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R A Prough

Publications and source records attributed to R A Prough.

At least 19 recordsLinked to original sources
PubMed2001

7,12-Dimethylbenz[a]anthracene inhibition of steroid production in MA-10 mouse Leydig tumor cells is not directly linked to induction of CYP1B1.

Testosterone, which is essential for spermatogenesis, is synthesized in the Leydig cells of the testis. This study addresses whether male reproductive toxicity from exposure to polycyclic or polychlorinated aromatic hydrocarbons, such as 7,12-dimethylbenz[a]anthracene (DMBA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), may be due to direct effects on Leydig cell function. Using a cell-based assay, the effects of TCDD, benz[a]anthracene (BA), and DMBA on steroid production and cytochrome P4501B1 (CYP1B1) expression in treated MA-10 mouse Leydig tumor cells or primary cultures of rat Leydig cells was determined. (Bu)(2)cAMP-stimulated steroid production was inhibited approximately 25% and approximately 80% by DMBA treatment of MA-10 cells and rat Leydig cells, respectively, while BA or TCDD were without effect. Conversely, male Sprague-Dawley rats treated with TCDD displayed a 75% decrease in serum testosterone levels, while DMBA-treated rats had circulating testosterone levels comparable to control rats. Injection of human chorionic gonadotropin (hCG) 1 h prior to euthanasia restored testosterone levels in TCDD-treated rats to 79% of the hCG-stimulated levels in control rats. Steady-state levels of CYP1B1 mRNA, as detected by RT-PCR, are present in the MA-10 cells and treatment with TCDD, BA, DMBA, or the cAMP analog (Bu)(2)cAMP induced CYP1B1 mRNA expression levels. CYP1B1 was constitutively expressed in rat testis, adrenal, liver, and kidney tissues while CYP1A1 was undetectable. TCDD treatment induced CYP1B1 expression in the adrenal and liver and CYP1A1 in the kidney and liver. DMBA treatment induced only CYP1A1 levels in kidney and liver. In sum, DMBA or a reactive DMBA metabolite, but not TCDD, has a direct effect on steroidogenesis in isolated Leydig cells. CYP1B1 expression levels, however, cannot be directly correlated to potential in vitro or in vivo toxic effects of TCDD or DMBA.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Short heterodimer partner (SHP) orphan nuclear receptor inhibits the transcriptional activity of aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT).

SHP (short heterodimer partner) is an orphan nuclear receptor lacking a DNA binding domain that interacts with nuclear receptors (NR) including thyroid receptor (TR), retinoic acid receptors (RAR and RXR), and estrogen receptors alpha and beta (ERalpha and ERbeta). SHP acts as a negative regulator of these receptors by inhibiting DNA binding and transcriptional activation. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) binds to arylhydrocarbon receptor (AHR), activating the AHR/AHR nuclear translocator (ARNT) heterodimer. We investigated the physical and functional interaction of SHP with AHR/ARNT. In RL95-2 human endometrial carcinoma cells, SHP inhibited TCDD-stimulated reporter activity from the AHR-responsive CYP1A1 and UGT1A6 gene promoters in a concentration-dependent manner. In GST pull-down assays, ARNT interacted directly with SHP in vitro, but AHR did not interact with GST-SHP. SHP inhibited AHR/ARNT-DNA binding in vitro. These results identify ARNT as a novel SHP target. We speculate a role for SHP in the suppression of agonist-activated AHR/ARNT activity.

Animals↗
PubMed2001

Metabolism of DHEA by cytochromes P450 in rat and human liver microsomal fractions.

Administration of dehydroepiandrosterone (DHEA) to rodents produces many unique biological responses, some of which may be due to metabolism of DHEA to more biologically active products. In the current study, DHEA metabolism was studied using human and rat liver microsomal fractions. In both species, DHEA was extensively metabolized to multiple products; formation of these products was potently inhibited in both species by miconazole, demonstrating a principal role for cytochrome P450. In the rat, use of P450 form-selective inhibitors suggested the participation of P4501A and 3A forms in DHEA metabolism. Human liver samples displayed interindividual differences in that one of five subjects metabolized DHEA to a much greater extent than the others. This difference correlated with the level of P4503A activity present in the human liver samples. For one subject, troleandomycin inhibited hepatic microsomal metabolism of DHEA by 78%, compared to 81% inhibition by miconazole, suggesting the importance of P4503A in these reactions. Form-selective inhibitors of P4502D6 and P4502E1 had a modest inhibitory effect, suggesting that these forms may also contribute to metabolism of DHEA in humans. Metabolites identified by LC-MS in both species included 16alpha-hydroxy-DHEA, 7alpha-hydroxy-DHEA, and 7-oxo-DHEA. While 16alpha-hydroxy-DHEA appeared to be the major metabolite produced in rat, the major metabolite produced in humans was a mono-hydroxylated DHEA species, whose position of hydroxylation is unknown.

