Regulation of cytochrome P-450c by glucocorticoids and polycyclic aromatic hydrocarbons in cultured fetal rat hepatocytes

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the synthesis of cytochrome P-450c (the major isozyme induced by polycyclic aromatic hydrocarbons) were investigated in fetal rat hepatocytes maintained in primary monolayer culture. Treatment of hepatocytes in culture with 1,2-benzanthracene resulted in a 50-fold increase in 7-ethoxycoumarin O-deethylase activity. The level of P-450c increased in the cells in a time-dependent fashion as determined by immunoelectrophoretic analysis. The inductive effect of BA was potentiated approximately 1.6- to 2.3-fold when 1 microM dexamethasone was included in the culture medium. However, dexamethasone alone had little or no effect on the induction of P-450c. The rate of synthesis of P-450c was examined by immunoisolation of the specific isozyme from total cellular proteins radiolabeled with [35S]methionine and from the protein products formed during in vitro translation of the isolated mRNA. In addition, the amount of mRNA specific for cytochrome P-450c was determined by Northern blot analysis of RNA extracted from cultured cells. The changes in the rates of synthesis and mRNA levels were found to parallel the changes in enzyme activity. The concentration of dexamethasone required to cause a half-maximal increase in P-450c content in the presence of 1,2-benzanthracene was between 10(-8) and 10(-7) M. It is concluded that glucocorticoids act synergistically with polycyclic aromatic hydrocarbons to increase the levels of P-450c expressed in the fetal rat liver, and that this action is likely mediated by the classical type II glucocorticoid receptor.     

Synergistic induction of monooxygenase activity by glucocorticoids and polycyclic aromatic hydrocarbons in human fetal hepatocytes in primary monolayer culture

The ability of polycyclic aromatic hydrocarbons and glucocorticoids to regulate monooxygenase activity of human fetal liver has been studied using hepatocytes prepared by collagenase digestion of liver samples from human abortuses of 13 to 19 weeks of gestational age, and maintained in primary monolayer culture for periods up to 5 days. Addition of 1,2-benzanthracene to the cells caused an increase in monooxygenase activity (3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin) in a time-and concentration-dependent fashion. The concentration of 1,2-benzanthracene required to achieve half-maximal induction was 5 microM. The inductive effect of the polycyclic hydrocarbon was potentiated approximately 2.5-fold when dexamethasone (250 nM) or other glucocorticoids were included in the culture medium. Dexamethasone alone had little or no effect on the induction of monooxygenase activity. The concentration of dexamethasone required for half-maximal stimulation of monooxygenase activity in the presence of 1,2-benzanthracene was 5-10 nM, and the action of dexamethasone was reversed by the addition of cortisol 21-mesylate, consistent with the concept that the action of dexamethasone was mediated by binding to a glucocorticoid receptor. These results are suggestive that glucocorticoids, which are produced by the fetal adrenal and have an important role in the regulation of fetal development, act synergistically with polycyclic aromatic hydrocarbons to induce the activity of liver monooxygenases in the human fetus.     

Dexamethasone-mediated potentiation of P450IA1 induction in H4IIEC3/T hepatoma cells is dependent on a time-consuming process and associated with induction of the Ah receptor

The synergistic effect of dexamethasone (DEX) and polycyclic aromatic hydrocarbons on the induction of cytochrome P450IA1 (P450IA1) was examined in H4IIEC3/T Reuber hepatoma cells. P450IA1 activity was determined by the hydroxylation of benzo[a]pyrene (AHH) and deethylation of 7-ethoxyresorufin (EROD). The amount of Ah receptor, i.e. the specific cytosolic binding protein of 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in H4IIEC3/T cells was characterized and quantitated by high performance gel filtration. Benz[a]anthracene and TCDD induced AHH and EROD activities, respectively, about 20-fold within 4 h. The increase was about 100-fold when cells were pretreated with DEX. The glucocorticoid alone induced P450IA1 activities 3-4 fold. DEX elicited half maximum AHH induction at a concentration of 20 nM in the presence or absence of benz[a]anthracene. Maximal potentiation of AHH induction required treatment with DEX for at least 32 h prior to the exposure to benz[a]anthracene. Treatment of H4IIEC3/T cells with DEX for 20 h caused a 2-3-fold increase in the amount of Ah receptor. The results suggest that the synergistic effect of DEX and polycyclic aromatic hydrocarbons on P450IA1 induction involves a time-consuming process which may consist of the synthesis or modification of a factor, possibly the Ah receptor.     

