Biochemical and immunochemical evidence for the induction of an ethanol-inducible cytochrome P-450 isozyme in male Syrian golden hamsters.
The effects of ethanol and of phenobarbital pretreatment on hamster microsomal metabolism of aniline and p-nitrophenol have been investigated. Hydroxylation of both compounds was increased over 2-fold by ethanol pretreatment, whereas phenobarbital pretreatment had little effect on either activity. Ethanol pretreatment had no effect on the specific content of total cytochrome P-450, while phenobarbital pretreatment increased the specific content 1.6-fold. Comparison of the specific activities for aniline hydroxylation and p-nitrophenol hydroxylation of individual microsomal samples from control, ethanol-pretreated and phenobarbital-pretreated animals showed a high degree of correlation (r2 = 0.98) consistent with the involvement of the same site for catalysis of these two compounds. Antibody to rabbit ethanol-inducible cytochrome P-450 (isozyme 3a) inhibited over 80% of the aniline (high affinity) and p-nitrophenol hydroxylase activities of microsomes from ethanol-treated hamsters. A comparison of the antibody-inhibitable rates for both hydroxylase activities with microsomes from untreated, ethanol- or phenobarbital-pretreated hamsters suggested that an isozyme homologous to rabbit isozyme 3a (hamster cytochrome P-450alc) was induced in hamsters about 3.5-fold by ethanol and was unaffected by phenobarbital. The induction of hamster cytochrome P-450alc was confirmed by immunoblot analysis of hamster microsomes. A single protein with a molecular weight of approximately 54,000 was recognized by antibody to the rabbit isozyme. Quantification of the immunoblots demonstrated that the hamster isozyme was increased about 3-fold, in good agreement with the induction determined by a comparison of the antibody-inhibitable rates. The results indicated that, although there was no change in the total spectrally observable cytochrome P-450, there was a marked change in the distribution of the isozymes of cytochrome P-450, with an increase in the alcohol-inducible form after 28-day ethanol consumption by chow-fed hamsters. This isozyme can be readily monitored by either high-affinity aniline or p-nitrophenol hydroxylation or by Western immunoblot analysis and appears to be the ethanol-inducible form of cytochrome P-450 in hamsters.
McCoy
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Department of Environmental Health Sciences, Case Western Reserve University, School of Medicine, Cleveland, OH 44106.
Koop
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D000815:Aniline Hydroxylase / Q000096:biosynthesis; D000818:Animals; D006224:Cricetinae; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D000431:Ethanol / Q000494:pharmacology*; D006900:Hydroxylation; D007527:Isoenzymes / Q000096:biosynthesis*; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000096:biosynthesis
D007527:Isoenzymes; D000431:Ethanol; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D000815:Aniline Hydroxylase