Adult↗
PubMed2001

Molecular regulation of genes encoding xenobiotic-metabolizing enzymes: mechanisms involving endogenous factors.

It is widely recognized that xenobiotic-metabolizing enzymes play a fundamental role in the basic processes of carcinogenesis and toxicity on one hand, and chemoprevention and drug efficacy on the other. Realization that different factors can profoundly affect the expression of these enzymes at the genome level has resulted in an enhanced appreciation of the importance these genes play in our modern industrialized age. There continues to be rapid proliferation of studies addressing the molecular regulation of these genes. The discovery of common signal transduction pathways and transcription factors that dictate tissue and developmental-specific expression, as well as variation in expression within a given tissue, suggest that there may be significant interaction among these various regulatory systems. This report is a summary of a symposium that was part of the Structure, Function and Regulation of Cytochromes P450 and Xenobiotic Metabolizing Enzymes satellite meeting of the 2000 joint meeting of the American Society for Biochemistry and Molecular Biology, the American Society for Pharmacology and Experimental Therapeutics, the French Pharmacological Society, and the Pharmacological Society of Canada held in Boston, Massachusetts. This symposium brought together several speakers who addressed specific receptor-mediated signal transduction pathways involved in the regulation of xenobiotic-metabolizing enzymes, as well as other molecular mechanisms whereby endogenous factors are involved in controlling tissue- and developmental-specific expression.

Enzymes↗
PubMed2001

Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: involvement of both the glucocorticoid and pregnane X receptors.

Glucocorticoids regulate the rat glutathione S-transferase A2 (GSTA2) gene in a biphasic manner in cultured hepatocytes that repress gene expression at low concentration (10--100 nM) but induce gene expression at high concentration (>1 microM). High concentrations of the glucocorticoid receptor (GR) antagonist RU38486 (5--10 microM) also induced the expression of GSTA2. These effects were reproduced in HepG2 cells transfected with a luciferase reporter containing 1.6 kilobase pairs of 5'-flanking sequence of GSTA2 and expression plasmids for either GR, pregnane X receptor (PXR) or a combination of both. Dexamethasone t-butylacetate (1 microM t-Bu-DEX) repressed gene expression between 60 to 75% when only GR was expressed. When PXR was expressed, both basal and t-Bu-DEX-dependent gene expression was increased over 2-fold, respectively. Biphasic regulation of gene expression was observed over a broad range of t-Bu-DEX concentrations when expression plasmids for both receptors were cotransfected. Other steroids of the pregnane class induced GSTA2 expression as expected for a PXR-dependent process. Because no canonical responsive element for the PXR-RXR alpha heterodimer was observed in the 5'-flanking region of the construct, deletion analysis was used to identify a pregnane responsive region between base pairs -700 and -683; this 20-bp region contains the antioxidant response element (ARE). When the ARE sequence was mutated, basal, t-butylhydroquinone- and 17 alpha-hydroxypregnenolone-inducible expression were all lost. These results suggest that PXR interacts with factors binding to the ARE to elicit the pregnane inductive response for GSTA2.

Animals↗
PubMed1999

Negative regulation of rat hepatic aldehyde dehydrogenase 3 by glucocorticoids.

