| Abstract
| - Toxicity induced by nitric oxide (NO•) has been extensively investigated in many in vitro and in vivoexperimental models. Recently, our laboratories found that both concentration and cumulative total doseare critical determinants of cell death caused by NO•. Here, we report results of studies designed todefine total dose thresholds and threshold effects for several NO•-induced toxicity and cellular responsesand to determine impacts of p53 on them. We exposed human lymphoblastoid TK6 cells harboring wild-type p53 and isogenic p53-null NH32 cells to NO• delivered by a membrane delivery system. Cells wereexposed at a steady state concentration of 0.6 μM for varying lengths of time to deliver increasingcumulative doses (expressed in units of μM min), and several end points of cytotoxicity and mutagenesiswere quantified. Threshold doses for NO•-induced cytotoxicity were 150 μM min in TK6 cells and 300μM min in NH32 cells, respectively. Threshold doses for NO•-induced apoptosis were identical to thosefor cytotoxicity, but mitochondrial depolarization thresholds were lower than those for cytotoxicity andapoptosis in both cell types. To gain insight into underlying mechanisms, cells of both types were exposedto sublethal (33% of cytotoxicity threshold), cytotoxicity threshold, or toxic (twice the cytotoxicitythreshold) doses of NO•. In TK6 cells (p53), the sublethal threshold dose induced DNA double-strandbreaks, but nucleobase deamination products (xanthine, hypoxanthine, and uracil) in DNA were increasedonly modestly (<50%) by toxic doses. Increased mutant fraction at the thymidine kinase gene (TK1)locus was observed only at the toxic dose of NO•. Treatment of NH32 cells with NO• at the threshold ortoxic dose elevated mutagenesis of the TK1 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 higherin p53-null NH32 cells than in wild-type TK6 cells. NO• treatment induced p53-independent cell cyclearrest predominately at the S phase. Akt signaling pathway and antioxidant proteins were involved in themodulation of toxic responses of NO•. These findings indicate that exposure to doses of NO• at or abovethe cytotoxicity threshold dose induces DNA double-strand breaks, mutagenesis, and protective cellularresponses to NO• damage. Furthermore, recombinational repair of DNA may contribute to resistance toNO• toxicity and potentially increase the risk of mutagenesis. The p53 plays a central role in these responsesin human lymphoblastoid cells.
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