There is increasing evidence that ethanol toxicity may be associated with elevated production of reactive oxygen intermediates. Among the mechanisms suggested by which ethanol produces oxidative stress, ethanol induction of the microsomal mixed-function oxidase system and cytochrome P450 2E1, and ethanol-derived NADH are of special interest. The specific objective of this application is to employ electron spin resonance (ESR) spectroscopy to determine production of radicals such as superoxide, hydroxyl and carbon center radicals such as the hydroxyethyl radical by liver cell organelles, particularly microsomes, and assess the influence of ethanol treatment on this production of reactive radical intermediates. The major advantages of ESR are that it provides unambiguous, direct determination of radicals, is highly sensitive, and is the only method to detect reactive intermediates such as HER. Experiments will be carried out which compare the effectiveness of NADH with that of NADPH in promoting radical generation and in interacting with a variety of iron complexes to catalyze radical generation by liver cell organelles such as microsomes, mitochondria, nuclei and plasma membranes. The role of P450 2E1 will be assessed using substrates, inhibitors and antibodies. The effects of anti-oxidants and redox cycling agents, and comparisons of results obtained by ESR and chemical detection, will be made. The effect of chronic ethanol treatment on ESR- detectable rates of oxygen and hydroxy radicals and HER production will be evaluated. Since ethanol toxicity originates in the perivenous zone, microsomes and other organelles will be isolated from periportal hepatocytes prepared from control and ethanol-treated rats, and production of reactive intermediates determined by ESR. To identify microsomal enzymes which play a role in the NADH-and NADPH-dependent generation of reactive radical intermediates, experiments with purified NADH-b5 reductase, NADPH-P450 reductase, b5 and P450 (especially P450 2E1) will be carried out, as will selected experiments with human liver microsomes and human liver P450 2E1.
A final aim will be to use ESR to evaluate production of glycerol and other polyhydroxylated alcohol radicals. It is anticipated that direct and specific ESR studies will provide new information on the generation of, and the role of, reactive radical intermediates in alcohol toxicity.
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