SIN-1-induced DNA damage in isolated human peripheral blood lymphocytes as assessed by single cell gel electrophoresis (comet assay)

Abstract

Human lymphocytes were exposed to increasing concentrations of SIN-1, which generates superoxide and nitric oxide, and the formation of single-strand breaks (SSB) in individual cells was determined by the single-cell gel electrophoresis assay (comet assay). A dose- and time-dependent increase in SSB formation was observed rapidly after the addition of SIN-1 (0.1–15 mM). Exposure of the cells to SIN-1 (5 mM) in the presence of excess of superoxide dismutase (0.375 mM) increased the formation of SSB significantly, whereas 1000 U/ml catalase significantly decreased the quantity of SSB. The simultaneous presence of both superoxide dismutase and catalase before the addition of SIN-1 brought the level of SSB to that of the untreated cells. Moreover, pretreatment of the cells with the intracellular Ca 2+-chelator BAPTA/AM inhibited SIN-1-induced DNA damage, indicating the involvement of intracellular Ca 2+ changes in this process. On the other hand, pretreatment of the same cells with ascorbate or dehydroascorbate did not offer any significant protection in this system. The data suggest that H 2O 2-induced changes in Ca 2+ homeostasis are the predominant pathway for the induction of SSB in human lymphocytes exposed to oxidants.