Erin G. Schuetz

17β-estradiol hydroxylation catalyzed by human cytochrome P450 1A1 : a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.

Biphasic regulation of cytochrome P450 2B1/2 mRNA expression by dexamethasone in primary cultures of adult rat hepatocytes maintained on matrigel

We have demonstrated recently that although rat hepatocytes rapidly lose their cytochrome P450 mRNA content following their introduction into primary culture, hepatocytes cultured on Matrigel, a reconstituted basement membrane, subsequently spontaneously reexpress the mRNAs of some constitutive P450 forms (Kocarek et al., Mol Pharmacol 43: 328-334, 1993). In the present study, we used the Matrigel cell culture system to examine the dose-dependent effects of dexamethasone (DEX) treatments on the mRNAs for two of the P450 forms that are reexpressed spontaneously between days 3 and 5 in culture, 2B1/2 and 2C6. Treatment of cultured hepatocytes with low doses of DEX (10(-9) to 10(-8) M) that induced the mRNA for tyrosine aminotransferase, a model glucocorticoid-inducible gene, suppressed the spontaneous appearance of 2B1/2 mRNA while having little or no effect on the level of 2C6 mRNA or on beta-actin mRNA. However, treatment of the hepatocyte cultures with high doses of DEX (10(-6) to 10(-5) M) that induced P450 3A1 mRNA increased the amounts of the 2B1/2 and 2C6 mRNAs (4.1- and 2.4-fold, respectively, at 10(-5) M DEX). In contrast to the suppressive effects on the spontaneous increases in 2B1/2 mRNA, low doses of DEX (10(-8) to 10(-7) M) enhanced the induction of 2B1/2 mRNA by phenobarbital (2.5-fold at 10(-7) M DEX). Treatment of the hepatocyte cultures with triamcinolone acetonide, another potent glucocorticoid, suppressed spontaneous 2B1/2 mRNA expression at low doses, but did not induce 2B1/2 mRNA at high doses. Treatments with steroids of other classes, including dihydrotestosterone, 17 alpha-ethinylestradiol, fludrocortisone or R-5020, failed to suppress 2B1/2 mRNA levels at low doses. Additionally, treatment with RU-486, a glucocorticoid/progestin receptor antagonist, induced 2B1/2 mRNA at high doses (10(-6) to 10(-5) M). The suppressive effects of DEX on spontaneous 2B1/2 mRNA expression observed at low doses are consistent with a classical glucocorticoid-mediated mechanism, while the high-dose inductive effects of DEX appear to be exerted through a nonclassical mechanism, perhaps akin to that for induction of 3A1.

Regulation of cytochrome P450 2B1/2 genes by diallyl sulfone, disulfiram, and other organosulfur compounds in primary cultures of rat hepatocytes

Our previous study demonstrated that diallyl sulfide (DAS), a compound derived from garlic, transcriptionally activated the P450 2B1/2 genes in rat liver. In the present study, rat primary hepatocytes were used to determine the effects of DAS and its metabolite, diallyl sulfone (DASO2), on the expression of the P450 2B1/2 genes. Freshly isolated adult rat hepatocytes were cultured in a serum-free medium on a reconstituted basement membrane matrix matrigel that enabled the hepatocytes to maintain expression of numerous liver-specific genes for more than 1 week. After 48-hr of acclimation, 0.1, 0.5, and 2.0 mM concentrations of DAS or DASO2 were added to the culture medium and the cells were harvested at 4, 12, 24, or 36 hr after the treatment for the preparation of microsomes and RNA. Cytotoxicity was not observed by morphological examinations after DAS and DASO2 treatments. In contrast to the in vivo results, there was only a slight increase in the levels of P450 2B1/2 mRNA and protein in DAS-treated cells. However, DASO2 treatment (2 mM) resulted in 11-, 21-, and 22-fold increases in P450 2B1/2 mRNA levels at 12, 24, and 36 hr after the treatment, respectively. P450 2B1/2 protein levels were also increased markedly in DASO2-treated cells. Co-incubation of the rat hepatocyte cultures with a physiological concentration of growth hormone significantly blocked the induction of P450 2B1/2 mRNA by DASO2. Northern blot analysis using oligonucleotide probes specific for 2B1 and 2B2 demonstrated that DASO2 induced mRNA levels of both 2B1 and 2B2, with a greater induction of 2B1 mRNA. For comparison, the effects of disulfiram (DSF) and its metabolite, diethyldithiocarbamate (DDTC), on P450 2B1/2 mRNA expression were also examined in the cultured rat hepatocytes. Both DSF and DDTC caused a significant increase in P450 2B1/2 mRNA level with the highest induction at 0.5 mM. Addition of growth hormone to the culture effectively suppressed the P450 2B1/2 mRNA induction by DSF but had little effect on the induction by DDTC. Neither mRNA nor protein levels of P450 2E1 in cultured hepatocytes were affected by all the organosulfur compounds tested. These results suggest that DASO2, DSF and DDTC selectively modulate P450 isozymes in cultured rat primary hepatocytes and that the induction of P450 2B1/2 by DAS in rat liver may be mediated by its metabolite, DASO2.

