Regulation of sulfotransferases by xenobiotic receptors.

The transcription factor networks that regulate basal and xenobiotic-modulated expression of the hepatic sulfotransferases affect the dynamics of xenobiotic detoxication, carcinogen bioactivation and metabolic homeostasis. Emerging evidence suggests that liver-enriched transcription factors, the aryl hydrocarbon (Ah) receptor and members of the nuclear receptor transcription factor superfamily all play integrated roles in the control of sulfotransferase gene transcription. Unlike the well known up-regulation of CYP1A1, expression of rat hepatic aryl (SULT1A1) and hydroxysteroid (SULT2A) sulfotransferase is suppressed in response to treatment with the prototypic Ah receptor ligand, 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin. Glucocorticoid-inducible rat hepatic SULT1A1 gene transcription occurs through a glucocorticoid receptor (GR)-mediated mechanism, while human hepatic SULT1A1 does not display GR-inducible expression. By comparison, liver-enriched transcription factors, such as CCAAT/enhancer binding protein, are essential for the maintenance of basal and GR-inducible rat hepatic SULT2A expression. The transcriptional control of rodent and human hepatic SULT2A expression is subject to trans-activation by the environmental sensor, pregnane X receptor (PXR). IR0 (inverted repeat with zero intervening bases) motifs located in the 5'-flanking regions of rodent SULT2A genes are required for transcriptional activation by PXR and other nuclear receptors, including constitutive androstane receptor, farnesoid X receptor and vitamin D receptor. Peroxisome proliferator activated receptor alpha (PPARalpha) mediates the induction of human, but not rat, hepatic SULT2A gene transcription, thus implicating a role for fatty acids as endogenous regulators of hepatic sulfonation in humans. This review focuses on the xenobiotic sensors and transcription factor systems that regulate sulfotransferase gene expression.

Random monoallelic expression of three genes clustered within 60 kb of mouse t complex genomic DNA.

Mammals achieve gene dosage control by (1) random X-chromosome inactivation in females, (2) parental origin-specific imprinting of selected autosomal genes, and (3) random autosomal inactivation. Genes belonging to the third category of epigenetic phenomenon are just now emerging, with only six identified so far. Here we report three additional genes, Nubp2, Igfals, and Jsap1, that show 50%-methylated CpG sites by Southern blot analyses and primarily monoallelic expression in single-cell allele-specific RT-PCR analysis of bone marrow stromal cells and hepatocytes. Furthermore, we show that, in contrast to X inactivation, alleles can switch between active and inactive states during the formation of daughter cells. These three genes are the first in their category to exist as a tight cluster, in the proximal region of mouse chromosome 17, providing a thus far unique example of a region of autosomal random monoallelic expression.

Transcriptional regulation of rat hepatic aryl sulfotransferase (SULT1A1) gene expression by glucocorticoids.

The 5'-flanking region [1892 base pairs (bp)] of the rat aryl sulfotransferase (SULT1A1) gene was cloned and the cis-acting sequences involved in glucocorticoid-inducible SULT1A1 gene transcription were characterized. SULT1A1 promoter and 5'-flanking sequences lacked a TATA box and a consensus glucocorticoid response element. Using a 5'-rapid amplification of cDNA ends approach, four SULT1A1 transcription start sites were identified. Transient transfection studies with SULT1A1-5':luciferase reporter constructs in primary cultured rat hepatocytes revealed that treatment with the potent glucocorticoid dexamethasone (10(-9)-10(-5) M) produced concentration-dependent increases in luciferase activity in constructs containing from 1892 to 119 bp of the SULT1A1 5'-flanking region. Relative to the most upstream SULT1A1 transcription start site, the minimal cis-acting sequences that were required for dexamethasone-inducible SULT1A1 expression were located between -84 and -69 bp. Treatment of transfectants with a panel of steroids, including dexamethasone, triamcinolone acetonide, hydrocortisone, dihydrotestosterone, 17beta-estradiol, and pregnenolone-16alpha-carbonitrile, revealed that steroid-inducible SULT1A1 gene expression was specific for glucocorticoid-class steroids. Concentration-response studies, coupled with a robust inhibition of glucocorticoid-inducible SULT1A1-5':luciferase reporter activity by antiglucocorticoid/antiprogestin RU-486, recapitulated earlier findings on endogenous SULT1A1 gene expression and implicated a major role for the glucocorticoid receptor transcription factor in the regulation of glucocorticoid-inducible SULT1A1 gene expression.

