1. GENERAL OVERVIEW OF THE COMET ASSAY AND ITS APPLICATIONS Diana Anderson University of Bradford United Kingdom

4. COMET ASSAY IS ONE OF THE MOST IMPORTANT ASSAYS IN TOXICOLOGY TODAY Standard method for measuring DNA strand breaks in single somatic and germ cells in vitro and in vivo. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt. Electrophoresis at high pHs results in structures resembling Comets, observed by fluorescence microscopy. Comet tail is formed by DNA fragments moving towards the anode. ASSAY CAN BE USED FOR: - Genotoxicity testing of novel compounds and ecotoxins. - Human biomonitoring and molecular epidemiology. - Basic research into DNA damage and repair.

5. GENOTOXICITY TESTING OF NOVEL COMPOUNDS AND ECOTOXINS This can be illustrated by work done in human lymphocytes and sperm with oestrogens. In CHO cells with halogenated acetic acids. Also by work in rodents with CP, EMS, EGME, BLM with bone marrow and testicular cells

9. HUMAN BIOMONITORING AND MOLECULAR EPIDEMIOLOGY We have examined: NaNO 2 in Diabetic patients because Type 2 diabetics, in an Epidemiology study, were linked to increased nitrate levels in drinking water Mothers and babies for differential sensitivity to the alkylating agent EMS Individuals exposed to food mutagens MeIQx and PhIP Diabetics exposed to Hydrogen Peroxide

12. Mean tail-moment values (± SE) of lymphocytes from mothers and babies (cordblood) in the Comet assay, with 1–40 mM of EMS (and negative control) n 0 EMS 1mM EMS 10mM EMS 20 mM EMS 30 mM EMS 40 mM EMS Mothers 22 6.7 ± 0.89* 10.12 ±1.12 14.87 ± 1.35 19.40 ± 1.38 28.96 ±2.13 46.60± 5.40 Babies 22 4.90 ±0.90 8.76 ±1.07 12.72 ±1.50 15.99 ± 1.38 24.90 ± 2.58 39.46 ± 3.92

13. DNA damage in lymphocytes from subjects below and above 45 years of age treated with MeIQx

16. BASIC RESEARCH INTO DNA DAMAGE AND REPAIR We investigated PBL from non-Hodgkins lymphoma patients. Those resistant to their standard chemotherapy regimen, CHOP, were found to be overexpressing mutant p53 protein (p53 + patient) and did not repair. Those who responded did not overexpress mutant p53 protein (p53 - patient) and did repair. Data could be used to select resistant patients for another treatment regimen involving verapamil (chemosensitizer and p-glycoprotein antagonist) and doxorubicin and cells from all patients showed increased sensitivity to treatment.

17. Immunocytochemical staining for p53 protein: positive population show intense brown nuclear staining. (A) cytospin of Raji cells used as p53 positive control. (B) cytospin of normal peripheral lymphocytes used as p53 negative control (C) cytospin of transformed AML in which more than 75% of cells were p53 positive. (D) cytospin of CLL case showing both p53 positive and p53 negative cells. A B CD

20. Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of healthy controls, lung cancer, COPD and asthma patient groups in the Comet assay after treatment with different TiO 2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H 2 O 2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

22. Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of male and female healthy controls, and male and female patient groups in the Comet assay after treatment with different TiO 2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H 2 O 2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

23. Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of healthy controls, aged 65 years in the Comet assay after treatment with different TiO 2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H 2 O 2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

24. Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of non smoker healthy controls and non smoker and smoker patient groups in the Comet assay after treatment with different TiO 2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H 2 O 2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

25. Histogram showing the means of Olive tail moment and % tail DNA in lymphocytes of Caucasian controls and patients and Asian controls andpatient groups in the Comet assay after treatment with different TiO 2 concentrations (10, 30 and 50 µg/ml), as well as the negative control of untreated lymphocytes (Nc) and the positive control of 80 µM (2.72 µg/ml) H 2 O 2 (Pc) for 30 minutes. Bars indicate standard errors. Not significant: ns, *p < 0.05, ** p < 0.01 and ***p < 0.001

28. ANALYSIS of RESULTS (cont) Also, to determine if differences could have a cancer diagnostic value via analysis of Receiver Operating Characteristic (ROC) curves in 208 people.

30. ANALYSIS of RESULTS (cont.) Sensitivity = TP X 100 Specificity = TN X 100 TP + FN FP + TN Where: TP = true positives, TN= true negatives, FN = false negatives, FP = false positives. The sensitivity and specificity of the assay was modelled using different threshold levels enabling optimum identification of only cancer patients and only healthy individuals using the above formula. Use as stand alone assay or in conjunction with other assays.

32. Regulatory Concerns The Comet assay is recommended for use in Regulatory guidelines by the UK in 2000m by the Committee on Mutagenicity of Chemicals in Food, Consumer Products...Committee on Mutagenicity of Chemicals in Food, Consumer Products... Also in - Mutagenicity testing for Chemical Risk Assessment: update of the WHO/IPCS Harmonised Scheme (Eastmond et al., 2009, Mutagenesis 24 341-349) ~It is used as an Indicator Test

34. Individual Guidelines to be found in: Mutation Research 463 (2000) 111-172 Whole issue is available free of charge: http://mutationresearch.com/mutat/29/43/25/23/article.pdf

35. Hartmann et al, 2003, Mutagenesis,18, 45-51 Tice et al, 2000, Environ Mol Mutagen, 35, 206-221 Speit and Hartmann, 2005, Methods Mol Biol, 291, 85- 95 Burlinson et al, 2007, Mutat Res, 627, 31-35 Smith et al, 2008, Mutagenesis, 23, 233-240 Frotschi, 2015, Mutagenesis, 30 51-57 USEFUL REFERENCES to help meet regulatory requirements