The alcohol-inducible forms of cytochrome P450 contribute to alcohol oxidation and are of biomedical importance because they are involved in the chemical toxicities, mutagenesis, and carcinogenesis associated with alcohol abuse. In addition, they catalyze the reductive cleavage of lipid hydroperoxides and may thereby cause a loss in the integrity of biological membranes. Our goals are as follows: 1. To determine the role of alcohol-inducible P450 oxygenases (rabbit liver microsomal isozymes 2E1 and 2E2) in the oxidative cleavage of esters and amides, with the eventual goal of finding physiologically important substrates for the reaction. Compounds of metabolic importance to be considered include retinyl esters, thiol esters, phosphate esters of carbohydrates, triglycerides, phospholipids, nucleotides, and various peptides. Xenobiotic esters and amides that form aldehydes or other potentially toxic products will also be examined as possible substrates. 2. To compare the activities of P450 2E1 and 2E2 in the reductive beta-scission of various lipid hydroperoxides and determine the metabolic fate and possible function of the products. The long-range aim of the work is to determine whether chronic exposure of animals to ethanol, by causing induction of P450s, leads to enhanced degradation of membrane lipids and eventual loss of membrane integrity. In addition to determining the metabolic fate of the products of reductive cleavage, we will look for the presence of hydroxylated fatty acids, which are alternative products of hydroperoxide reduction. 3. To study the inactivation of ethanol-inducible P450 and other microsomal P450 isozymes by trans-4-hydroxy-2-nonenal (HNE) and related products of membrane lipid peroxidation. We will also study the biosynthesis of HNE in mammalian tissues and examine the possible inhibitory effects on P450s 2E1 and 2E2 of a series of other alpha, beta-unsaturated carbonyl compounds produced by ionizing radiation and drug metabolism as well as by lipid peroxidation. 4. To determine the mechanism of induction of P450 2E1 in diabetes, which may involve release of down-regulation of gene expression by insulin. We will first establish a cell line with stably expressed P450 2E1 under the control of its own promoter, in Hep G2, which does not express detectable P450 2E1 endogeneously, and then investigate the effect of insulin in the transfected Hep G2 cells. The mechanism of regulation by insulin will also be examined by nuclear run-on assays and mRNA half-life determination. In addition, we will determine whether induction in animals by the diabetic state and by alcohol administration give additive effects on the P450 2E level in liver microsomes. If so, the oxidative damage and chemical pathology (including nitrosamine activation and acetaminophen toxicity) associated with these cytochromes may be comparably elevated.
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