Sex-dependent regulation by dexamethasone of murine hydroxysteroid sulfotransferase gene expression.

To determine whether glucocorticoid-inducible expression of hepatic hydroxysteroid sulfotransferase is conserved in mouse, the effects of dexamethasone (DEX) on hydroxysteroid sulfotransferase (mSULT2A) gene expression were investigated in primary cultured hepatocytes prepared from C57BL/6J mice. In female mouse hepatocytes, DEX (10(-7) and 10(-5) M, respectively) produced 8.2- and 17.8-fold increases, respectively, in the amounts of mSULT2A mRNA relative to control. By contrast, mSULT2A mRNA levels were undetectable in male mouse hepatocytes. Female-predominant mSULT2A mRNA expression was also observed in liver samples from C57BL/6J and three other mouse strains. Treatment of primary cultured female mouse hepatocytes with dihydrotestosterone in the presence of DEX suppressed mSULT2 expression. Transfection of primary cultured male or female mouse hepatocytes with a rat SULT2-40/41 reporter construct revealed that hepatocytes of both sexes have sufficient machinery to achieve DEX-inducible SULT2 transcription. However, treatment with the potent histone deacetylase inhibitor trichostatin A failed to elicit mSULT2A expression in male mouse hepatocytes.

Post-transcriptional regulation of rat CYP2E1 expression: role of CYP2E1 mRNA untranslated regions in control of translational efficiency and message stability.

Altered expression of hepatic CYP2E1 by xenobiotic or physiological stimuli is largely mediated through post-transcriptional mechanisms that may include altered CYP2E1 mRNA translation and/or protein degradation. Examination of the polyribosomal distribution of rat hepatic P450 mRNAs indicated that, whereas nearly all of the CYP2B, CYP3A, and CYP4A mRNAs were recovered in the polysomal fractions, indicating active translation, approximately 30-40% of CYP2E1 mRNA was not associated with polysomes and therefore not actively engaged in protein synthesis. To examine the CYP2E1 mRNA molecule for sequences that might affect its translational efficiency, a series of CYP2E1 recombinant RNAs (rcRNAs) with modified 5' or 3' untranslated regions (UTRs) was translated in vitro using the rabbit reticulocyte lysate system. Deletion of most of the CYP2E1 5' UTR, which was predicted to contain secondary structure, increased in vitro CYP2E1 protein synthesis. Polysomal distribution analyses of 5'-modified rcRNAs demonstrated that, as seen for hepatic CYP2E1 mRNA, a substantial fraction of each CYP2E1 rcRNA was not associated with polysomes. The polysomal distribution analyses of the CYP2E1 rcRNAs also confirmed that the observed changes in CYP2E1 protein synthesis were associated with altered ribosomal loading. Deletion of the poly(A) tail, and partial or complete deletion of the 3' UTR, decreased CYP2E1 protein synthesis. These changes in protein synthesis were accompanied by increased degradation of the CYP2E1 rcRNAs. Incubation with translational inhibitors, but not increased levels of RNase inhibitor, decreased the degradation of the rcRNAs during in vitro translation. In conclusion, these studies suggest that secondary structure in the 5' UTR of CYP2E1 mRNA is at least partially responsible for the inefficient translation of this mRNA. The poly(A) tail and sequences contained within the 3' UTR appear to be important for protecting CYP2E1 mRNA from RNase activity associated with the translation machinery.

Identification of a ubiquitination-Target/Substrate-interaction domain of cytochrome P-450 (CYP) 2E1.