The expression of the aldehyde dehydrogenase 3 gene is known to be controlled by multiple regulatory processes. In liver, inducible expression appears to be mediated by two AhRE sequences which allow regulation of this gene by xenobiotic compounds which are ligands for the Ah receptor (Takimoto et al., 1994; this work). Constitutive expression of ALDH3 in tissues such as the cornea also involves the -3,500 region which contains an AhRE (Boesch et al., 1996; Boesch et al, 1998). However, the constellation of transcription factors which appear to interact with the AhRE in constitutively expressing corneal cells does not include either the Ah receptor nor the prototypical ARNT protein (Boesch et al., 1998). For both inducible and constitutive ALDH3 expression the more distal 5' flanking region sequences appear to interact with more proximal regulatory elements. Of particular interest is the region near -1 kb which includes the GC (-930 to -910) and cAMP (-1057 to -991) responsive elements as well as the 2 NF1 sites (-916 to -815), all of which appear to act as negative modulators of ALDH3 expression. A second putative ALDH3 negative regulatory region lies even more distal than -3,500 bp. To date, this region has been little studied, but appears to be involved in regulating both inducible and constitutive ALDH3 expression. This region may also be responsible for some of the tissue-specificity of ALDH3 expression. With respect to the work described here, in both isolated hepatocytes and HepG2 cells, no consistent negative regulation by glucocorticoids was observed in the basal expression of ALDH3. This indicates that the mechanism of GC-mediated negative regulation involves direct interference with ALDH3 gene activation mediated by the Ah receptor. Our results suggest a complex interplay between multiple transcription factors, including the GC and Ah receptors, regulates the hepatic expression of the ALDH3 gene. Active recruitment of transcription factors needed for gene transactivation, amelioration of the actions of negative regulatory trans-acting factors or cis-acting elements and/or chromatin remodeling may be required for achieve proper regulation of the aldehyde dehydrogenase 3 gene.

Aldehyde Dehydrogenase↗
PubMed1999

The negative regulation of the rat aldehyde dehydrogenase 3 gene by glucocorticoids: involvement of a single imperfect palindromic glucocorticoid responsive element.

Glucocorticoids repressed the polycyclic aromatic hydrocarbon-dependent induction of Class 3 aldehyde dehydrogenase (ALDH3) enzyme activity and mRNA levels in isolated rat hepatocytes by more than 50 to 80%, with a concentration-dependence consistent with the involvement of the glucocorticoid receptor (GR). No consistent effect on the low basal transcription rate was observed. This effect of glucocorticoids (GC) on polycyclic aromatic hydrocarbon induction was effectively antagonized at the mRNA and protein level by the GR antagonist RU38486. The response was cycloheximide-sensitive, because the protein synthesis inhibitor caused a GC-dependent superinduction of ALDH3 mRNA levels. This suggests that the effects of GC on this gene are complex and both positive and negative gene regulation is possible. The GC-response was recapitulated in HepG2 cells using transient transfection experiments with CAT reporter constructs containing 3.5 kb of 5'-flanking region from ALDH3. This ligand-dependent response was also observed when a chimeric GR (GR DNA-binding domain and peroxisome proliferator-activated receptor ligand-binding domain) was used in place of GR in the presence of the peroxisome proliferator, nafenopin. A putative palindromic glucocorticoid-responsive element exists between -930 and -910 base pairs relative to the transcription start site. If this element was either deleted or mutated, the negative GC-response was completely lost, which suggests that this sequence is responsible, in part, for the negative regulation of the gene. Electrophoretic mobility shift analysis demonstrated that this palindromic glucocorticoid-responsive element is capable of forming a specific DNA-protein complex with human glucocorticoid receptor. In conclusion, the negative regulation of ALDH3 in rat liver is probably mediated through direct GR binding to its canonical responsive element.

5' Untranslated Regions↗
PubMed1999

Cytochrome P-450 mRNAs are modulated by dehydroepiandrosterone, nafenopin, and triiodothyronine.

Dehydroepiandrosterone (DHEA) is the only known naturally occurring compound that promotes peroxisome proliferation in rodent liver, and stimulates transcriptional induction of genes involved in lipid metabolism and peroxisomal beta-oxidation. Therefore, we examined mRNA for several such genes in rat liver, specifically acyl-CoA oxidase and the cytochromes P-450 (CYP4A1, CYP4A3, and CYP3A23), after 5 to 6 day treatments with either DHEA, or nafenopin, a known peroxisome proliferator. Acyl-CoA oxidase and CYP4A1 were induced nearly identically by DHEA and nafenopin, with induction being more pronounced in female rats. However, CYP3A23 was induced only by DHEA, suggesting an induction mechanism independent of the peroxisome proliferator activated receptor. Previously, we observed triiodothyronine (T3) suppression of peroxisome proliferator induced CYP4As and we sought to determine whether CYP3A23 might be regulated in a different manner. T3 was found to also suppress DHEA-dependent induction of CYP3A23. CYP4A2 expression in kidney was also negatively regulated by T3. To characterize a putative negative thyroid hormone response element (nTRE) in the 5' flanking region of this gene, a luciferase reporter gene containing a rat CYP4A2 flanking sequence extending to -1865 bp was transfected into HepG2 cells along with human thryroid hormone receptor expression vector. Expression of luciferase activity was unaffected by T3, suggesting the absence of a functional nTRE within this portion of CYP4A2. These data demonstrate gene regulatory activity by DHEA different from that of nafenopin, and a suppressive effect of T3, consistent with indirect regulatory mechanisms not involving an nTRE.

