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David S Riddick - One of the best experts on this subject based on the ideXlab platform.
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Cytochrome P450 2C11 5 flanking region and promoter mediate in vivo suppression by 3 methylcholanthrene
Drug Metabolism and Disposition, 2008Co-Authors: Rana M Sawaya, David S RiddickAbstract:Aromatic hydrocarbons such as 3-methylcholanthrene (MC) elicit toxic and adaptive responses through the aryl hydrocarbon receptor (AHR). Aromatic hydrocarbons act via an unknown mechanism to suppress the transcription of CYP2C11 , a growth hormone-regulated gene encoding the male-specific rat hepatic Cytochrome P450 2C11. We hypothesize that suppression of CYP2C11 by aromatic hydrocarbons is mediated by the gene9s promoter and 5′-flank. Using hydrodynamics-based injections to deliver plasmid DNA to the liver of live rats, we studied the MC responsiveness of luciferase constructs containing 10.1, 5.6, and 2.4 kilobases (kb) of the CYP2C11 5′-flank. MC suppressed CYP2C11 -luciferase activity of the 10.1- and 5.6-kb constructs to less than 50% of vehicle levels by 24 and 72 h. Luciferase activity of the 2.4-kb CYP2C11 construct was decreased to 63% of vehicle levels 24 h after MC treatment, but no suppression was detected by 72 h. Negative regulatory element(s) responsible for CYP2C11 reporter suppression by MC exist in the proximal 2.4 kb of the 5′-flank; however, additional cis -acting elements located between –5.6 and –2.4 kb mediate persistent reporter suppression. As a positive control for AHR activation, MC dramatically induced the luciferase activity of a Cyp1a1 -driven luciferase plasmid under AHR control. Modulation of reporter gene activity by MC was accompanied by induction of endogenous CYP1A1 and suppression of endogenous CYP2C11 mRNA/protein. This is the first demonstration of aromatic hydrocarbon-mediated suppression of a CYP2C11 -luciferase construct, and this finding suggests that the 5′-flanking region and promoter mediate down-regulation of this gene in the intact rat.
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Cytochrome P450 2C11 5 flanking region and promoter regulation by aromatic hydrocarbons in vitro
Toxicology, 2008Co-Authors: Rana M Sawaya, David S RiddickAbstract:Aromatic hydrocarbons elicit toxic and adaptive responses via the aryl hydrocarbon receptor (AHR). Aromatic hydrocarbons suppress the transcription of the growth hormone-regulated, male-specific rat hepatic Cytochrome P450 2C11 gene (CYP2C11) in vivo via an unknown mechanism. We hypothesize that the suppression of CYP2C11 by aromatic hydrocarbons is mediated by the gene's promoter and 5'-flanking region. Following bioinformatic analysis of putative transcription factor (TF) binding sites, we cloned extended lengths of the CYP2C11 5'-flanking region into a promoterless luciferase plasmid. Suppression of CYP2C11 constructs was not observed upon treatment of transfected rat 5L, BP8 or mouse Hepa-1 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 3-methylcholanthrene. In human HepG2 cells, the 10.1-kb construct displayed a pronounced 6- to 8-fold induction by TCDD. Deletion analysis localized the paradoxical induction response to a region between -1.8 kb and -1.3 kb, which contains a dioxin-responsive element (DRE) previously shown by us to be capable of binding activated AHR. This was confirmed by site-directed mutagenesis of the DRE. Induction of the 10.1-kb construct by TCDD in HepG2 cells was blocked by alpha-naphthoflavone, an AHR antagonist/partial agonist. The AHR is likely involved in the induction of CYP2C11-luciferase activity by TCDD in HepG2 cells and this response is at least partly DRE-mediated. Although CYP2C11 is suppressed by aromatic hydrocarbons in vivo, CYP2C11-luciferase constructs display a potentially misleading paradoxical induction in vitro that is cell-specific. Regulation of CYP2C11-luciferase plasmids is being studied in vivo in rat liver, where an intact endocrine system and the full complement of TFs needed for CYP2C11 suppression are present.
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The 2001 Veylien Henderson Award of the Society of Toxicology of Canada. Positive and negative transcriptional regulation of Cytochromes P450 by polycyclic aromatic hydrocarbons.