Cytochrome P-450 (CYP) 2E1, the alcohol-inducible form of CYP, metabolizes a wide variety of endogenous substrates, therapeutic agents, protoxicants, and procarcinogens. CYP2E1 levels are post-transcriptionally elevated in response to certain xenobiotic inducers (e.g., pyridine), and proposed mechanisms include increased translational efficiency and protection of the enzyme from ubiquitin-dependent proteolysis. Molecular modeling of a predicted cytosolic domain of CYP2E1 resulted in identification of a putative ubiquitination-target/substrate-interaction structure (residues 317-340). An affinity-purified antibody reactive to this domain quenched CYP2E1 ubiquitination in a concentration-dependent manner in a rabbit reticulocyte lysate-based ubiquitination assay. The same antibody also inhibited rat liver microsomal chlorzoxazone 6-hydroxylase activity, a marker of CYP2E1 catalytic activity, in an equivalent concentration-dependent manner. These two observations suggest an association between the CYP2E1 cytosolic domain involved in catalysis and its serving as a target for ubiquitination. Thus, these results provide a plausible mechanistic explanation for the observation that substrate binding shields the CYP2E1 protein from turnover by the ubiquitin-proteasome-dependent machinery.

Regulation of rat hepatic hydroxysteroid sulfotransferase (SULT2-40/41) gene expression by glucocorticoids: evidence for a dual mechanism of transcriptional control.

Glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2-40/41) gene transcription was investigated in primary cultured rat hepatocytes transiently transfected with a series of SULT2-40/41 5'-flanking region-luciferase reporter constructs, with emphasis on examining the functional role of an inverted repeat-0 nuclear receptor motif (IR0). Treatment of transfected cultures with any of four glucocorticoids activated luciferase expression from a construct containing 1938 base pairs (bp) of the SULT2-40/41 gene 5'-flanking sequence, whereas deletion of bp -227 to -158 (containing the IR0 motif) largely abolished the effect. On closer analysis, treatment of hepatocyte cultures with either of the potent glucocorticoids dexamethasone [strong cytochrome P-450 3A (CYP3A) inducer] or triamcinolone acetonide (weak CYP3A inducer) produced dose-dependent increases in luciferase activity when hepatocytes were transiently transfected with a construct containing as little as 158 bp of 5'-flanking sequence or containing a mutated IR0 motif. The dexamethasone dose-dependent increase in luciferase activity continued through a dose of 10(-6) M when the transfected construct contained the IR0 motif, but was maximal at 10(-7) M when the transfected construct lacked the IR0 motif. In contrast, triamcinolone acetonide-induced luciferase activity was maximal at a dose of 10(-7) M, irrespective of the presence or absence of the IR0 motif. Coincubation of transfected hepatocytes with 10(-8) M dexamethasone and the antiglucocorticoid RU486 inhibited luciferase expression. Luciferase induction by the prototypical CYP3A inducer pregnenolone 16alpha-carbonitrile was restricted to constructs containing the IR0 motif. These data suggest that glucocorticoid-inducible SULT2-40/41 gene expression occurs through a dual mechanism, whose components possibly involve the glucocorticoid receptor and the pregnane X receptor.

Regulation of rat hepatic cytochrome P450 expression by sterol biosynthesis inhibition: inhibitors of squalene synthase are potent inducers of CYP2B expression in primary cultured rat hepatocytes and rat liver.

