Recommendations to enhance reproducibility and reliability in Comet assay Rashini Yasara Baragama-arachchi 1,2 Dr. Jagath Weerasena 1 Dr. Shiroma Handunnetti 1 Dr. Radhika Samarasekara 2 1 Institute of Biochemistry, Molecular Biology and Biotechnology, University of Colombo, Sri Lanka 2 Industrial Technology Institute, Sri Lanka

Introduction Comet assay is a technique predominantly use in the field of toxicology to assess the DNA damages in single cell suspensions (Nandhakumar et al, 2011) Also known as Single cell gel electrophoresis assay (SCGE assay) The concept of Single cell gel electrophoresis assay was first introduced by Ostling and Johanson in 1984 Later it was developed by N.P. Singh in 1988

Advantages It can be applied both in vitro and in vivo to virtually any cell type or cell line from prokaryotic and eukaryotic organisms Require only small numbers of cells per sample (<10,000) Sensitivity for detecting low levels of DNA damage Does not require cumbersome techniques like radiolabelling Low cost Flexible Rapid

Applications 1. Genetic toxicology For screening and regulatory testing of industrial chemicals, pharmaceuticals, biocides, cosmetics and various herbal extracts use for different purposes. 2. Human biomonitoring Occupational exposure to hazardous chemicals, pollutants and radiation 3. Ecogenotoxicology Monitoring contamination of the environment by genotoxic agents

Applications 4. Mechanistic studies DNA damage & repair 5.Nutrition Harmful and beneficial effects of diet and dietary components 6. Clinical Diagnosis of disease and monitoring effects of treatment 7. Molecular epidemiology Assessing inter-individual differences in susceptibility to DNA damage and capacity to repair

Principle Alkaline unwindingElectrophoresis

Justification Method of choice for evaluation of potential genotoxicity of various chemicals and prospective therapeutics Accepted as a part of battery of assays used for regulatory submissions in genetic toxicology by regulatory authorities (Collins et al, 2008) Yet the major drawback of this technique is the unreliability in making reproducible data, due to miscellaneous conditions used in different laboratories and due to lack of understanding of the critical steps (Azqueta et al, 2011)

Objectives of the study To optimize the Comet assay to enhance reproducibility and reliability

Preparation of cells for comet assay Cell culture Human lymphocytes were extracted from whole blood obtained from healthy volunteers Ethical approval was obtained Initial viable cell count was determined by performing Trypan blue dye exclusion assay cells were seeded in 6-well plates at 2x 10 5 cells/well Lymphocytes were incubated for 1 hour at 37ºC with hydrogen peroxide (H 2 O 2, 200 µM) as positive control and 1xPBS as negative control Cell viability after the treatment was evaluated by performing Trypan blue dye exclusion assay

Preparation of base slides Store at RT Absolute methanol Hot Normal melting agarose (NMA)

Preparation of micro-gel slides a) Preparation of 0.5% Low melting agarose (LMPA) The required amount of LMPA was made freshly during the day of the assay without microwaving Instead the tube containing the LMPA and PBS was placed in a boiling water bath until LMPA dissolved and placed in a 37 °C water bath for 20 min before use Critical step

Preparation of micro-gel slides LMPA at 37 °C Cell suspension (80 µl ) 100 µl 2 nd agarose coat 90 µl Refrigerated for 30 min 3 rd agarose coat Refrigerated for 30 min 90 µl Remove coverslip b) Embedding cells in LMPA and coating of base slides

Cell lysis and Alkaline unwinding of DNA Cell lysis Cells were lysed for 2 h at 4 ˚C Slides were gently washed with chilled distilled water to remove traces of detergent Poland and McLeish, 2008 Alkaline unwinding of DNA Slides were placed on the middle of the platform in an electrophoresis tank Slides were covered with chilled electrophoresis buffer Incubated for 30 min to allow for unwinding of the DNA and to expose of ALS

Electrophoresis Electrophoresed at 17 V and 164 mA for 45 min at 4 °C A software developed by Gunnar Brunborg from National Institute of Public Health, N-0462 Oslo, Norway was used to calculate the accurate voltage for electrophoresis (Collins et al; 2008) Spreadsheet to calculate voltages and currents in an electrophoresis tank.docxSpreadsheet to calculate voltages and currents in an electrophoresis tank.docx Critical step