Canadian Journal of Physiology and Pharmacology, 2003Co-Authors: David S Riddick, Anahita Bhathena, Yoav E TimsitAbstract:Most responses to aromatic hydrocarbons such as 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin are mediated by the aromatic hydrocarbon receptor (AHR). The AHR regulates induction of drug-metabolizing enzymes such as Cytochrome P450 1A1. However, the expression of several genes of biological significance is decreased by these chemicals. We are examining the mechanisms by which aromatic hydrocarbons suppress constitutive hepatic Cytochromes P450, especially the male-specific rat liver Cytochrome P450 2C11 (CYP2C11), which is regulated by pulsatile growth hormone (GH) secretion. Aromatic hydrocarbons suppress CYP2C11 via a transcriptional mechanism both in vivo and in cultured hepatocytes, and the AHR appears to be involved; however, studies of proteinDNA interactions and reporter genes driven by the CYP2C11 5'-flanking region have not provided a definitive mechanism for this response. MC attenuates the ability of GH to stimulate hepatic CYP2C11 expression in hypophysectomized (hypx) mal...
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suppression of Cytochrome P450 2C11 by aromatic hydrocarbons mechanistic insights from studies of the 5 flanking region of the cyp2C11 gene
Drug Metabolism and Disposition, 2002Co-Authors: Anahita Bhathena, Chunja Lee, David S RiddickAbstract:The aromatic hydrocarbon receptor (AHR) functions as a ligand-activated transcription factor that mediates responses to aromatic hydrocarbons (AHs). Induction of Cytochrome P450 1A1 (CYP1A1) is the most fully characterized response and is mediated by binding of the activated AHR complex to dioxin-responsive elements (DREs) located in the 5′-flanking region of the gene. In contrast to CYP1A1 induction, several other genes including the rat male-specific constitutive hepatic CYP2C11 are suppressed by AHs. Our aim was to determine whether CYP2C11 suppression by AHs is mediated by the AHR via interaction with DRE-like sequences. 2,3,7,8-Tetrachlorodibenzo- p- dioxin (TCDD) suppressed CYP2C11 mRNA in primary rat hepatocytes without altering the mRNA half-life. We identified five regions in the CYP2C11 5′-flank containing the DRE invariant core; electrophoretic gel retardation assays showed that at least one of these DREs is a potential binding site for the AHR. To test the function of the CYP2C11 -DREs, Hepa-1, BRL 5637, and HepG2 cells were transfected with reporter constructs containing regions of the CYP2C11 5′-flank and promoter. No decrease in luciferase activity was found following TCDD treatment. In primary rat hepatocytes, the luciferase reporter vectors were suppressed by interleukin-1β but not by TCDD. In vitro footprinting showed protein binding at several sites in the CYP2C11 5′-flank, but the pattern was not altered by in vivo 3-methylcholanthrene treatment. These studies imply that AHs down-regulate CYP2C11 by a negative transcriptional mechanism that is not simply due to AHR binding to an identified DRE-like sequence and that is distinct from that used by inflammatory cytokines.
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stimulation of hepatic signal transducer and activator of transcription 5b by gh is not altered by 3 methylcholanthrene
Endocrinology, 2002Co-Authors: Yoav E Timsit, David S RiddickAbstract:We are investigating the mechanisms by which aromatic hydrocarbons, such as 3-methylcholanthrene (MC), suppress hepatic Cytochrome P450 2C11 (CYP2C11) gene expression. CYP2C11 is an enzyme expressed in the liver of male rats and is regulated by a pulsatile pattern of GH secretion. We have previously shown that MC attenuates the stimulatory effect of GH on CYP2C11 expression in hypophysectomized male rats. In follow-up studies we evaluated the effect of MC on GH-stimulated signal transducer and activator of transcription 5b (STAT5b) phosphorylation, nuclear translocation, and DNA-binding activity. GH-stimulated increases in hepatic nuclear STAT5b and phospho-STAT5b levels were not different between groups of hypophysectomized rats receiving MC or vehicle. This observation was corroborated at the DNA-binding level by EMSA. We also measured GH-induced STAT5b activation in the H4IIE rat hepatoma cell line. STAT5b DNA-binding activity detected in GH-treated cells was not affected by MC. Immunocytochemistry experiments revealed no effect of MC on GH-stimulated STAT5b nuclear translocation in H4IIE cells. These in vivo and in vitro data suggest that interference with GH-stimulated STAT5b activation does not constitute a mechanism by which MC attenuates the stimulatory effect of GH on CYP2C11 gene expression.
