugens and effectiveness of exogenous metabolic activation system Examples: MitomycinC ; Cytosine arabinoside Negative controls : consists of solvent alone doesn’t requires any metabolic activators Oecd guideline for the testing of chemicals; guideline 487

20. Procedure : I n-vitro Cell lines + appr.culture media acc. to test Incubate at 37 ° C cells are taken from the stock to prepare suspension Harvested Monolayer of cells Stock Step:1

21. Blood sample + Lithium Heparin Separation of lymphocytes 44-48hrs Step:2 Phytohaemaglutinin – induces cell division This culture is added to cell lines along with test chemical

22. Without S9 fraction After 3-6hrs With S9 fraction Aroclor 1254- induces enzymes Cells should be exposed to test 3-6hrs Metabolic activation Remove the S9 and treatment medium by washing Freshmedia ; C ytochalasinB Harvest for 1.5- 2 cell cycle length Cells should be exposed to test 3-6hrs Remove the treatment medium by washing F resh media; C ytochalasinB a nd Harvest for 1.5- 2 cell cycle length Step: 3

23. Principle : In-Vivo Rodents are treated with the test agent by appropriate route, bone marrow extracted at appropriate times after treatment, smear slides are prepared either with whole bone marrow or cellulose column-fractionated cell suspension, stained, coded, and analyzed for the toxicity (PCE to NCE ratio) and micro nucleated cell frequency. Makoto Hayashi et.al (2000) Invivo Rodent Erythrocyte MicronucleusAssayII .,Environmental and Molecular Mutagenesis 35:234–252

24. In-vivo micronucleus: They are done using following Peripheral blood Bone marrow Krishna and Hayashi, (2000 ),Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 455, 1 155-166

25. Procedure: In-Vivo 5 animals should be treated with the test chemical. Dosing done at 3levels (low, medium , high doses) Aft er 24-48hrs animals are sacrificed by carbon dioxide euthanasia cut the ends of bone; flush the bonemarrow with isotonic solution Centrifuge the marrow suspension Preparation of smear of sample collected

26. Staining: Slides are stained using Giemsa or F luorescent DNA specific dyes Example of fluorescent stains Acridine orange Hoechst + pyronin Y May - G runwald May - G runwald + Giemsa Ren , Atyah , Chen and Zhou, (2017 ), Journal of translational medicine, 15, 1 110

27. Analysis: Flow cytometry Laser scanning cytometry Image analysis

28. Applications: Distinguish aneugens from clastogens Perform long-term toxicological studies Examining the radioactive properties of H 2 receptor antagonists Evaluation of radio sensitivity of human glioma cells Warheit and Donner, Nanotoxicology , 4, 4 409-413.

29. Conclusion: Micronucleus assay has wide spread applications in the area of toxicology for assessing risk assessment of drugs , pollutants, NCE etc Micronucleus assay results using tissues other than hematopoietic tissue have become increasing in toxicological studies to evaluate the multi organ toxicity caused by different genotoxic agents