5' Untranslated Regions↗
PubMed1998

Purification and characterization of hamster liver microsomal 7alpha-hydroxycholesterol dehydrogenase. Similarity to type I 11beta-hydroxysteroid dehydrogenase.

While studying the bile acid synthetic pathway of hamsters, we discovered an NADP+-dependent liver microsomal 7alpha-hydroxycholesterol dehydrogenase (7alpha-HCD) activity that was not observed in rat liver microsomal fractions. The hamster liver microsomal 7alpha-HCD was purified to homogeneity using 2', 5'-ADP and cholic acid-agarose affinity chromatography. 7alpha-HCD displayed a molecular weight of approximately 34,000 on SDS-polyacrylamide gel electrophoresis; it is an intrinsic membrane protein of the hamster liver endoplasmic reticulum and exists as a multimeric aggregate in pure form. Partial N-terminal amino acid sequence analysis showed that 7alpha-HCD had high sequence similarity to human 11beta-hydroxysteroid dehydrogenase (11beta-HSD; 24/30 amino acid identity). The Km values for corticosterone and 7alpha-hydroxycholesterol were 1.2 and 1.9 microM, respectively, for purified 7alpha-HCD; both reactions displayed identical Vmax values (approximately 170 nmol/min/mg of protein). The IC50 of carbenoxolone, a competitive inhibitor of 11beta-HSD, was 75 nM for 7alpha-hydroxycholesterol dehydrogenation and 210 nM for corticosterone dehydrogenation. The tissue-specific expression in hamster was as follows: adrenal >/= liver > kidney > testis >> brain > lung. Microsomal 7alpha-HCD is uniquely expressed in hamster liver and to some extent in human liver but not in rat liver. Western blot analysis with two antibodies elicited against an N-terminal peptide of the human 11beta-HSD and purified hamster liver 7alpha-HCD, respectively, suggested the presence of multiple forms of 7alpha-HCD in hamster liver, most likely due to the existence of a family of 11beta-HSD proteins. Since 7-oxocholesterol is a potent inhibitor of cholesterol 7alpha-hydroxylase, alternative mechanisms for regulation of bile acid synthesis may exist in human and hamster liver due to production of this metabolite and its potential as an oxysterol.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1998

Modulation of methylnitrosourea-induced breast cancer in Sprague Dawley rats by dehydroepiandrosterone: dose-dependent inhibition, effects of limited exposure, effects on peroxisomal enzymes, and lack of effects on levels of Ha-Ras mutations.