Michael Murray - One of the best experts on this subject based on the ideXlab platform.
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pretranslational up regulation of the hepatic microsomal δ4 3 oxosteroid 5α oxidoreductase in male rat liver by all trans retinoic acid
Biochemical Pharmacology, 1999Co-Authors: Michael Murray, Alison M ButlerAbstract:Abstract Administration of all- trans -retinoic acid (ATRA; 60 mg/kg daily for 3 days) to male rats increased the rate of 5α-dihydrotestosterone (5α-DHT) formation from testosterone in microsomal fractions in vitro . The formation of androstane-3α,17β-diol from testosterone was also increased because of the higher concentration of 5α-DHT produced in microsomal incubations. Northern analysis confirmed that the increased rate of 5α-DHT formation was due to the pretranslational up-regulation in Δ 4 -3-oxosteroid 5α-oxidoreductase (EC 1.3.99.5) mRNA expression in ATRA-treated male rat liver. Thus, ATRA elicited in male rat liver a partial feminization of the expression of this enzyme, which normally exhibits a female-selective distribution in the rat. Subsequent experiments evaluated whether the administration of human chorionic gonadotropin or thyroxine to ATRA-treated male rats decreases 5α-reductase activity to that observed in untreated male rat liver. Although these treatments did not decrease 5α-reductase to untreated male levels, it was found that administration of ATRA to gonadectomized male rats produced complete feminization of the enzyme. Again, up-regulation was confirmed at the mRNA level. The activity of the male-specific Cytochrome P450 2C11 (as reflected by microsomal testosterone 16α-hydroxylation activity) was correspondingly decreased by treatments that increased steroid 5α-reductase activity. Thus, gonadectomy in combination with ATRA administration effected a more pronounced decrease in 16α-hydroxylation activity than either treatment alone. These findings suggest that ATRA is a novel positive regulator of the 5α-reductase that in combination with the removal of circulating androgen, which normally suppresses 5α-reductase levels, feminizes the expression of this enzyme in rat liver.
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restoration of Cytochrome P450 2C11 in vitamin a deficient rat liver by exogenous androgen
The FASEB Journal, 1996Co-Authors: Michael Murray, Alison M Butler, Cynthia AgusAbstract:Down-regulation of microsomal androgen-dependent CYP2C11 is produced in male rat liver by dietary vitamin A deficiency. Decreased circulating androgen concentrations also occur in vitamin A-deficient male rats. Both effects are prevented by addition of all-trans-retinoic acid to the diet. The present study evaluated directly whether androgen deficiency may be responsible for the down-regulation of 2C11 in vitamin A-deficient male rats. The major finding was that subcutaneous administration of the androgen methyltrienolone (MT) during the final week of the study restored CYP2C11 protein and its associated steroid 16alpha-hydroxylation activities to control levels; CYP2C11 mRNA was also restored. Despite the efficient restoration of CYP2C11 at a pretranslational level, no alteration in vitamin A status was apparent and animals remained vitamin A deficient after MT treatment. The possibility was assessed that vitamin A can maintain the microsomal content of CYP2C11 in normal liver. However, in contrast to MT...