The effects of treatment with squalestatin 1, a potent inhibitor of squalene synthase, the first committed enzyme of sterol biosynthesis, were examined on cytochrome P450 expression in primary cultured rat hepatocytes and rat liver. Incubation of cultured hepatocytes with squalestatin 1 caused marked accumulations (maximal elevations that were approximately 25-100% of phenobarbital-elicited increases) of CYP2B mRNA and immunoreactive protein but not of CYP1A, CYP3A, or CYP4A. Squalestatin 1 treatment increased CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA content in hepatocyte cultures with comparable potencies (ED50 = 5.0 and 18 nM, respectively), and significantly induced CYP2B (mRNA, immunoreactive protein, and pentoxyresorufin O-dealkylase activity) in the livers of treated rats, producing maximal increases at a dose of 25 mg/kg/day that were approximately 32-87% of phenobarbital-induced increases. Squalestatin 1 treatment induced both CYP2B1 and CYP2B2 and activated reporter gene expression in cultured hepatocytes transiently transfected with a plasmid containing approximately 2.4 kb of CYP2B1 gene 5'-flanking region or containing a previously described phenobarbital-responsive region. Coincubation of cultured hepatocytes with 25-hydroxycholesterol suppressed squalestatin 1-mediated CYP2B and 3-hydroxy-3-methylglutaryl coenzyme A mRNA induction with approximately the same potency. Treatment of cultures with SQ-34919, a structurally distinct squalene synthase inhibitor, produced the same selective CYP2B mRNA induction as did squalestatin 1. These results suggest that inhibition of hepatic sterol synthesis activates processes that culminate in increased CYP2B gene transcription.

Differential regulation of individual sulfotransferase isoforms by phenobarbital in male rat liver.

Xenobiotics that induce the cytochromes P450 also produce changes in rat hepatic sulfotransferase (SULT) gene expression. In the present study, male Sprague-Dawley rats were treated for 3 consecutive days with doses of phenobarbital (PB) that induce cytochrome P450 2B1/2 expression. The effects of PB treatment on hepatic aryl SULT (SULT1) and hydroxysteroid SULT (SULT2) mRNA and immunoreactive protein levels and on mRNA expression of individual SULT1 and SULT2 enzyme isoforms were characterized. PB suppressed SULT1A1 mRNA levels, increased the expression of the SULT-Dopa/tyrosine isoform, and did not produce significant changes in SULT1C1 and SULT1E2 mRNA expression. In rats injected with the highest test dose of PB (100 mg/kg), hepatic SULT1A1 mRNA levels were decreased to approximately 42% of control levels and SULT-Dopa/tyrosine mRNA levels were increased to approximately 417% of vehicle-treated control levels. Like the SULT1 subfamily, individual members of the SULT2 gene subfamily were differentially affected by PB treatment. PB (35, 80, and 100 mg/kg) suppressed SULT20/21 mRNA expression to approximately 61, approximately 30, and approximately 41% of vehicle-treated control levels, respectively. In contrast, SULT60 mRNA levels were increased to approximately 162% of control levels and SULT40/41 mRNA levels were increased to approximately 416% of vehicle-treated control levels in rats treated with 100 mg/kg PB. These studies support a complex role for PB-mediated effects on the SULT multigene family in rat liver. Because individual SULT1 and SULT2 enzyme isoforms are known to metabolize a variety of potentially toxic substrates, varied responses to PB among members of the SULT multigene family might have important implications for xenobiotic hepatotoxicity.

Comparative pharmacodynamics of CYP2B induction by DDT, DDE, and DDD in male rat liver and cultured rat hepatocytes.

In this study the pharmacodynamics were characterized of rat hepatic cytochrome P-450 2B (CYP2B) induction by the pesticide DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane] and its metabolites DDE [1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene], which is bioretained, and DDD [1,1-dichloro-2,2-bis(p-chlorophenyl)ethane], which is metabolized further and therefore less prone to bioaccumulate. DDT, DDE, and DDD were each found to be pure phenobarbital-type cytochrome P-450 inducers in the male F344/NCr rat, causing induction of hepatic CYP2B and CYP3A, but not CYP1A. The ED50 values for CYP2B induction (benzyloxyresorufin O-dealkylation) by DDT, DDE, and DDD were, respectively, 103, 88, and > or = 620 ppm in diet (14 d of exposure). The efficacies (Emax values) for induction of benzyloxyresorufin O-dealkylation by DDT, DDE, and DDD were 24-, 22-, and > or = 1-fold, respectively, compared to control values. The potencies of the three congeners for CYP2B induction appeared also to be similar, with EC50 values (based on total serum DDT equivalents) of 1.5, 1.8, and > or = 0.51 microM, respectively. The EC50 values based on DDT equivalents in hepatic tissue were 15, 16, and > or = 5.9 micromol/kg liver tissue, respectively. In primary cultures of adult rat hepatocytes, DDT, DDE, and DDD each displayed ability to induce total cellular RNA coding for CYP2B (ED50 values of 0.98, 0.83, and > or = 2.7 microM, respectively). These results suggest that DDT, DDE, and DDD each possess a high degree of intrinsic CYP2B-inducing ability for rat liver, despite marked differences in bioretention among the congeners.

