Optimized noninvasive procedures to measure DNA damage in comet assay.
The comet assay is a well-established, simple and sensitive method to measure DNA damage in single cell and is commonly used in human trials to investigate the effects of pollution, occupational hazards and potential genoprotective agents. Peripheral blood lymphocytes are most commonly used in human biomonitoring studies, but lymphocytes collected from the mouth offer a potentially attractive, noninvasive alternative. The aim of the current study was to develop a buccal cell lymphocyte comet assay procedure. Cells were collected from mouthwash of three healthy volunteers and tested individually. The comet assay was performed under different pH and times of alkaline treatment, electrophoresis run times and hydrogen peroxide concentrations. Optimal conditions for buccal lymphocytes in comet assay were found to be pH >13 for unwinding and electrophoresis buffers, 10-min alkaline unwinding treatment and 20-min electrophoresis run time. We successfully utilized our optimized assay conditions to demonstrate the genoprotective activity of quercetin. This newly established procedure offers an alternative noninvasive sampling method for the investigation of DNA protection and/or damaging effect.
Department of Applied Science, Hong Kong Institute of Vocational Education (Shatin), Sha Tin, New Territories, Hong Kong. savio.yim.tong.szeto@inet.polyu.edu.hk
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D000328:Adult; D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D020552:Comet Assay / Q000379:methods*; D004249:DNA Damage; D004586:Electrophoresis; D005260:Female; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008214:Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009061:Mouth Mucosa / Q000166:cytology*; D016877:Oxidants / Q000494:pharmacology; D011794:Quercetin / Q000494:pharmacology
D000975:Antioxidants; D016877:Oxidants; D011794:Quercetin; D006861:Hydrogen Peroxide