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pretranslational down regulation of Cytochrome P450 2C11 in vitamin a deficient male rat liver prevention by dietary inclusion of retinoic acid
Journal of Pharmacology and Experimental Therapeutics, 1995Co-Authors: Robert Martini, Alison M Butler, Xingmai Jiang, Michael MurrayAbstract:Manipulation of vitamin A intake has been associated with altered rates of Cytochrome P450 (P450)-mediated microsomal drug oxidation. Dietary vitamin A deficiency reportedly results in decreased rates of P450-dependent substrate oxidation, but the mechanisms underlying these changes remain unclear. In this study, the effects of dietary vitamin A modulation, as well as dietary inclusion of all-trans-retinoic acid (ATRA), on major constitutive P450s were defined. Total microsomal P450 in deficient male rats was decreased to 72% of control (0.63 +/- 0.07 vs. 0.88 +/- 0.08 nmol/mg of protein; P < .05); this was prevented by inclusion of ATRA (12 micrograms/g) in the deficient diet. Dietary vitamin A deficiency decreased rates of P450 2C11-mediated testosterone 2 alpha- and 16 alpha-hydroxylation in rat liver to 44 and 47% of respective adequate control, whereas rates of 6 beta- and 7 alpha-hydroxylation of the steroid were unaltered; inclusion of ATRA into the deficient diet prevented the loss of 2C11 activities. Immunoblot and RNA analysis revealed decreases in P450 2C11 apoprotein and its corresponding mRNA in liver from deficient rats that was prevented by inclusion of ATRA in the deficient diet. Serum testosterone concentrations were reduced in deficient rats and this also was prevented by dietary ATRA. To discern whether this was a direct effect of vitamin A on P450 2C11 regulation, further experiments evaluated the effect of ATRA administration to male rats maintained on standard rat chow (vitamin A-adequate). Dose- and time-dependent decreases in P450 2C11 activity were observed.(ABSTRACT TRUNCATED AT 250 WORDS)
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suppression of the constitutive microsomal Cytochrome P450 2C11 in male rat liver during dietary vitamin a deficiency
Biochemical Pharmacology, 1994Co-Authors: Robert Martini, Michael MurrayAbstract:Abstract The effect of dietary vitamin A deficiency on hepatic microsomal Cytochrome P450 (P450) and associated oxidase activities was examined in the male rat. Intake of a deficient diet by weanling rats over 10 weeks led to a pronounced decline in hepatic and serum vitamin A concentrations to levels that were beneath the limits of detection. These changes occurred concurrently with a decrease in total microsomal P450 to 77% of vitamin A adequate control. Measurement of microsomal androstenedione metabolism revealed respective decreases in 16α- and 6β-hydroxylation pathways to 61 and 71% of adequate control; 7α-hydroxylation was not significantly decreased. Immunoquantitation of the principal catalyst of steroid 16α-hydroxylation, the androgen-dependent P450 2C11, indicated a significant decrease in the microsomal content of the enzyme to 78% of control (13.7 ± 0.9 ng/μg protein in deficient rat liver versus 17.5 ± 0.5 in adequate control; P
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metabolite intermediate complexation of microsomal Cytochrome P450 2C11 in male rat liver by nortriptyline
Molecular Pharmacology, 1992Co-Authors: Michael MurrayAbstract:Antidepressant drugs that contain alkylaminoalkyl substituents have been associated with serious pharmacokinetic interactions in humans that may be related to the inhibition of Cytochrome P450 (P450) enzymes. In this study, the propensity of the tricyclic antidepressant nortriptyline (NOR) to inhibit individual microsomal P450 enzymes in rat liver was investigated to provide a mechanistic explanation for these pharmacokinetic interactions. Enzyme kinetic studies revealed that NOR inhibited steroid 2 alpha-, 6 beta, 7 alpha-, and 16 alpha-hydroxylation in untreated rat liver with Km/Ki ratios of 0.53, 0.59, 0.25, and 0.29, respectively. When the drug was preincubated with microsomes and NADPH before testosterone hydroxylation was conducted, marked increases in the Km/Ki ratios were observed (to 8.8, 3.9, 0.62, and 13, respectively). Thus, enzymic oxidation of NOR enhanced its inhibition capacity against P450 activities. Indeed, the altered Km/Ki ratios indicate 17-, 6.6-, 2.5-, and 47-fold increases in inhibition of the four pathways of testosterone hydroxylation after the biotransformation of NOR to its metabolites. From these experiments it was apparent that testosterone 2 alpha- and 16 alpha-hydroxylations, catalyzed predominantly by P450 2C11, were subject to the most pronounced increase in inhibition. Under these conditions, the apparent content of microsomal P450 was decreased, thus suggesting the formation of a NOR metabolite intermediate (MI) complex with the Cytochrome. Further, optical difference spectroscopy of NADPH-supported metabolism of NOR in microsomes and in a reconstituted system incorporating purified P450 2C11 indicated the appearance of an absorbance peak near 454 nm, similar to those produced by triacetyloleandomycin, SKF 525-A, and orphenadrine. Formation of this absorbance peak in microsomes was inhibited by an antibody raised against the male-specific P450 2C11. Because oxidative metabolism of NOR to inhibitory products would not necessarily involve MI complexation, additional experiments were undertaken in which NOR-related free metabolites produced in microsomal incubations were removed on Sep-Pak mini-C18 columns before estimation of testosterone hydroxylation. The principal finding from this experiment was that P450 3A2-dependent steroid 6 beta-hydroxylase activity was inhibited to a much lesser extent after removal of unbound NOR metabolites on Sep-Pak columns (25% inhibition after Sep-Pak extraction, compared with 82% inhibition observed when all NOR metabolites were present during subsequent testosterone hydroxylation); inhibition of P450 2C11-mediated 2 alpha- and 16 alpha-hydroxylation was not noticeably different after Sep-Pak treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
David J Waxman - One of the best experts on this subject based on the ideXlab platform.
