Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.