Negative regulation by dexamethasone of fluvastatin-inducible CYP2B expression in primary cultures of rat hepatocytes: role of CYP3A.

Fluvastatin (Fluva), a synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, induces CYP2B1/2 in rat liver and primary cultured rat hepatocytes. However, the overall profile of CYP induction, which includes induction of CYP4A, suggests that Fluva is not a typical "phenobarbital (PB)-like" inducer. Several treatments affecting diverse cell signaling pathways have been reported to modify PB-inducible CYP2B expression in primary cultured rat hepatocytes. We examined the effects of selected treatments on the ability of Fluva to induce CYP2B1/2 mRNA. Only dexamethasone (Dex) produced effects on Fluva-inducible CYP2B1/2 mRNA expression that differed from those produced on PB-inducible CYP2B1/2 mRNA expression. Dex concentrations up to 10(-7) M of potentiated PB (10(-4) M)-mediated CYP2B1/2 mRNA induction, while higher Dex concentrations produced a progressive reduction in PB-induced CYP2B1/2 mRNA levels. By contrast, Dex concentrations up to 10(-8) M had no effect on Fluva (3 x 10(-5) M)-induced CYP2B1/2 mRNA levels, while Dex concentrations of 10(-7) M and higher markedly suppressed Fluva-mediated CYP2B1/2 mRNA induction. The concentrations of several glucocorticoids that produced suppression of Fluva-induced CYP2B1/2 mRNA levels were the same concentrations that induced CYP3A mRNA. Treatment with pregnenolone 16 alpha-carbonitrile also produced a concentration-dependent suppression of Fluva-induced CYP2B1/2 mRNA levels. Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA was concentration-dependently reversed when hepatocytes were cotreated with troleandomycin, a selective CYP3A inhibitor. The amounts of Fluva detected in culture medium and cells were reduced significantly when hepatocytes were incubated with Dex. However, Dex-mediated suppression of Fluva-induced CYP2B1/2 mRNA expression was not overcome when hepatocytes were incubated with Fluva concentrations greater than 3 x 10(-5) M, suggesting that mechanisms other than CYP3A-catalyzed metabolism may contribute to Dex-mediated suppression of Fluva-induced CYP2B1/2 expression.

Regulation of cytochrome P450 expression by inhibitors of hydroxymethylglutaryl-coenzyme A reductase in primary cultured rat hepatocytes and in rat liver.