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1 2 chloroethyl 3 cyclohexyl 1 nitrosourea ccnu modulates rat liver microsomal cyclophosphamide and ifosphamide activation by suppressing Cytochrome P450 2C11 messenger rna levels
Drug Metabolism and Disposition, 1994Co-Authors: T K H Chang, Haiyan Chen, David J WaxmanAbstract:The alkylating anticancer drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU; lomustine) is frequently administered to cancer patients as part of a combination chemotherapy regimen. Previous studies have indicated that CCNU treatment of adult male rats leads to prolonged decreases in liver Cytochrome P450 (CYP)-mediated enzyme activities. Because the alkylating agent prodrugs cyclophosphamide and ifosphamide are known to be activated by liver Cytochrome P450 enzymes, the potential for interaction between CCNU and these oxazaphosphorines was examined. Treatment of adult male rats with a single dose of CCNU (30 mg/kg i.p.) resulted in a progressive loss of liver microsomal cyclophosphamide and ifosphamide hydroxylation activities in vitro (30-60% decrease after 7-27 days). The individual liver P450 forms modulated by CCNU were then identified using P450 form-specific microsomal testosterone hydroxylase assays. CCNU treatment was found to decrease substantially CYP2C11-dependent testosterone 2 alpha-hydroxylase activity (80-90% decrease after 14 or 27 days), but it did not affect CYP3A2-dependent testosterone 6 beta-hydroxylase activity. It only modestly decreased CYP2A2-mediated testosterone 15 alpha-hydroxylase activity. The reduction in CYP2C11 activity was not associated with a decline in liver microsomal NADPH-Cytochrome P450 reductase activity, but rather was caused by a complete suppression of CYP2C11 mRNA levels. In contrast to other alkylating agents, such as cisplatin, which is known to feminize the overall expression profile of gender-specific liver enzymes, CCNU did not increase levels of the female-predominant liver enzymes steroid 5 alpha-reductase and CYP2C7, nor did it deplete circulating testosterone levels.(ABSTRACT TRUNCATED AT 250 WORDS)
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cyclophosphamide modulates rat hepatic Cytochrome P450 2C11 and steroid 5α reductase activity and messenger rna levels through the combined action of acrolein and phosphoramide mustard
Cancer Research, 1993Co-Authors: Thomas Chang, David J WaxmanAbstract:Abstract Cyclophosphamide treatment of adult male rats leads to sustained decreases in several liver microsomal Cytochrome P450 (CYP) activities, including CYP 2C11-catalyzed cyclophosphamide activation, via a process that is associated with a feminization of the overall pattern of liver enzyme expression (G. A. LeBlanc and D. J. Waxman, Cancer Res., 50: 5720–5726, 1990). The present study compares the effects of cyclophosphamide and its isomeric analogue ifosphamide on the gender-dependent expression of hepatic CYP 2C11 and steroid 5α-reductase in adult male rats and also examines the role of the cyclophosphamide metabolites acrolein and phosphoramide mustard in feminizing the expression of these liver enzymes. Ifosphamide (a) suppressed the male-specific CYP 2C11 mRNA and CYP 2C11-catalyzed liver microsomal testosterone 2α-hydroxylation and cyclophosphamide and ifosphamide 4-hydroxylation and (b) elevated the female-dominant liver enzyme steroid 5α-reductase and its mRNA 7–9 days after drug treatment, both occurring in a manner similar to that of cyclophosphamide, but requiring a 50% higher dose (180 mg/kg, single i.p. injection) to achieve these effects. This pattern of response could not be achieved by treatment of rats with acrolein or with cyclophosphamide analogues that decompose to acrolein without formation of phosphoramide mustard. In contrast, phosphoramide mustard treatment (100 mg/kg) did modulate microsomal CYP 2C11 and steroid 5α-reductase activities. Treatment with a lower dose (50 mg/kg) of phosphoramide mustard or with the acrolein precursor 4-hydroperoxydechlorocyclophosphamide (200 mg/kg) alone did not affect liver enzyme expression, whereas the combination of these agents produced an overall pattern of response that was similar to that conferred by cyclophosphamide. These studies establish that ifosphamide is less potent than cyclophosphamide in modulating the pattern of Cytochrome P450 and steroid 5α-reductase expression and that phosphoramide mustard is responsible for the modulation of liver enzyme expression by cyclophosphamide, with acrolein potentiating the modulating activity of the mustard.