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, is an effective chemopreventive agent in the N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor model. Dietary DHEA (5-600 ppm; 600 mg/kg diet) was administered beginning 1 week before MNU and administered continually throughout the duration of the experiment. The highest dose of DHEA (600 ppm) significantly decreased tumor incidence from 95 to 45% and increased tumor latency and decreased tumor multiplicity from 4.1 to 0.5 tumors/rat. Lower doses of DHEA (5, 24, and 120 ppm) were also effective, decreasing tumor multiplicity by 28, 40, and 55%, respectively, increasing tumor latency in a dose-dependent manner but only minimally affecting final tumor incidence. DHEA in the diet caused a dose-dependent increase in serum levels of DHEA. The 120-ppm dietary dose of DHEA resulted in serum levels of DHEA of approximately 42 pmol/ml levels, similar to those seen in young humans. When we examined whole mounts of mammary glands derived from rats exposed to higher levels of DHEA (600 ppm), we observed a striking increase in lobular development. The doses of DHEA used in these studies (< or =600 ppm) had minimal effects on the induction of fatty acid CoA synthetase, a peroxisome-associated enzyme. In contrast, a dose of 2000 ppm substantially increased levels of peroxisome-associated fatty acid CoA synthetase. The varied and striking efficacy of DHEA was achieved in the absence of any significant effect on body weight gain in the treated rats. Furthermore, tumors from rats treated with MNU alone or rats treated with MNU plus DHEA were examined for the presence of mutations in the Ha-Ras oncogene. There was a slight decrease in the percentage of tumors bearing Ha-Ras mutations in tumors derived from MNU-control rats as contrasted with tumors from MNU-DHEA (120 and 600 ppm)-treated rats. Based on the striking chemopreventive efficacy of continual exposure to DHEA, we examined the effects of more limited exposure to DHEA. Rats were treated with DHEA for a period of 7 weeks immediately before and after MNU injection. Rats were then placed on the control diet for the ensuing 15 weeks. Even this limited exposure to DHEA for a period of 7 weeks profoundly decreased final tumor incidence and multiplicity. Additionally, we examined the effects of intermittent dosing with DHEA. Rats were treated alternatively at 3-week intervals either with diet containing DHEA or with control diet. It was found that this intermittent dosing with DHEA also substantially inhibited the formation of mammary tumors.

Animals↗
PubMed1998

Negative regulation of the rat glutathione S-transferase A2 gene by glucocorticoids involves a canonical glucocorticoid consensus sequence.

Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathione S-transferase Ya1 gene (gstA2) in isolated rat hepatocytes and rat liver in vivo. Transient transfection experiments with HepG2 cells were used to identify GC-responsive elements (GREs). With cotransfected GC receptor, chloramphenicol acetyltransferase (CAT) constructs containing a palindromic GRE (pGRE) and three GRE hexanucleotide half-sites between -1.6 and -1.1 kb of the 5'-flanking region of gstA2 were repressed >50% by GC when induced with polyaromatic hydrocarbon. This pGRE, if either mutated or deleted, significantly reduces GC responsiveness of the gene to 20-30%; no effect of GC was observed with CAT constructs containing -1.15 kb of the 5'-flanking region. The dexamethasone concentration dependence of the repression was consistent with involvement of the GC receptor and was antagonized by RU38486. Electrophoretic mobility shift assays demonstrated that pGRE formed a specific DNA/protein complex, which was prevented by the addition of excess unlabeled or mouse mammary tumor virus GRE but not by unrelated or mutated gstA2 GRE double-stranded oligonucleotides. This complex was supershifted by incubation of nuclear extracts containing GC receptor with anti-GC receptor globulins. Constructs containing multiple copies of pGRE sequence were either nonresponsive or positively responsive (three copies) to GC. Luciferase constructs containing -1.62 to -1.03 kb of the 5'-flanking region also were regulated positively by GC. Chimeric GC-peroxisome proliferator activated receptor activated the constructs that were positively responsive to GC but did not mediate the negative effect in constructs containing 1.6 kb of 5'-flanking region. We conclude that pGRE and half-site GREs of gstA2 participate in regulation of this gene; however, a second unidentified responsive element must exist between -1.03 and -0.164 kb, resulting in repression of gstA2 expression.

Animals↗
PubMed1997

cAMP-dependent negative regulation of rat aldehyde dehydrogenase class 3 gene expression.

