Immunochemical evidence for induction of the alcohol-oxidizing cytochrome P-450 of rabbit liver microsomes by diverse agents: ethanol, imidazole, trichloroethylene, acetone, pyrazole, and isoniazid.

Abstract

Isozyme 3a of rabbit liver microsomal cytochrome P-450, also termed P-450ALC, was previously isolated in this laboratory from animals administered ethanol or imidazole, and the purified cytochrome was shown to function in the reconstituted system as an oxygenase in catalyzing the oxidation of ethanol and other alcohols. Although liver microsomes from animals treated in various ways exhibit increased alcohol-oxidizing activity, evidence was not available as to whether this was due to enzyme induction or to other factors influencing the activity. Immunochemical quantitation of P-450 isozyme 3a has now been achieved by use of purified antibody to this cytochrome in NaDodSO4/PAGE/blotting and dot-blotting techniques. The specific content of isozyme 3a in liver microsomes was found to be increased from 2-to >4-fold by administration of the following agents, in increasing order of effectiveness as inducers: isoniazid, trichloroethylene, pyrazole, ethanol, imidazole, and acetone. Isozyme 3a represents about 5% of the total P-450 in control animals and is increased to as high as 27% by acetone treatment. Isozyme 3a-dependent butanol-oxidation activity, determined by the inhibitory effect of antibody on the various microsomal preparations, was found to increase proportionally with increased content of this cytochrome.Chronic ethanol consumption by rats results in a proliferation of the hepatic smooth endoplasmic reticulum and an increase in NADPH-cytochrome P-450 reductase and spectrally observable cytochrome P-450 (1-3). The induction of P-450 by ethanol is associated with an increase in the metabolism and, in some instances, in the toxicity of many compounds, including N-nitrosodimethylamine (4-6), acetaminophen (7-9), aniline (10-12), ethanol (13), and carbon tetrachloride (14-16). The treatment of rabbits with ethanol also results in an increase in hepatic microsomal aniline and ethanol hydroxylation, but the specific content of microsomal P-450 is not always significantly increased (17). Several years ago in this laboratory, a unique form of P-450, designated isozyme 3a on the basis of its relative electrophoretic mobility, was isolated from ethanol-treated rabbits and characterized (17, 18). More recently, we have purified the same cytochrome from rabbits treated with imidazole (19). Purified isozyme 3a, also referred to as P-450ALC (20), exhibits the highest activity of six purified rabbit liver isozymes toward ethanol and other alcohols as well as aniline (18), converts acetaminophen to a reactive intermediate (21), and is the only P-450 isozyme we have studied that is active in the demethylation of N-nitrosodimethylamine at low substrate concentrations (22). Isozyme 3a is responsible for the increase in oxidation of alcohols and aniline in liver microsomes from ethanol-treated rabbits, as indicated by inhibition of the induced activity with antibody prepared to the purified enzyme (23). Furthermore, the microsomal demethylation of N-nitrosodimethylamine at low substrate concentrations is completely inhi...

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