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irreversible suppression of growth hormone dependent Cytochrome P450 2C11 in adult rats neonatally treated with monosodium glutamate
Journal of Pharmacology and Experimental Therapeutics, 1993Co-Authors: Bernard H Shapiro, Nisar A Pampori, Prabha A Ram, David J WaxmanAbstract:Neonatal exposure to monosodium glutamate (MSG) permanently blocks growth hormone (GH) secretion, which results in the development of a well-defined syndrome characterized by stunted body growth, obesity and impaired drug metabolism. We have found that restoration of the normal masculine circulating profile of GH (i.e., six daily pulses) by use of an external pumping apparatus is ineffective in restoring the normal expression of hepatic Cytochrome P450 2C11, a major GH-dependent drug and steroid metabolizing enzyme that is eliminated by MSG treatment. Moreover, administering GH at two, four or seven plasma pulses per day with amplitudes ranging from physiologic to 7 times normal were similarly ineffective in restoring the expression (at both an activity and mRNA level) of the Cytochrome. Additionally, multiCytochrome P450-dependent hexobarbital hydroxylase was also unresponsive to GH administration in the MSG-treated rats. Because GH replacement was unable to correct the enzyme defects, our results suggest that the developmental abnormalities produced by neonatal MSG are not simply a result of a GH deficiency per se, but are due to an irreversible insensitivity of the target cell to GH.
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sex specific growth hormone regulated transcription of the Cytochrome P450 2C11 and 2c12 genes
Journal of Biological Chemistry, 1992Co-Authors: Scott S Sundseth, John A Alberta, David J WaxmanAbstract:Growth hormone (GH) differentially regulates the expression of several male-specific and female-specific liver Cytochrome P450 mRNAs as a function of its sex-dependent ultradian secretory pattern. Pulsatile GH release stimulates expression of the male-specific P450 2C11, while a continuous GH secretion pattern suppresses expression of 2C11 and stimulates the expression of the female-specific P450 2C12. To help define the level at which GH regulates the expression of 2C11 and 2C12 mRNA, liver nuclear RNA samples isolated from rats differing in GH status were analyzed for 2C11 and 2C12 hnRNAs by hybridization to 2C11 and 2C12 gene-specific exonic oligonucleotide probes, as well as exon/intron junction probes. The 2C11 and 2C12 hnRNAs were found to be responsive to circulating GH profiles in a manner indistinguishable from the corresponding mature, cytoplasmic mRNAs, with no 2C12 mRNA precursors found in untreated male or hypophysectomized female liver nuclei, and no 2C11 mRNA precursors in untreated female or hypophysectomized male liver nuclei. Thus, transport of 2C11 and 2C12 RNA to the cytoplasm and cytoplasmic mRNA stability are unlikely to be important GH-regulated control points for sex-specific P450 RNA expression. Run-on transcription analysis further established that GH regulates the sex-specific expression of the 2C11 and 2C12 genes at the level of transcript initiation. Transcription was also shown to be the major step for regulation of the male-specific P450 2A2 RNA, whose expression, unlike 2C11, is not obligatorily dependent on pulsatile GH release. In vitro footprinting analysis of 2C11 and 2C12 promoter fragments incubated with liver nuclear proteins isolated from rats differing in GH status revealed several sex- and GH-dependent differences in DNase cleavage patterns ("hypersensitivity sites"), demonstrating that GH can regulate specific protein-DNA interactions in the 5'-flanking sequences of these two genes. In vitro transcription assays driven by 2C11 and 2C12 5'-flanking DNA sequences fused to TATAA box-G-less cassette template constructs did not, however, faithfully mimic the sex-specific transcription of the 2C11 and 2C12 genes, indicating that additional cis-elements or trans-acting factors may be required to achieve the transcriptional regulation of these genes that occurs in vivo.
