Nitric Oxide Inhibits Iron–Induced Lipid Peroxidation in HL-60 Cells

Abstract

Nitric oxide (•NO) can protect cells against the detrimental effects of reactive oxygen species. Using low-density lipoprotein as well as model systems, it has been demonstrated that •NO can serve as a chain-breaking antioxidant to blunt lipid peroxidation. To test the hypothesis that •NO can serve as a chain-breaking antioxidant in cell membranes, we examined the effect of •NO on iron-induced lipid peroxidation in human leukemia cells. We exposed HL-60 cells to an oxidative stress (20 μM Fe2+) and monitored the consumption of oxygen as a measure of lipid peroxidation. Oxygen consumption was arrested by the addition of •NO as a saturated aqueous solution. The duration of inhibition of oxygen consumption by •NO was concentration-dependent in the 0.4–1.8 μM range. The inhibition ended upon depletion of •NO. The addition of •NO prior to initiation of peroxidation delayed the onset of peroxidation; the nearer in time it was before Fe2+ addition, the longer the inhibition. Depletion of cellular glutathione levels by d,l-buthionine-S,R-sulfoximine prior to Fe2+ addition resulted in a more rapid initial rate of oxygen depletion and a shorter time for the •NO-induced inhibition of oxygen consumption. Complementary studies of this iron-induced lipid peroxidation, using thiobarbituric acid reactive substances as a marker, also demonstrated the protective effects of •NO. This protection of cells against lipid peroxidation also manifested itself as a reduction in trypan blue uptake, an observation demonstrating the protective effects of •NO on membrane integrity. We conclude that •NO protects HL-60 human leukemia cells from lipid peroxidation and that this protection ameliorates the toxicity of the oxidation processes initiated by Fe2+ and dioxygen.