NADPH-dependent production of oxy radicals by purified components of the rat liver mixed function oxidase system. II. Role in microsomal oxidation of ethanol.

The oxidation of ethanol by a reconstituted system containing NADPH-cytochrome P-450 reductase and cytochrome P-450 from phenobarbital-treated rats was characterized especially with regard to the role of oxygen radicals in the mechanism of ethanol oxidation. Results described in the preceding manuscript demonstrated that a strong oxidizing species with properties similar to that of the hydroxyl radical was generated by a NADPH-cytochrome P-450 reductase system. Ethanol was oxidized to acetaldehyde in the presence of the reductase-NADPH system in the absence of cytochrome P-450, similar to results found with typical hydroxyl radical scavenging agents. However, in contrast to results with the scavengers, the addition of cytochrome P-450 to the assay mixture resulted in a 2to %fold increase in the rate of ethanol oxidation over that of the reductase-dependent rate. The oxidation of ethanol was dependent on both the amount of reductase and the amount of cytochrome P-450. Ethanol oxidation by the reductase-dependent pathway was inhibited 66 and 46% by dimethyl sulfoxide and benzoate, respectively. When cytochrome P-450 was present, the ability of these competing scavengers to inhibit ethanol oxidation was attenuated such that the inhibition was accountable by the reductase-dependent component. Inhibition of ethanol oxidation by superoxide dismutase in the presence and absence of cytochrome P-450 also appeared to reflect an effect upon the reductase-dependent pathway, with no effect by superoxide dismutase on the increased rate of ethanol oxidation produced by the addition of cytochrome P-450. The reductase-dependent oxidation of ethanol was inhibited by desferrioxamine and stimulated by either EDTA or iron, suggesting that ethanol was oxidized by interaction with hydroxyl radicals generated via an iron-catalyzed Haber-Weiss reaction. Desferrioxamine did not inhibit the increase in ethanol oxidation produced by the addition of cytochrome P450. The insensitivity of the cytochrome P-450-stimulated rate of ethanol oxidation to competing scavengers, superoxide dismutase, and desferrioxamine suggests little or no role for hydroxyl radicals in the cyto-