Gail D. Bellward - One of the best experts on this subject based on the ideXlab platform.
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Impact of tamoxifen on peripubertal Androgen imprinting of rat hepatic Cytochrome P450 2C11, Cytochrome P450 3A2, and steroid 5α-reductase
Biochemical pharmacology, 1996Co-Authors: Thomas K. H. Chang, Maureen M.y. Chan, Susan L. Holsmer, Stelvio M. Bandiera, Gail D. BellwardAbstract:Abstract Expression of sex-dependent rat hepatic Cytochromes P450 and steroid 5α-reductase is regulated mainly by the sex-specific pattern of growth hormone (GH) secretion and is subject to androgen imprinting. Since tamoxifen suppresses GH pulse amplitude and nadir levels, we investigated the effect of tamoxifen on peripubertal testosterone imprinting of hepatic CYP2C11, CYP3A2, CYP2A1, and steroid 5α-reductase. Prepubertal tamoxifen administration (5 mg once daily s.c. on days 28 and 29 of age) to non-ovariectomized female Sprague-Dawley rats did not affect hepatic microsomal CYP2C11-dependent testosterone 2α-hydroxylase, CYP3A-mediated testosterone 6β-hydroxylase, CYP2A1-dependent testosterone 7α-hydroxylase, or steroid 5αreductase activity in adult rats. Testosterone treatment (5 μmol/kg, s.c., once daily) of intact female rats during either puberty (days 35–49 of age) or adult life (days 69–77 of age) had no effect on these enzyme activities in adult (78-day-old) female rats, but the same treatment given during both of these periods induced the male-specific testosterone 2α- and 6β-hydroxylase activities and suppressed the female-predominant testosterone 7α-hydroxylase and steroid 5α-reductase activities, indicating that peripubertal testosterone administration imprints the adult androgen responsiveness but not the basal levels of these enzyme activities in non-ovariectomized female rats. However, peripubertal androgen imprinting of the basal levels of testosterone 2α-hydroxylase and steroid 5α-reductase activities was observed in female rats administered tamoxifen prepubertally. Tamoxifen pretreatment also enhanced testosterone imprinting of the adult androgen responsiveness of testosterone 2α- and 6β-hydroxylase and steroid 5α-reductase activities. The enhanced testosterone hydroxylase activities were, however, not associated with an increase in microsomal NADPH-Cytochrome P450 reductase activity, but were accompanied by elevated hepatic CYP2C11 and CYP3A2 protein levels. Overall, the present study indicates that prepubertal tamoxifen administration does not interfere with the normal sex differentiation of the gender-dependent hepatic Cytochromes P450 and steroid 5α-reductase, but this drug modulates peripubertal androgen imprinting of CYP2C11, CYP3A2, and steroid 5α-reductase in adult female rats.
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Peripubertal androgen imprinting of rat hepatic Cytochrome P450 2C11 and steroid 5 alpha-reductase: pretranslational regulation and impact on microsomal drug activation.
