Induction of cytochrome P-450 mRNAs quantitated by in vitro translation and immunoprecipitation

Abstract

Cytochrome P-450 mRNAs were quantitated by in vitro translation of liver RNA followed by immunoprecipitation with antibodies specific for cytochromes P-450. The kinetics of cytochrome P-450 mRNA induction by 3-methylcholanthrene and by phenobarbital were examined, and differences in the types of cytochrome P-450 mRNAs induced by 3-methylcholanthrene (MC), β-napthoflavone (BNF), and phenobarbital (PB) in male and female rats were determined. Phenobarbital strongly induced the mRNA encoding a single peptide antigenically related to the phenobarbital-induced cytochrome P-450 PB-B. Male rat liver contained 62% more translatable mRNA for this peptide than did female. 3-Methylcholanthrene and β-napthoflavone, but not phenobarbital, induced mRNAs encoding three peptides that were immunologically related to cytochrome P-450 BNF MC-B , which is induced by 3-methylcholanthrene. The levels of translatable mRNA coding for these peptides were twice as high in females as in males. Striking sex differences were observed in the levels of translatable mRNAs for peptides related to cytochrome P-450 PB PCN-E , which is induced by phenobarbital and by pregnenolone-16-α-carbonitrile. In females, only RNA preparations from the livers of phenobarbital-treated rats had significant levels of mRNAs encoding these peptides. In contrast, significant levels of these RNAs were observed even in untreated males, and the levels of these mRNAs increased markedly following phenobarbital treatment. All cytochrome P-450 inducers examined caused a 50 to 70% decrease in translatable albumin mRNA. This effect was specific for albumin mRNA, since levels of total translatable mRNA were not generally altered by these inducers. The kinetics of induction of cytochrome P-450 mRNA differed from those of induction of aryl hydrocarbon hydroxylase (AHH) activity. Translatable cytochrome P-450 mRNA was increased as early as 4 h after phenobarbital treatment, peaked between 24 and 36 h, and dropped back to control levels by 120 h. The induction of AHH lagged behind the increase in translatable mRNA, remaining at control levels well after levels of translatable mRNA began to increase but then decreasing roughly in parallel with translatable mRNA. These findings suggest that transcription was not rate limiting for regulation of PB-inducible cytochrome P-450 activity. 3-Methylcholanthrene caused parallel increases in AHH activity and translatable cytochrome P-450 mRNA, but when translatable mRNA began to decrease after about 24 h, AHH activity remained high, suggesting that this P-450 mRNA was less stable than the enzyme for which it coded.