Divergent effects of cycloheximide on the induction of Class II and Class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes

We have previously reported that when hepatocytes isolated from adult male rats are cultured in serum-free medium on matrigel, a reconstituted basement membrane gel, it is possible to elicit a stimulation of gene expression for both Class II cytochrome P450b/e and Class III cytochrome P450p by phenobarbital treatment (E.G. Schuetz et al., 1990 J. Biol. Chem. 265, 1188-1192). In the present study, an investigation of the requirement of protein synthesis for the rise in mRNAs for these cytochromes, pretreatment of the cells with cycloheximide prior to adding phenobarbital or phenobarbital-like inducers to the culture medium inhibited induction of P450b/e mRNA (46-90%), whereas the accumulation of P450p mRNA was enhanced (2- to 19-fold). Heme depletion did not appear to explain these observations because the inhibitory effects of cycloheximide on the induction of P450b/e mRNA were not overcome by supplementation of the medium with exogenous heme or with delta-aminolevulinic acid. Because Class IIIA P450s are regulated by gender as well as by phenobarbital, we examined the basal expression of P450p mRNA in cultures of hepatocytes derived from male rats and found that cycloheximide treatment was without effect. However, in cultures of hepatocytes isolated from female rats, where P450p mRNA is barely detectable, cycloheximide treatment greatly enhanced expression of P450p mRNA. As was observed in the cultured cells, the treatment of living female rats with cycloheximide also increased the amounts of P450p mRNA to levels comparable to those found in livers of untreated male rats. Analysis of Northern blots hybridized with oligonucleotides specific for P450PCN1(IIIA1) and P450PCN2(IIIA2), respectively, revealed that untreated male rat liver and cultures of hepatocytes prepared from these animals expressed readily detectable amounts of P450PCN1(IIIA1) mRNA. Such analyses confirmed that cycloheximide treatment selectively increased P450PCN1(IIIA1) mRNA in female rat liver, whereas the amount of mRNA for P450PCN2(IIIA2), a closely related male-specific family member, was unaffected. We conclude that the pathways for the induction of P450b/e and P450p by phenobarbital, and the pathways for the gender-specific basal expression of P450PCN1(IIIA1) and P450PCN2(IIIA2) are not the same and can be distinguished by their differential response to inhibition of ongoing protein synthesis.

Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes

SummaryBecause the metabolic environment can alter gene expression in cultured cells, we examined the effects of change of medium on the levels of several cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on Matrigel. The amounts of P450 1A2, 2B1/2, or 3A1 mRNA were unaffected by changing the medium. In contrast, P450 1A1 mRNA levels were increased 1 to 2 h after media change, reached maximum levels by 6 h, and declined to baseline by 24 h. Supplementing day-old media with components of the medium revealed that only addition of amino acids resulted in 1A1 mRNA induction. From the results of direct additions and omissions, we showed that tryptophan, but not histidine, was largely responsible for the 1A1 mRNA induction. Moreover, mild photoactivation of the tryptophan resulted in a substantially increased magnitude of 1A1 mRNA induction. The time course for 1A1 mRNA induction by treatment with photoactivated tryptophan was identical to that observed after medium change. Treatment of hepatocyte cultures with β-naphthoflavone, which is metabolized by 1A1, also resulted in a transient 1A1 mRNA induction time-course profile over a 24-h period, whereas treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which is relatively stable to metabolic transformation, produced sustained elevations of 1A1 mRNA, suggesting that the transient response to tryptophan also may involve metabolism of the inducer. Our results extend previous data showing that oxidized products of tryptophan induce 1A1, and suggest that the transient nature of the induction may be due to elimination of the activated tryptophan molecule.

Regulation of cytochrome P450 2B1/2 mRNAs by Kepone (chlordecone) and potent estrogens in primary cultures of adult rat hepatocytes on Matrigel.

We previously reported that when primary cultures of rat hepatocytes were treated with phenobarbital (PB) or one of several organochlorine pesticides, including Mirex, there was co-induction of cytochrome P450 2B1 and 2B2 mRNAs and immunoreactive proteins, whereas Kepone selectively induced 2B2 (Kocarek et al. (1991) Mol. Pharmacol. 40, 203-210). Indeed, Kepone treatment actively suppressed induction of 2B1 and 2B2 mRNAs in hepatocytes cotreated with phenobarbital. Because Kepone differs chemically from Mirex only in the replacement of 2 chlorine atoms with a ketone group, which exists in aqueous solution as a gem-diol and appears to confer weak estrogenic properties, we treated hepatocyte cultures with one of 3 potent estrogens, beta-estradiol, 17 alpha-ethinylestradiol or diethylstilbestrol. Treatment with each of these estrogens induced 2B1 and 2B2 mRNA only at very high doses (10(-4) M). Beta-Estradiol (10(-4) M) treatment also induced 2B1/2 mRNA in hepatocyte cultures prepared from a prepubescent female rat. The anti-estrogen tamoxifen failed to reverse 2B1/2 mRNA induction following beta-estradiol or Kepone treatment of adult hepatocyte cultures. High doses of beta-estradiol or 17 alpha-ethinylestradiol failed to induce 2B1/2 mRNA in treated rats. We also examined the effects of chloral hydrate, a simple gem-diol, on 2B1/2 mRNA induction in the hepatocyte cultures. Treatment with chloral hydrate (3 x 10(-3) M), like Kepone (10(-5) M), suppressed 2B1/2 mRNA induction following phenobarbital (10(-4) M) treatment, while Kepone alcohol (10(-5) M), which is not a gem-diol, produced less suppression. Our results suggest that selective induction by Kepone of 2B2 is unlikely related to its effects as a weak classical estrogen, while the ability of Kepone to suppress induction of 2B1 and 2B2 by PB may be related to its properties as a gem-diol.