Evidence for involvement of cAMP-dependent pathway in the phenobarbital-induced expression of a novel hamster cytochrome P450, CYP3A31.

Recently, we isolated a novel Syrian hamster cDNA clone that encodes a protein which has been named CYP3A31. In primary hepatocyte cultures, CYP3A31 is dramatically induced by phenobarbital. To elucidate the mechanism of this induction, we first studied the effects of cAMP on phenobarbital-induced CYP3A31 expression using forskolin and N6,O2'-dibutyryl cAMP in hepatocyte cultures. At 100 microM, forskolin significantly inhibited both the phenobarbital-induced CYP3A31 mRNAs expression and the testosterone 6beta-hydroxylation activity related to the CYP3A subfamily in rats, whereas 0.1 microM forskolin potentiated the phenobarbital induction of CYP3A31 mRNA and the testosterone 6beta-hydroxylation activity. Treatment with N6,O2'-dibutyryl cAMP resulted in an inhibition of phenobarbital-induced CYP3A31 gene expression and testosterone 6beta-hydroxylation activity. Increasing amounts of transfected cAMP-response element binding proteins (CREB) or CREB-binding proteins in hamster hepatocytes reduced the phenobarbital-induction of CYP3A31 mRNAs expression. These results suggest that in vitro induction of CYP3A31 by phenobarbital in Syrian hamster hepatocytes is regulated by a cAMP-dependent pathway.

Modulation of snake hepatic cytochrome P450 by 3-methylcholanthrene and phenobarbital.

Induction mode of the hepatic drug-metabolizing enzymes was studied in Corn snake (Elaphe guttata emoryi). Treatment of snakes with 3-methylcholanthrene or phenobarbital produced no effects on liver weight and total content of cytochromes P450 and b5. Treatment with 3-methylcholanthrene significantly induced the activities of arylhydrocarbon hydroxylase, 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase, whereas those of ethoxycoumarin O-deethylase, benzphetamine N-demethylase, erythromycin N-demethylase and testosterone hydroxylases were not affected. 3-Methylcholanthrene-induced activities of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase were inhibited by 20 microM alpha-naphthoflavone by 98% and 73%, respectively. Phenobarbital-treatment caused a significant induction of the activities of erythromycin N-demethylase and testosterone 6 beta-hydroxylase, but did not affect those of the other phase I enzymes and the other testosterone hydroxylases. The activities of UDP-glucuronyltransferase and glutathione S-transferase were not affected by either 3-methylcholanthrene or phenobarbital administration. Immunoblotting showed that 3-methylcholanthrene-treatment induced a protein band related to hamster CYP1A2, and decreased the intensity of the two bands detected with anti-rat CYP2B1. Phenobarbital-treatment did not affect the intensity of CYP2B-related proteins. The results suggest that snake liver has multiple forms of cytochrome P450, notably those inducible by 3-methylcholanthrene.

cDNA cloning and expression of a novel CYP3A from the Syrian hamster, CYP3A31.

A clone, encoding a cytochrome P450 protein consisting of 501 amino acids, was isolated from a cDNA library constructed from mRNA of Syrian hamster liver. The deduced amino acid sequence of this clone showed a high homology (65 to 81%) with other mammalian CYP3As and hence, this novel isozyme was named CYP3A31. By Northern blotting, using an oligonucleotide specific to CYP3A31, the mRNA for this isozyme was shown to be expressed constitutively in liver and induced by treatment with phenobarbital but repressed by 3-methylcholanthrene or dexamethasone treatments. The increase in mRNA expression by phenobarbital and decrease by dexamethasone corresponded to changes in CYP3A protein as analysed by Western blotting. These indicate that CYP3A31 might constitute one of the major CYP3A isozymes in the hamster.

Expression of cytochrome P450 isozymes in Syrian hamster after dietary vitamin A supplementation and deficiency.

Expression of hepatic cytochrome P450 isozymes by vitamin A-deficient or -supplemented diet was studied in Syrian hamsters. In male hamsters given a vitamin A-supplemented diet at a level of 250 IU/g-diet for 6 weeks, a marked increase in the activity of testosterone 7 alpha-hydroxylase was observed, accompanied by an elevation in the level of protein immunorelated to CYP2A1. In the hamsters fed a vitamin A-deficient diet for 6 weeks, a decrease was obtained in the total cytochrome P450 content and in the activities of most drug-metabolizing enzymes in livers.

Differential modulation of hepatic cytochrome P-450 enzymes in rat and Syrian hamster by 4'-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl.

The effects of a single injection (40 mg/kg) of 4'-trifluoromethyl-2,3,4,5-tetrachlorobiphenyl (CF3) on hepatic cytochrome P-450 monooxygenases were assessed in rat and syrian hamster. The CF3 treatment significantly increased the total amount of cytochrome P-450 in both species. In rats, CF3 treatment caused marked increases in ethoxyresorufin O-deethylase (EROD), arylhydrocarbon hydroxylase (AHH), and testosterone 7 alpha-hydroxylase activities but significantly reduced the activities of benzphetamine N-demethylase (BzND), erythromycin N-demethylase (ErND), testosterone 6 beta, 16 alpha, and 16 beta-hydroxylase, and formation of androstenedione. Administration of CF3 to hamsters strongly induced the activities of EROD, AHH, BzND, testosterone 15 alpha, and 16 alpha-hydroxylases, and androstenedione production, whereas ErND, testosterone 6 beta, and 7 alpha-hydroxylases were decreased. Administration of CF3 to rats induced the CYP1A family proteins and CYP2A1, while CF3 reduced the level of CYP2B1, and, to a lesser extent, of CYP6 beta 2. In hamsters, CF3 treatment significantly induced the CYP1A2, CYP2A1, CYP2A8, and CYP2B1 isozymes, whereas the CYP6 beta 2 level was decreased. The ability of hepatic microsomes to activate aflatoxin B1 and benzo(a)pyrene was elevated by CF3 treatment in hamsters, while activation of aflatoxin B1 was decreased in microsomes from CF3-treated rats. These results showed differences in the CF3-induced pattern of rat and hamster cytochrome P-450 monooxygenases.