It was previously demonstrated that treatment of primary cultured rat hepatocytes with lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, induced the mRNAs for several cytochromes P450 (P450s), including CYP2B1/2, CYP3A1/2, and CYP4A. In this study, we have compared the effects of lovastatin with those of three additional HMG-CoA reductase inhibitors (simvastatin, pravastatin, and the structurally dissimilar drug fluvastatin) on P450 expression in primary cultured rat hepatocytes, and we have also characterized the effects of in vivo treatment with fluvastatin on P450 expression in rat liver. Treatment of cultured hepatocytes with lovastatin, simvastatin, or fluvastatin increased CYP2B1/2, CYP3A1/2, and CYP4A mRNA and immunoreactive protein levels over the dose range (3 x 10(-6) to 3 x 10(-5) M) required to increase the amount of HMG-CoA reductase mRNA. The increases in CYP2B1/2 levels produced by 3 x 10(-5) M fluvastatin treatment were larger than those produced by lovastatin or simvastatin treatment or by treatment with 10(-4) M phenobarbital. In contrast, treatment of cultured hepatocytes with 3 x 10(-5) M lovastatin, simvastatin, or fluvastatin increased CYP3A1/2 and CYP4A mRNA and immunoreactive protein to lower levels than those produced by treatment with 10(-5) M dexamethasone or 10(-4) M ciprofibrate. Treatment of cultured hepatocytes with pravastatin had little or no effect on the amount of any of the P450s examined, although this drug induced HMG-CoA reductase mRNA as effectively as did fluvastatin. Incubation of hepatocytes with 10(-4) M fluvastatin increased CYP1A1 mRNA to 67% of the level induced by treatment with 10(-5) M beta-naphthoflavone. Doses of 50 or 100 mg/ kg/day fluvastatin administered for 3 days to rats increased the hepatic levels of CYP2B1/2 and CYP4A mRNA and immunoreactive protein, although to much lower levels than those produced by treatment with phenobarbital or ciprofibrate, respectively. Treatment of rats with fluvastatin had no effect on hepatic levels of CYP3A1/2 mRNA or immunoreactive protein. However, treatment with 50 mg/kg/day fluvastatin induced CYP1A1 mRNA and protein. The effects of fluvastatin treatment on P450 expression seen in primary cultured rat hepatocytes thus largely recapitulated the effects seen in vivo. The differences in effects among the HMG-CoA reductase inhibitors suggest that simple inhibition of HMG-CoA reductase cannot explain all of the effects of these drugs on P450 expression.

Regulation of rat hepatic sulfotransferase gene expression by glucocorticoid hormones.

Because hormones have been implicated in the molecular regulation of the sulfotransferase multigene family, the effects of glucocorticoid and antiglucocorticoid hormones on rat hepatic hydroxysteroid sulfotransferase-a and aryl sulfotransferase IV gene expression were investigated in vivo and in primary rat hepatocyte culture. Adult male Sprague-Dawley rats were treated for three consecutive days with 2% Tween-20 vehicle, or 100 mg/kg of dexamethasone, betamethasone, hydrocortisone, or triamcinolone acetonide. Betamethasone and triamcinolone acetonide significantly increased hepatic aryl sulfotransferase IV mRNA to levels that were approximately 252% and approximately 452% of control, respectively. Dexamethasone significantly increased hydroxysteroid sulfotransferase-a mRNA and protein to levels that were approximately 150% and approximately 316% of control, respectively. In contrast, in vivo treatment with hydroxysteroid sulfotransferase-a substrate dehydroepiandrosterone significantly decreased hydroxysteroid sulfotransferase-a mRNA levels (by approximately 55% relative to control). To determine if glucocorticoid mediated changes in sulfotransferase expression occurred as a result of direct effects on the hepatocyte, studies were performed in primary rat hepatocyte culture. Triamcinolone acetonide and betamethasone increased sulfotransferase mRNA expression in hepatocyte culture and hydrocortisone proved to be a less effective inducer. Effects of glucocorticoids on sulfotransferase gene expression were compared with glucocorticoid effects on tyrosine aminotransferase expression, a gene known to be regulated by a classical glucocorticoid receptor-mediated mechanism. Dexamethasone produced maximal increases in aryl sulfotransferase IV and tyrosine aminotransferase mRNA levels when added to culture medium at a concentration of 10(-7) M, whereas hydroxysteroid sulfotransferase-a mRNA levels continued to increase through a dexamethasone concentration of 10(-5) M. Treatment of hepatocytes with the antiglucocorticoid RU-486 (10(-5) M) inhibited dexamethasone-stimulated aryl sulfotransferase and tyrosine amino-transferase mRNA expression by approximately 48% and approximately 35%, respectively, but had less effect on hydroxysteroid sulfotransferase mRNA expression. These results suggest that glucocorticoids regulate rat hepatic aryl sulfotransferase IV and hydroxysteroid sulfotransferase-a via classical glucocorticoid receptor-mediated and non-classical mechanisms, respectively.

Xenobiotic-enhanced expression of cytochrome P450 2E1 and 2B1/2B2 in primary cultured rat hepatocytes.

