Oxygen radical generation by microsomes: Role of iron and implications for alcohol metabolism and toxicity

Abstract

Experiments were carried out to evaluate whether the molecular mechanism for ethanol oxidation by mcirosomes, a major pathway of alcohol metabolism, involved generation of hydroxyl radical (·OH). Microsomes oxidized chemical ·OH scavengers (KMB, DMSO, t-butyl alchol, benzoate) by a reaction sensitive to catalase, but not SOD. Iron was required for microsomal ·OH generation in view of the potent inhibition by desferrioxamine; however, the chelated form of iron was important. Microsomal ·OH production was effectively stimulated by ferric EDTA or ferric DTPA, but poorly increased with ferric ATP, ferric citrate, or ferric ammonium sulfate. By contrast, the latter ferric complexes effectively increased microsomal chemiluminescence and lipid peroxidation, whereas ferric EDTA and ferric DTPA were inhibitory. Under conditions that minimize ·OH production (absence of EDTA, iron) ethanol was oxidized by a cytochrome P-450-dependent process independent of reactive oxygen intermediates. Under conditions that promote microsomal ·OH production, the oxidation of ethanol by ·OH becomes more significant in contributing to the overall oxidation of ethanol by microsomes. Experiments with inhibitors and reconstituted systems containing P-450 reductase indicated that the reductase is the critical enzymes locus for interacting with iron and catalyzing production of reactive oxygen species. Mirosomes isolated from rats chronically fed ethanol catalyzed oxidation ·OH scavengers, light emission and inactivation of added metabolic enzymes at elevated rates, and displayed an increase in ethanol oxidation by a ·OH-dependent and a P-450-dependent pathway. It is possible that enhanced generation of reactive oxygen intermediates by microsomes may contribute to teh hepatotoxic effects of ethanol.