In addition to alcohol dehydrogenase and catalase, the ability of hepatic microsomal cytochrome P-450-dependent monoxygenases to metabolize ethanol to acetaldehyde has now been firmly established. However, the mechanism underlying cytochrome P-450-dependent ethanol oxidation as well as the role of this multicomponent enzyme system in the metabolism of ethanol in vitro and in vivo are poorly understood. Therefore, an ethanol-induced cytochrome P-450 isozymes(s) will be purified from hamster liver. The isolated hemeprotein(s) will be characterized in terms of spectral properties, molecular weight, peptide mapping and amino acid composition. These characteristics will then be compared to isozymic forms of cytochrome P-450 isolated from hamsters treated with phenobarbital. The distinctness of these various purified isozymes will be determined by examining immunological cross-reactivity. Ethanol oxidation will be investigated in a reconstituted monooxygenase system conmsisting of synthetic phospholipid, purified cytochrome P-450 reductase and either the ethanol-induced or phenobarbital-induced P-450 isozymes. The substrate specificity of the individual isozymes will first be compared, especially with regard to the oxidation of ethanol and other alcohols. End-point stoichiometric experiments will then be performed employing each of the purified isozymes in order to gain insight into the mechanism of ethanol oxidation catalyzed by ethanol-induced versus phenobarbital-induced forms of cytochrome P-450. Mechanistic studies will also involve assessment of the role of hydroxyl radicals (OH.) in ethanol oxidation by the different reconstituted monooxygenase systems. Such experiments will employ compounds known to affect NADPH-dependent microsomal OH. production, such as OH. scavengers and iron chelators, and will also involve determination of superoxide and hydrogen peroxide production (precursors of OH.) catalyzed by the various cytochrome P-450 isozymes when reconstituted with flavoprotein and lipid. Finally, the involvement of cytochrome P-450-dependent monooxygenases in the total microsomal metabolism of ethanol will be examined using antibodies produced against the ethanol-induced form(s) of this hemeprotein. Investigation of the various changes produced in hepatic microsomal cytochrome P-450 following ethanol consumption may further our understanding of the pharmacological and/or toxicological aberrations observed after chronic ingestion of this compound. Such studies may also aid in elucidation of the mechanism of metabolic tolerance to ethanol observed in alcoholics.
| Ardies, C M; Lasker, J M; Lieber, C S (1987) Characterization of the cytochrome P-450 monooxygenase system of hamster liver microsomes. Effects of prior treatment with ethanol and other xenobiotics. Biochem Pharmacol 36:3613-9 |
| Ardies, C M; Lasker, J M; Bloswick, B P et al. (1987) Purification of NADPH:cytochrome c (cytochrome P-450) reductase from hamster liver microsomes by detergent extraction and affinity chromatography. Anal Biochem 162:39-46 |
| Lasker, J M; Raucy, J; Kubota, S et al. (1987) Purification and characterization of human liver cytochrome P-450-ALC. Biochem Biophys Res Commun 148:232-8 |
| Nouchi, T; Lasker, J M; Lieber, C S (1986) Activation of acetaminophen oxidation in rat liver microsomes by caffeine. Toxicol Lett 32:1-8 |
