Nitric oxide increases toxicity of hydrogen peroxide against rat liver endothelial cells and hepatocytes by inhibition of hydrogen peroxide degradation

Abstract

Nitric oxide (NO) and hydrogen peroxide (H2O2) show cooperativity in their cytotoxic action. The present study was performed to decipher the mechanisms underlying this phenomenon. In cultured liver endothelial cells and in cultured, glutathione-depleted hepatocytes, the combined exposure to NO (released by spermine NONOate, 1 mM) and H2O2 (released by glucose oxidase) induced cell injury that was far higher than the injury elicited by NO or H2O2 alone. In both cell types, the addition of the NO donor increased H2O2 steady-state levels, although with different kinetics: in hepatocytes, the increase in H2O2 levels was already evident at early time points while in liver endothelial cells it became evident after ≥2 h of incubation. NO exposure inhibited H2O2 degradation, assessed after addition of 50 μM, 200 μM, or 4 mM authentic H2O2, significantly in both cell types. However, again, early and delayed inhibition was observed. The late inhibition of H2O2 degradation in endothelial cells was paralleled by a decrease in glutathione peroxidase activity. Glutathione peroxidase inactivation was prevented by hypoxia or by ascorbate, suggesting inactivation by reactive nitrogen oxide species (NOx). Early inhibition of H2O2 degradation by NO, in contrast, could be mimicked by the catalase inhibitor azide. Together, these results suggest that the cooperative effect of NO and H2O2 is due to inhibition of H2O2 degradation by NO, namely to inhibition of catalase by NO itself (predominant in hepatocytes) and/or to inhibition of glutathione peroxidase by NOx (prevailing in endothelial cells).