Investigation of the posttranscriptional mechanisms involved in the xenobiotic-mediated enhancement of cytochrome P450 2E1 (CYP2E1) expression has been limited by a lack of a functional primary hepatocyte cell culture system. We examined the effects of ciprofibrate (CIPRO) and pyridine (PYR) treatment on the expression of CYP2E1, P450 4A (CYP4A), and P450 2B (CYP2B) in primary rat hepatocytes cultured on Vitrogen or Matrigel substratum and in the presence of Chee's medium. Cells were cultured for 72 hr or longer before initiation of treatment. Northern blot analyses indicated that 24-hr CIPRO treatment enhanced the expression of CYP4A, and CYP2B mRNA in a concentration-dependent manner, with maximal induction of CYP2E1 mRNA (2- to 3-fold) and CYP4A1 mRNA (up to approximately 15-fold) monitored at 30-300 microM CIPRO. Maximal CYP2B mRNA levels (7- to 8-fold) were monitored at 300-1000 microM CIPRO. Treatment of hepatocytes for 24, 48, and 72 hr with 30 microM CIPRO showed progressive increases in CYP2B and CYP4A mRNA levels, with approximately 13- and 60-fold elevations in the respective mRNAs occurring at 72 hr posttreatment. In contrast, CYP2E1 mRNA levels were maximally elevated between 2- and 3-fold at both 24 and 48 hr and were returning to basal levels by 72 hr. Western blot analyses revealed that 24-hr PYR (25 mM) treatment of CIPRO-treated cells, in the absence of any further increase in CYP2E1 mRNA levels, increased CYP2E1 protein levels approximately 6- to 8-fold. PYR treatment also increased CYP2B mRNA and CYP2B1/2B2 protein levels approximately 16-fold relative to cells treated only with CIPRO.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the poly(A) tail lengths of a single mRNA species in total hepatic RNA.

Poly(A) tail length is important in the stability and translation of mRNA. We describe procedures for the rapid and reproducible analysis of poly(A) tail length of a single mRNA species contained in a sample of total hepatic RNA. A short 3' fragment of a specific mRNA is prepared by RNase H digestion of the targeted mRNA region annealed to a short DNA oligonucleotide. The length of the poly(A) tail of the 3' fragment is then determined by running the sample on a polyacrylamide gel, by electrophoretic transfer, by probing with a radiolabeled cDNA and by comparing the size of the detected region with a specific RNA ladder or a DNA ladder.

Comparative analysis of cytochrome P4503A induction in primary cultures of rat, rabbit, and human hepatocytes.

We previously demonstrated that induction of hepatic cytochrome P4503A (CYP3A) immunoreactive protein is a response in rats, but not rabbits, treated with the antiglucocorticoid, pregnenolone 16 alpha-carbonitrile and in rabbits, but not rats, treated with rifampicin. These striking interspecies differences in response to CYP3A inducers prompted us to compare the effects of a variety of agents on CYP3A expression in primary cultures of hepatocytes from rats, rabbits, and humans, maintained under nearly identified conditions on Matrigel. We used treatment with dexamethasone, the most effective inducer of CYP3A mRNA and CYP3A immunoreactive protein in cultures of rat hepatocytes, to define the 100% response. As expected from their effects in vivo, incubations of cultures with medium containing pregnenolone 16 alpha-carbonitrile or rifampicin induced CYP3A mRNA to high levels exclusively in rat hepatocytes or rabbit hepatocytes, respectively. Pregnenolone 16 alpha-carbonitrile treatment also did not induce CYP3A immunoreactive protein in rabbit hepatocytes, although rifampicin treatment did increase CYP3A immunoreactive protein levels in rat hepatocyte cultures. Additions of phenobarbital to the cultures induced CYP3A mRNA and CYP3A immunoreactive protein to a greater extent in rabbit hepatocytes (94-108% of the dexamethasone response) than in rat hepatocytes (38-57% of the dexamethasone response). In primary cultures of human hepatocytes, dexamethasone and phenobarbital treatments induced CYP3A mRNA (> or = 4.4- and 1.9-fold, respectively, over the amounts of CYP3A mRNA in incubated control cultures).(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

Regulation of phenobarbital-inducible cytochrome P450 2B1/2 mRNA by lovastatin and oxysterols in primary cultures of adult rat hepatocytes.