We investigated the inhibitory effects of intracellular cyclic adenosine monophosphate (cAMP) levels in regulating class 3 aldehyde dehydrogenase (aldh3) gene expression using cultures of primary rat hepatocytes and transient transfection experiments with HepG2 cells. In addition to regulation by an Ah receptor-dependent mechanism, expression of many members of the Ah gene battery have been shown to be negatively regulated. As was seen for the cytochrome P450 (cyp1A1) gene, aldh3 is transcriptionally inducible by polycyclic aromatic hydrocarbons (PAH), and this induction involving function of the arylhydrocarbon (Ah) receptor is inhibited by the protein kinase C (PKC) inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine di-HCl (H7) and staurosporine. However, PAH induction of ALDH-3 activity, protein, and mRNA was potentiated 2-4-fold by addition of the protein kinase A (PKA) inhibitors, N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide di-HCl (H8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide HCl (HA1004). These PKA inhibitors had no effect on the PAH induction of the cyp1A1. Protein kinase A activity of cultured hepatocytes was specifically inhibited by H8 and HA1004 in a concentration-dependent manner, but not by H7, and there was an inverse correlation observed between potentiation of PAH-induced aldh3 gene expression and inhibition of specific PKA activity by the PKA inhibitors. The cAMP analog dibutyryl cAMP, the adenylate cyclase activator forskolin, and the protein phosphatase 1 and 2A inhibitor okadaic acid all dramatically inhibited both PAH induction and H8 potentiation of PAH induction of aldh3 expression but had no effect on induction of cyp1A1 expression in cultured hepatocytes. Both basal and PAH-dependent expression of a chloramphenicol acetyltransferase expression plasmid containing approximately 3.5 kilobase pairs of the 5'-flanking region of aldh3 (pALDH3.5CAT) were enhanced 3-4-fold by the PKA inhibitor H8 but not by the PKC inhibitor H7 (>20 microM). cAMP analogs, activators of PKA activity, or protein phosphatase inhibitors diminished expression of the reporter gene in a manner identical to the native gene in cultured rat hepatocytes. Using deletion analysis of the pALDH3.5CAT construct, we demonstrated the existence of a negative regulatory region in the 5'-flanking region between -1057 and -991 base pairs which appears to be responsible for the cAMP-dependent regulation of this gene under both basal and PAH-induced conditions. At least two apparently independent mechanisms which involve protein phosphorylation regulate aldh3 expression. One involves function of the Ah receptor which requires PKC protein phosphorylation to positively regulate both aldh3 and cyp1A1 gene expression and the other a cAMP-responsive process which allows PKA activity to negatively regulate expression of aldh3 under either basal or inducible conditions.

Aldehyde Dehydrogenase↗
PubMed1997

Pharmacogenetics: a laboratory tool for optimizing therapeutic efficiency.

Pharmacogenetics is the study of the linkage between an individual's genotype and that individual's ability to metabolize a foreign compound. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Phenotypes exhibiting poor and ultraextensive metabolism result from genetic variance (polymorphism) of enzymes involved in metabolism. Thus, in pharmacogenetic studies one applies genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug metabolism phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic efficiency. More than 25 commonly prescribed medicines are metabolized by the cytochrome P-4502D6 (CYP2D6) isoenzyme, and polymorphism of the CYP2D6 gene affects the therapeutic management of up to 17% of individuals in some ethnic groups. In this review, we summarize and update information concerning drug-metabolizing genotypes with emphasis on CYP2D6 genotyping techniques that can be applied by the clinical laboratory for linking human genetics to therapeutic management.

Cytochrome P-450 CYP2D6↗
PubMed1997

Human NAD(P)H:quinone oxidoreductase induction in human hepatoma cells after exposure to industrial acrylates, phenolics, and metals.

Induction of the endogenous human NAD(P)H:quinone oxidoreductase (HQOR1) gene in the human hepatoma cell line HepG2 was measured at both the enzyme activity and RNA levels after exposure to a variety of industrial compounds. An RNA probe was designed that was complementary to portions of both the coding region and the 3'-nontranslated region unique to the largest (2.7-kilobase) HQOR1 transcript. Induction by three strong inducers of HQOR1 verified the utility of the antisense RNA probe. Ten industrial chemicals were evaluated as potential inducers, i.e. acrylonitrile, Sb2O3, BaO, CdCl2, CuCl, ethyl acrylate, methyl acrylate, MoO3, phenol, and toluene. Induction at the RNA level was about 2-fold higher than at the enzyme activity level except in the case of acrylonitrile, for which induction at the enzyme activity and RNA levels was similar. There was no preferential induction of the 2.7-kilobase transcript for any chemical tested, including 2,3,7,8-tetrachloro-dibenzo-p-dioxin, which had previously been reported to preferentially induce this transcript. Six of the 10 industrial chemicals, including four previously untested chemicals (phenol, Sb2O3, CuCl, and MoO3), were found to induce the HQOR1 gene. By comparison, previous studies in rodent systems failed to accurately predict the human HQOR1 gene response. Two chemicals previously shown to be inducers in rodent systems (methyl acrylate and CdCl2 failed to induce the HQOR1 gene. These results emphasize the importance of analyzing induction of the endogenous human gene, rather than simply extrapolating from rodent systems or gene fusion experiments.

