Threshold effects of nitric oxide-induced toxicity and cellular responses in wild-type and p53-null human lymphoblastoid cells

Toxicity induced by nitric oxide (NO center dot) has been extensively investigated in many in vitro and in vivo experimental models. Recently, our laboratories found that both concentration and cumulative total dose are critical determinants of cell death caused by NO center dot. Here, we report results of studies designed to define total dose thresholds and threshold effects for several NO center dot-induced toxicity and cellular responses and to determine impacts of p53 on them. We exposed human lymphoblastoid TK6 cells harboring wildtype p53 and isogenic p53-null NH32 cells to NO center dot delivered by a membrane delivery system. Cells were exposed at a steady state concentration of 0.6 mu M for varying lengths of time to deliver increasing cumulative doses (expressed in units of mu M min), and several end points of cytotoxicity and mutagenesis were quantified. Threshold doses for NO center dot-induced cytotoxicity were 150 mu M min in TK6 cells and 300 mu M min in NH32 cells, respectively. Threshold doses for NO center dot-induced apoptosis were identical to those for cytotoxicity, but mitochondrial depolarization thresholds were lower than those for cytotoxicity and apoptosis in both cell types. To gain insight into underlying mechanisms, cells of both types were exposed to sublethal (33% of cytotoxicity threshold), cytotoxicity threshold, or toxic (twice the cytotoxicity threshold) doses of NO center dot. In TK6 cells (p53), the sublethal threshold dose induced DNA double-strand breaks, but nucleobase deamination products (xanthine, hypoxanthine, and uracil) in DNA were increased only modestly (< 50%) by toxic doses. Increased mutant fraction at the thymidine kinase gene (TKI) focus was observed only at the toxic dose of NO center dot. Treatment of NH32 cells with NO center dot at the threshold or toxic dose elevated mutagenesis of the TKI gene, but did not cause detectable levels of DNA double-strand breaks. At similar levels of cell viability, the frequency of DNA recombinational repair was higher in p53-null NH32 cells than in wild-type TK6 cells. NO center dot treatment induced p53-independent cell cycle arrest predominately at the S phase. Akt signaling pathway and antioxidant proteins were involved in the modulation of toxic responses of NO center dot. These findings indicate that exposure to doses of NO center dot at or above the cytotoxicity threshold dose induces DNA double-strand breaks, mutagenesis, and protective cellular responses to NO center dot damage. Furthermore, recombinational repair of DNA may contribute to resistance to NO center dot toxicity and potentially increase the risk of mutagenesis. The p53 plays a central role in these responses in human lymphoblastoid cells.