NADPH-dependent production of oxy radicals by purified components of the rat liver mixed function oxidase system. I. Oxidation of hydroxyl radical scavenging agents.

Isolated microsomes catalyze an NADPH-dependent oxidation of typical hydroxyl radical scavenging agents. To determine which microsomal components participate in the oxidation of the scavengers, experiments were carried out with purified NADPH-cytochrome P-450 reductase and cytochrome P-450 isolated from phenobarbital-treated rats. The production of ethylene from 4-ketothiomethylbutyrate or of formaldehyde from either dimethyl sulfoxide or tertiary butyl alcohol was measured in the presence of NADPH plus reductase or NADPH plus reductase plus cytochrome P-450. The reductase-dependent system itself catalyzed the oxidation of the three scavengers. Addition of cytochrome P-450 had no effect on the rates of oxidation of the scavengers. Relative rates of oxidation of the scavengers reflected the amount of the reductase in the assay system. Varying the amount of cytochrome P-450 at a fixed concentration of the reductase did not result in rates different from that observed in the absence of cytochrome P-450. Dilauroyl phosphatidylcholine appeared to have a stimulatory role in these oxidations, whereas cytochrome P-450 reductase and NADPH were obligatory components. The reductase-dependent oxidation of the scavengers was inhibited by superoxide dismutase, catalase, and competing hydroxyl radical scavenging agents suggesting that superoxide, hydrogen peroxide, and an oxygen radical with the oxidizing power of the hydroxyl radical played a role in these oxidations. Further, these oxidations were inhibited by the iron-chelator desferrioxamine but stimulated by either EDTA or by iron. These results suggest that an iron-catalyzed Haber-Weiss reaction might be involved in the mechanism by which purified cytochrome P-450 reductase mediates the oxidation of typical hydroxyl radical scavengers. The results demonstrated that NADPH-cytochrome P-450 reductase may represent an important locus of oxygen activation leading to the production of a highly oxidizing species characteristic of the hydroxyl radical.