Repair kinetics of specific types of nitroso-induced DNA damage using the comet assay in human cells

The comet assay is sensitive and can detect DNA damage frequencies less than 1 in 10(7) bases. We have previously shown that several types of DNA damage associated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific pro-mutagen, can be investigated with some specificity using this technique. Little is known about their repair. We verified the ability of the comet assay to quantify the repair kinetics of specific types of damage in normal fibroblasts, e.g., dimethylsulfate-induced 7-methylguanines (7-mG) and UVB-induced cyclobutane pyrimidine dimers. The time course, formation and repair, of DNA damage after acute doses of NNK reactive metabolites, were then compared in normal human cells (fibroblasts and lymphocytes) and in cells proficient for activating NNK (U937 and NCI-H23). NNK can be activated in cells into reactive metabolites that can either methylate or pyridyloxobutylate DNA. The 7-mG generated by methylation gave post-treatment patterns that were sufficiently different between cell types to conclude that repair of 7-mG in U937 cells was fast, repair in lymphocytes was slow, and repair in NCI-H23 cells and fibroblasts displayed intermediate rates. Pyridyloxobutylation generated formamidopyrimidine (fapy) glycosylase (fpg)-sensitive sites that could be the fapy form of 7-pyridyloxobutylguanines produced in cells. For this type of adducts, the post-treatment patterns of adduct frequency as a function of time depended even more clearly on the cell type: fibroblasts and NCI-H23 cells showed an initial rapid increase in fpg-sensitive damage frequency that did not occur in lymphocytes and U937 cells. This increase seemed associated with p53 proficiency in fibroblasts. Our results show that repair kinetics can be investigated with the comet assay and that differences between cell types can be observed with that technique. But it seems that pro-mutagen activation and/or the way a type of adducts is formed can affect the quantification of the repair.