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interrelationship between RU38486 and the P450 activities in rat liver

Microsomal P450 monooxygenases contribute actively to the biotransformation of the antiglucocorticoid RU38486, an 11 beta-substituted nor-steroid. Pretreatment of adult rats by inducers of specific forms, belonging to different P450 subfamilies, affects the ability of liver microsomes to metabolize RU38486. Phenobarbital and pregnenolone 16 alpha-carbonitrile increase the metabolic activity of liver microsomes whereas methylcholanthrene decreases their capacity to oxidize the steroid. Thus P450 forms IIIA, IIB1,2 and IIC7 are good candidates to be involved in the degradation of this peculiar molecule. Our study has been completed by investigating whether RU38486 would influence the P450 spectrum. Whereas the treatment of rats with either a glucocorticoid (cortisol, dexamethasone) or an antiglucocorticoid (pregnenolone 16 alpha-carbonitrile) has been shown to induce the P450 activity by increasing the hepatic concentration of form IIIA, we observed a slight decrease of the P450 activity by treating the animals with RU38486. Moreover RU38486 was able to antagonize the P450 induction by the other steroids as well as it inhibits the synthesis of various liver enzymes induced by glucocorticoids (for instance tyrosine aminotransferase). These findings may be important for the therapeutic use of RU38486 since its inhibitory effect on P450 activity may be at the origin of drug interactions by modifying the endogenous hormonal status.     

The murine Ah locus. Comparison of the complete cytochrome P1-450 and P3-450 cDNA nucleotide and amino acid sequences

Mouse cytochrome P1-450 and P3-450 are most closely associated with induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.1) and acetanilide 4-hydroxylase activity, respectively. Full-length cDNA clones of P1-450 and P3-450 were generated from mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mouse liver. P1-450 cDNA is 2620 nucleotides in length and has a coding region (base 110 to 1,675) that produces a protein with 521 residues (Mr = 58,914). P3-450 cDNA is 1,894 nucleotides in length and yields a protein with 513 residues (Mr = 58,183). P1-450 mRNA is the first reported example in mouse in which UAG is used as the termination codon. P1-450 and P3-450, both induced by polycyclic hydrocarbons and regulated by the Ah receptor, exhibit overall nucleotide and protein homology of 68, and 73%, respectively. Segments of high homology, interspersed with regions of low homology, support the hypothesis of gene conversion or unequal crossing over as possible mechanisms for divergence of these two genes. Mouse P1-450 and P3-450 cDNAs were compared with previously published data on rat P-450e cDNA and rabbit form 2 protein, corresponding to two P-450 genes from the "phenobarbital inducible" P-450 gene subfamily. Nucleotide homology between a member of either gene subfamily is about 30%, and protein homology is about 15%, suggesting that the Ah locus-associated P-450 gene subfamily diverged from the phenobarbital inducible P-450 subfamily more than 200 million years ago. An N-terminal and a C-terminal cysteinyl fragment corresponding to the regions around P1-450 Cys-158 and Cys-458, respectively, are the only two cysteinyl peptides conserved among all four proteins compared. Because of greater homology in the C-terminal conserved cysteinyl fragment between the two gene subfamilies and a greater hydrophobic pocket in the C-terminal conserved cysteinyl fragment, the data favor this cysteine as the more likely candidate for the thiolate ligand to the heme iron in the P-450 enzyme active-site.     

Macrolide antibiotics inhibit the degradation of the glucocorticoid-responsive cytochrome P-450p in rat hepatocytes in vivo and in primary monolayer culture

Treatment of rats with macrolide antibiotics such as triacetyloleandomycin (TAO) dramatically increases the hepatic concentration of a cytochrome P-450 indistinguishable from P-450p, the major liver cytochrome induced by glucocorticoids such as dexamethasone (Wrighton, S. A., Maurel, P., Schuetz, E. G., Watkins, P. B., Young, B., and Guzelian, P. S. (1985) Biochemistry 24, 2171-2178). To investigate the mechanism of induction of P-450p, we treated rats for 4 days with these agents and found that dexamethasone and TAO induced the synthesis of P-450p at least 70- and 35-fold over control values, respectively, as estimated from measurements of P-450p mRNA translatable in a cell-free system. However, the accumulation of P-450p holocytochrome (measured as TAO-metabolite spectral complex) or P-450p protein (measured by quantitative immunoblotting) increased at least 150-fold by TAO but only 32-fold by dexamethasone. The possibility that TAO decreases the degradation of P-450p was supported by the observation that administration of TAO to dexamethasone-treated rats labeled with NaH[14C]O3 and [3H]-delta-aminolevulinic acid retarded the decay of radioactive immunoprecipitable P-450p protein (t1/2 = 60 versus 14 h) and heme (t1/2 = 73 versus 10 h). To confirm these results, P-450p protein synthesis was measured as radioactivity incorporated into immunoprecipitable P-450p in primary monolayer cultures of adult rat hepatocytes incubated with [3H]leucine. Dexamethasone treatment of the cultures stimulated P-450p synthesis by at least 30-fold whereas macrolides were without effect. However, macrolide antibiotics but not dexamethasone inhibited the disappearance of radiolabeled P-450p from cultured hepatocytes similar to the results obtained in vivo. We conclude that macrolide antibiotics induce P-450p, the most rapidly turning over cytochrome yet reported, by stimulating its synthesis indirectly and by blocking its degradation, significantly.