Acrylonitrile↗
PubMed1997

In vivo induction and in vitro inhibition of hepatic cytochrome P450 activity by the benzodiazepine anticonvulsants clonazepam and diazepam.

The ability of the benzodiazepines, as a chemical class, to cause the induction and/or inhibition of cytochromes P450 has not been well characterized. In the present study, the induction of the cytochrome P450 2B subfamily (CYP2B) in vivo and the inhibition of CYP2B activity in vitro by selected benzodiazepines was examined in hepatic tissues derived from male F344/NCr rats. Initial studies of the in vivo induction or in vitro inhibition of benzyloxyresorufin O-dealkylation activity revealed that both clonazepam and diazepam were relatively effective in vivo inducers of CYP2B when administered in the diet at 500 ppm for 5 days and also were fairly potent inhibitors of the activity of these hemoproteins in vitro. Oxazepam, in contrast, was ineffective as an inducer or an inhibitor of this activity. Further studies were performed to characterize the subfamily selectivity of the P450 induction and inhibition displayed by clonazepam. Specifically, microsomes from rats treated with clonazepam (1000 or 1800 ppm in the diet for 5 days) were found to be highly induced with respect to catalytic activities mediated by CYP2B, including benzyloxyresorufin and pentoxyresorufin O-dealkylation or testosterone 16 beta-hydroxylation, but other CYP proteins were minimally induced. In addition to inducing the CYP2B subfamily, clonazepam also induced the RNA encoding other drug metabolizing enzymes (e.g., epoxide hydrolase and the glutathione S-transferase alpha-subfamily) that are typically induced by phenobarbital-type inducers. Finally, clonazepam proved to be a potent noncompetitive or "mixed-type" competitive inhibitor of catalytic activities mediated by CYP2B, but not by other CYP proteins (e.g. CYP2A, CYP3A) in microsomes derived from phenobarbital-pretreated rats.

Animals↗
PubMed1996

Endogenous 7-oxocholesterol is an enzymatic product: characterization of 7 alpha-hydroxycholesterol dehydrogenase activity of hamster liver microsomes.

Previously, we described a new metabolite derived from endogenous cholesterol in the presence of hamster liver microsomal protein and NADPH (Song et al., 1991, Biochem. Pharmacol. 41, 1439-1447). Through gas chromatography/mass spectral analysis of the metabolite and its methoxime-3-dimethyl-t-butylsilyl ether derivative, this metabolite has been definitively identified as 7-oxocholesterol. Isotope incorporation experiments using molecular 18O2 demonstrated that no oxygen atoms from molecular oxygen were incorporated into the product, 7-oxocholesterol, when 7 alpha-hydroxycholesterol was used as substrate. In contrast, one atom of 18O was incorporated into cholesterol from 18O2 during its metabolism to form 7 alpha-hydroxycholesterol. Formation of 7-oxocholesterol was dependent upon the presence of NADP+, 7 alpha-hydroxycholesterol, and hamster liver microsomes. This enzyme appears to be a membrane-bound protein and its activity was most abundant in liver microsomal fractions and to a lesser extent in mitochondrial fractions; little or no activity was observed in nuclei or cytosol. The enzyme activity was present in highest content in the livers of hamsters and was also observed in human and bovine liver microsomes, but not those of mouse, rabbit, or rat. The reaction was inhibited by 2'-AMP, but not by anti-NADPH:cytochrome-P450 oxidoreductase globulin, carbon monoxide, metyrapone, nor miconazole. In contrast to the previously characterized 3 beta-hydroxy-delta 5-C27-steroid oxidoreductase activity, NAD+ did not serve as an effective cofactor for 7-oxocholesterol formation. The ability of NADPH to partially serve as a cofactor in this reaction was shown to be due to a high NADPH-oxidase activity of hamster liver microsomes, thereby providing sufficient NADP+ to serve as the oxidizing pyridine nucleotide for the reaction. These results document the existence of a non-P450, NADP(+)-dependent 7 alpha-hydroxycholesterol dehydrogenase in liver microsomes which catalyzes this reaction. The product, 7-oxocholesterol, is produced enzymatically in the livers of hamsters and other mammals and may regulate bile acid metabolism or other processes due to its action as an oxysterol.

Animals↗