Identification of P-450 ALC in microsomes from alcohol dehydrogenase-deficient deermice: Contribution to ethanol elimination in vivo

Abstract

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450 ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450 ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH −) and -positive (ADH +) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH − than from naive ADH + animals. Ethanol treatment induced the protein about 3-fold in the ADH + strain and about 4-fold in the ADH − strain. The antibody to rabbit P-450 ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450 ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450 ALC homolog, increased about 3-fold upon ethanol treatment in the ADH + strain and about 4-fold in the ADH − strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH + strain and 1.9-fold and 3.3-fold, respectively, in the ADH − strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450 ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450 ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH + and ADH − strains, respectively. After chronic ethanol treatment, P-450 ALC could account maximally for 8% of the total ethanol elimination in the ADH + strain and 22% in the ADH − strain. Further, cytochrome P-450 ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450 ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant. Peroxisomal β-oxidation capacity was increased 40% over control values by ethanol treatment, consistent with the hypothesis that the increase in ethanol elimination in the ADH-negative deermouse is mediated predominantly via catalase-H 2O 2.