Role of hydroxyl radicals in the iron-ethylenediaminetetraacetic acid mediated stimulation of microsomal oxidation of ethanol

The microsomal oxidation of ethanol or 1-butanol was increased by ferrous ammonium sulfate-ethylenediaminetetraacetic acid (1:2) (Fe-EDTA) (3.4-50 microM). The increase was blocked by hydroxyl radical scavenging agents such as dimethyl sulfoxide or mannitol. The activities of aminopyrine demethylase or aniline hydroxylase were not affected by Fe-EDTA. The accumulation of H2O2 was decreased in the presence of Fe-EDTA, consistent with an increased utilization of H2O2. Other investigators have shown that Fe-EDTA increases the formation of hydroxyl radicals in systems where superoxide radicals are generated. The stimulation by Fe-EDTA appears to represent a pathway involving hydroxyl radicals rather than catalase because (1) stimulation occurred in the presence of azide, which inhibits catalase, (2) stimulation occurred in the presence of 1-butanol, which is not an effective substrate for catalase, and (3) stimulation was blocked by hydroxyl radical scavenging agents, which do not affect catalase-mediated oxidation of ethanol. A possible role for contaminating iron in the H2O or buffers could be ruled out since similar results were obtained with or without chelex-100 treatment of these solutions. The stimulatory effect by Fe-EDTA required microsomal electron transfer with NADPH, and H2O2 could not replace the NADPH-generating system. In the absence of microsomes or catalase, Fe-EDTA also stimulated the coupled oxidation of ethanol during the oxidation of xanthine by xanthine oxidase. These results suggest that during microsomal electrom transfer, conditions may be appropriate for a Fenton type or a modified Haber-Weiss type of reaction to occur, leading to the production of hydroxyl radicals.