Mechanisms of toxicity induced by nitric oxide in human lymphoblastoid cells expressing wild-type and null p53

767 Nitric Oxide (NO) induces cell death and mutagenesis in many in vitro and in vivo experimental models. Biochemical mechanisms through which NO induces cytotoxicity and genotoxicity are unclear. In this study, human lymphoblastoid TK6 cells harboring wild-type p53 and isogenic p53 null NH32 cells were exposed for 1.5 to 16 hours to NO, delivered into the culture medium by diffusion through permeable tubing, at a steady state concentration of 0.6 μM, similar to levels estimated to occur in vivo in inflamed tissues. Thresholds for NO-induced cell death were shown to be 150 μM min in TK6 cells and 300 μM min in NH32 cells. In further experiments, three NO doses for each cell type, namely, 50% of threshold (sublethal), threshold, and 2X threshold (toxic) were used to investigate mechanisms of cell death. In TK6 cells, threshold (150 μM min) or higher (300 μM min), but not sublethal (50 μM min) doses, induced extensive DNA double-strand breaks. In contrast, no DNA double-strand breaks were detected in NH32 cells, even after treatment with a toxic dose (600 μM min). Threshold and toxic doses of NO induced mutagenesis of the TK1 gene in NH32 cells, whereas only the toxic dose was effective in TK6 cells. Increases in TK1 mutant fraction in NH32 cells were substantially greater than in TK6 cells. Mitochondrial membrane potential loss and apoptosis were lower in NH32 cells than in TK6 cells. NO treatment caused depletion of cellular glutathione (GSH) in both cell lines in a dose- and time-dependent manner. In both cell lines, sublethal doses caused GSH depletion but no loss in viability. By comparison, treatment of TK6 cells with 125 μM BSO (L-buthionine SR-sulfoximine) also caused virtually complete GSH depletion, but no cell death up to 48 hours after treatment. Intracellular GSH level partially recovered during 24 to 48 hours following depletion. Prior GSH depletion by BSO did not increase sensitivity to NO-induced cell killing. Toxic NO doses caused reduced Cu/Zn-superoxide dismutase (SOD1) protein expression, measured by Western blot, in TK6 cells, but increased expression in NH32 cells. Under the same conditions, levels of catalase protein were upregulated only in TK6 cells, and GSH peroxidase levels were unchanged in both cell types. Survival signaling proteins NF-κB p50 and p65, and Akt were suppressed only in TK6 cells. Taken together, these data indicate that mechanisms of toxicity induced by NO in cells expressing wild-type p53 differ from those in isogenic p53 null cells, and that p53 modulates cell death through regulation of oxidative stress and survival signaling proteins. SOD1 and catalase, but not GSH peroxidase, may be p53 target proteins that play important roles in NO-induced stress responses. Although NO effectively induces GSH depletion, this alteration does not appear to be a critical initiating factor for cell death in these human lymphoblastoid cell lines.