Neutralization and visualization Neutralization and fixing of slides Slides were dipped in cold neutralization buffer, air dry and fixed with absolute methanol Staining & visualization of slides Slides were stained with 45 µl of Ethidium bromide [EtBr] (20 µg/ ml), left for 5 min and then dipped in chilled distilled water to remove excess stain Visualized under 40x objective of the fluorescent microscope

Comet scoring and statistical analysis 100 cells per slide were assessed. “Casp 1.2.3b.1” image analysis software was used to assess the quantitative and qualitative extent of DNA damage in the cell Results were analyzed using SPSS statistical software (version17.0) The results were considered to be significantly different at P < 0.05

Measured parameters ParameterDefinition Percentage of DNA in the tail Fraction of DNA in the tail as compared to the whole image (Albertini et al, 2000) Tail moment (TM) Tail length X Fraction of DNA in the tail (Lovell et al, 2008)

Cell viability after treatment Cell viability was >80 % after treatments

Optimized conditions Optimization of conditions for comet assay to achieve reproducible and reliable data 1.Optimization of conditions for preparation of base slides 2.LMPA preparation method 3.Solidification times of 2 nd & 3 rd agarose layers 4.Lysis duration 5.Electrophoresis conditions – Voltage – Duration

Optimization of conditions for preparation of base slides Repeated heating of agarose No significant effect on the overall process Agarose concentration Alteration of actual concentration of agarose (1%) Alteration of actual concentration of agarose (1%)

LMPA preparation method Method 01 No migration of DNA after electrophoresis No migration of DNA after electrophoresis Prepared in bulk and re-melted by microwaving Method 02 Prepared freshly on the day of assay using a boiling water bath Proper migration of DNA after electrophoresis

Solidification times of 2nd & 3rd agarose layers 30 min 20 min 10 min Solidification times Detachment of agarose layers when removing the cover slips Adequately solidified agarose layers Adequately solidified agarose layers

Lysis duration 2 hoursOvernight 1 hour Insuficiently lysed Fully lysed cells Cells were lysed and DNA dispersed

Electrophoresis conditions Alkaline unwinding Optimal unwinding time was found to be 30 min Electrophoresis Parameter Optimized condition Voltage24V, 17 V Duration30, 45, 60 min

Comet formation Positive Control (C+) 200 µM H 2 O 2 Negative Control (C-) PBS

Genotoxic potential of H 2 O 2 * p < 0.05 when compared to Negative control * * Negative control – Vehicle (PBS) Positive control – 200 µM H 2 O 2 Mean values of TM, OTM and Tail DNA percentage of Comets (n=100); Error bars indicate: Mean ± SEM Negative control Positive control

Conclusions Concentration of NMA does not affect final outcome as it only provide a better anchorage for subsequent agarose layers The most critical parameters are 1.Concentration of LMPA, as DNA migrate through LMPA 2.Electrophoresis voltage. It MUST be 1V/cm, not 24 V Comet assay was a very sensitive technique Sensitivity means here is not that its ability to detect low level of DNA damages, but the extreme care that has to be taken when performing each and every step to achieve better reproducible results

References 1.Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F et al. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. (2000) Mutation Research :463 ;111–172 2.Azqueta A, Gutzkow KB, Brunborg G and Collins AR. Towards a more reliable comet assay: Optimising agarose concentration, unwinding time and electrophoresis conditions (2011) Mutation Research: 724; Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. (2004b) Molecular Biotechnology: 26(3); Collins AR, Oscozi AA,Brunborg G, Gaiva I, Giovannelli L, Kruszewski M et al. REVIEW:The comet assay: topical issues. (2008) Mutagenesis: 23 (3 ) ;143–151 5.Hartmann A, Agurell E, Beevers C, Brendler-Schwaab S, Burlinson B, Clay P et al. Recommendations for conducting the in vivo alkaline Comet assay. (2003) Mutagenesis:18(1); 45–51 6.Morley N, Rapp A, Dittmar H, Salter L, Gould D, Greulich KO et al. UVA-induced apoptosis studied by the new apo/necro-Comet-assay which distinguishes viable, apoptotic and necrotic cells. (2006) Mutagenesis: 21( 2 ); 105–114

References 7.Nandhakumar S, Parasuraman S, Shanmugam M, Rao KR, Chand P and BhatBV. Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay). (2011) Journal of Pharmacology and Pharmacotherapeutics: 2(2); 107–111 8.Singh N, Lai H. 60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells. (1998) Mutation Research: 400(1-2); Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H et al. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. (2000) Environmental and Molecular Mutagenesis: 35;

Acknowledgement National Science Foundation of Sri Lanka