Lovastatin (LOVA) is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase widely used in clinical practice. We treated primary cultures of adult rat hepatocytes, maintained in a minimal, serum-free medium on Matrigel, a reconstituted basement membrane, with this drug, and found that the amounts of P450 2B2 mRNA detected on Northern blots were increased at the same doses (10(-5) to 3 x 10(-5) M) required for induction of HMG-CoA reductase mRNA, a gene known to be under oxysterol regulatory control. LOVA treatment produced selective effects increasing also the mRNA levels for P450s 2C6, 2C7, 3A1, and 4A1 but not for 1A1, 2A1/2, or NADPH-cytochrome P450 oxidoreductase. LOVA treatment increased the induction of 2B1/2 mRNA in cells cotreated with either phenobarbital (PB; 10(-4) M) or clotrimazole (CTZ; 10(-5) M), or of 3A1 mRNA in cells cotreated with PB (2 x 10(-3) M), but not dexamethasone (10(-5) M. LOVA treatment did not potentiate the induction of 1A1 or 4A1 mRNA in cells cotreated with beta-naphthoflavone (10(-5) M) or ciprofibrate (10(-4) M), respectively. In contrast to the potentiation of 2B1/2 mRNA induction produced by treatments with LOVA in combination with PB or CTZ, cotreatment of hepatocytes with PB and CTZ did not result in increased induction relative to that seen in cells treated with either agent alone. Treatment of hepatocyte cultures with either mevalonate (3 x 10(-4) to 3 x 10(-3) M), the immediate product of HMG-CoA reductase, or 25-hydroxycholesterol (10(-6) to 10(-5) M), a model oxysterol, resulted in dose-dependent suppression of 2B1/2 mRNA induction in cells treated with PB-like inducers. Taken together, our results demonstrate that LOVA is a unique inducer of P450 mRNA in cultured rat hepatocytes and implicate oxysterols as potential intracellular modulators of 2B1/2 induction. We conclude that endogenous metabolic factors including those related to cholesterol biosynthesis are critical in induction of liver cytochromes P450 2B1 and 2B2 by PB and "PB-like" agents.

Expression of multiple forms of cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on matrigel.

Freshly isolated rat hepatocytes rapidly lose their cytochrome P450 (P450) proteins and mRNAs, with no evidence of subsequent restoration, after placement into traditional systems of primary culture on type I collagen. We examined the patterns of expression of 10 constitutively expressed P450 mRNAs in rat hepatocytes cultured for up to 5 days on a matrix of Matrigel, a reconstituted basement membrane that allows inducible expression of some P450s, and compared these patterns with those seen in hepatocytes cultured on type I collagen (Vitrogen). mRNA for each P450 was detected on Northern blots in samples prepared from freshly isolated male rat hepatocytes, and the amount of each mRNA decreased markedly during the first 2 days of culture in cells maintained on either matrix (decreases of 53-97% on Matrigel and 62 to > 99% on Vitrogen), in contrast to the level of NADPH-P450 oxidoreductase mRNA, which increased during this interval. On subsequent days, hepatocytes cultured on Matrigel expressed the individual P450 mRNAs in one of the following four patterns. 1) P450 1A2, 2A2, 2E1, and 3A1/2 mRNAs remained low throughout the 5-day culture period. 2) 2A1 mRNA increased between days 2 and 4 but then decreased on day 5. 3) 2B1/2, 2C6, 2C7, and 4A1 mRNAs increased continually between days 2 and 5, 4) The total mRNAs detected with a 2D probe remained at constant levels between culture days 2 and 5. Our results show that rat hepatocytes cultured on Matrigel in serum-free medium spontaneously reexpress the mRNAs for several constitutive P450s in form-specific patterns, and they suggest that the Matrigel culture system will be useful for identifying the underlying regulatory mechanisms.