Single-cell gel (comet) assay detects primary DNA damage in nonneoplastic urothelial cells of smokers and ex-smokers

A protocol for DNA damage assessment by the single-cell gel (SCG)/comet ass ay in human urinary bladder washing cells was established. Modifications of the standard alkaline protocol included an increase to 2% of sodium sarcos inate in the lysis solution, a reduction in the glass-slide area for comet analysis, and a cutoff value for comet head diameter of at least 30 mum, to exclude contaminating leukocytes. Distinguishing cell populations is cruci al, because significant differential migration was demonstrated for transit ional and nontransitional cells, phenomena that may confound the results. W hen applying the modified protocol to urinary bladder cells from smokers wi thout urinary bladder neoplasia, it was possible to detect a significant (P = 0.03) increase in DNA damage as depicted by the tail moment (6.39 +/- 3. 23; mean 95% confidence interval; n = 18) when compared with nonsmokers (1. 94 +/- 1.41; n = 12). No significant differences were observed between ex-s mokers and current smokers regarding comet parameters. Inflammation was not a confounding factor, but DNA migration increased significantly with age i n nonsmokers (r = 0.68; P = 0.014). Thus, age matching should be a concern when transitional cells are analyzed in the SCG assay. As it is well known, DNA damage may trigger genomic instability, a crucial step in carcinogenes is. Therefore, the present data directly support the classification of indi viduals with smoking history as patients at high risk for urinary bladder c ancer.