The oxidation of ethanol by a reconstituted system containing NADPH-cytochrome P-450 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 2- to 3-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 P-450. 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 cytochrome P-450-dependent pathway of ethanol oxidation. Cumene and t-butyl hydroperoxide supported the oxidation of aminopyrine and ethanol or 1-butanol when added directly to cytochrome P-450 in the absence of NADPH and the reductase. The organic hydroperoxide-supported oxidation of ethanol was inhibited by metyrapone, but not by dimethyl sulfoxide. Typical hydroxyl radical scavengers were not oxidized by the hydroperoxide-supported system. These results suggest that two independent pathways are operative in supporting NADPH-dependent microsomal oxidation of ethanol. One pathway involves hydroxyl radicals which can be generated by the reductase, whereas the other pathway required the combined presence of both the reductase and cytochrome P-450 and appears to be independent of oxygen radicals.
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - 1983|