The Journal of pharmacology and experimental therapeutics, 1996Co-Authors: Thomas K. H. Chang, Gail D. BellwardAbstract:To characterize the dose response and time course of peripubertal testosterone imprinting of rat hepatic CYP2C11 and steroid 5 alpha-reductase and to gain further insights into the mechanism and consequences of peripubertal androgen imprinting of these enzymes, prepubertally gonadectomized female rats were injected s.c. with testosterone enanthate (5 mumol/kg/day) on days 35 to 49 (peripubertal period) or days 81 to 89 (adulthood) and then sacrificed on day 90. Androgen treatment during the peripubertal or adult period increased hepatic microsomal testosterone 2 alpha-hydroxylase activity by 4- to 5-fold and decreased steroid 5 alpha-reductase activity by 30 to 50%. By comparison, androgen administration during both periods completely masculinized these two enzyme activities. Whereas shortening the duration of treatment to 5 days during the peripubertal and adult periods resulted in only a partial masculinization of these activities, reducing the dosage of testosterone enanthate from 5 mumol/kg/day to 2.5 mumol/kg/day during both the peripubertal (15 days) and adult periods (9 days) still fully masculinized testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities. Northern blot analysis showed that peripubertal and adult testosterone treatment of female rats increased hepatic CYP2C11 mRNA levels, decreased steroid 5 alpha-reductase mRNA levels and did not change CYP2C6 mRNA levels. Enhanced cyclophosphamide 4-hydroxylation and ifosfamide 4-hydroxylation was found in liver microsomes isolated from adult female rats exposed to testosterone during puberty and adult life. In contrast to once daily subcutaneous injections, continuous testosterone release via subcutaneous implant was ineffective in producing the long-term changes in testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities. Overall, the present study establishes that peripubertal androgen imprinting of CYP2C11 and steroid 5 alpha-reductase can be achieved after daily subcutaneous testosterone administration. This occurs by a pretranslational mechanism(s), which lead to long-lasting effects on microsomal drug activation.
Thomas K. H. Chang - One of the best experts on this subject based on the ideXlab platform.
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Impact of tamoxifen on peripubertal Androgen imprinting of rat hepatic Cytochrome P450 2C11, Cytochrome P450 3A2, and steroid 5α-reductase
Biochemical pharmacology, 1996Co-Authors: Thomas K. H. Chang, Maureen M.y. Chan, Susan L. Holsmer, Stelvio M. Bandiera, Gail D. BellwardAbstract:Abstract Expression of sex-dependent rat hepatic Cytochromes P450 and steroid 5α-reductase is regulated mainly by the sex-specific pattern of growth hormone (GH) secretion and is subject to androgen imprinting. Since tamoxifen suppresses GH pulse amplitude and nadir levels, we investigated the effect of tamoxifen on peripubertal testosterone imprinting of hepatic CYP2C11, CYP3A2, CYP2A1, and steroid 5α-reductase. Prepubertal tamoxifen administration (5 mg once daily s.c. on days 28 and 29 of age) to non-ovariectomized female Sprague-Dawley rats did not affect hepatic microsomal CYP2C11-dependent testosterone 2α-hydroxylase, CYP3A-mediated testosterone 6β-hydroxylase, CYP2A1-dependent testosterone 7α-hydroxylase, or steroid 5αreductase activity in adult rats. Testosterone treatment (5 μmol/kg, s.c., once daily) of intact female rats during either puberty (days 35–49 of age) or adult life (days 69–77 of age) had no effect on these enzyme activities in adult (78-day-old) female rats, but the same treatment given during both of these periods induced the male-specific testosterone 2α- and 6β-hydroxylase activities and suppressed the female-predominant testosterone 7α-hydroxylase and steroid 5α-reductase activities, indicating that peripubertal testosterone administration imprints the adult androgen responsiveness but not the basal levels of these enzyme activities in non-ovariectomized female rats. However, peripubertal androgen imprinting of the basal levels of testosterone 2α-hydroxylase and steroid 5α-reductase activities was observed in female rats administered tamoxifen prepubertally. Tamoxifen pretreatment also enhanced testosterone imprinting of the adult androgen responsiveness of testosterone 2α- and 6β-hydroxylase and steroid 5α-reductase activities. The enhanced testosterone hydroxylase activities were, however, not associated with an increase in microsomal NADPH-Cytochrome P450 reductase activity, but were accompanied by elevated hepatic CYP2C11 and CYP3A2 protein levels. Overall, the present study indicates that prepubertal tamoxifen administration does not interfere with the normal sex differentiation of the gender-dependent hepatic Cytochromes P450 and steroid 5α-reductase, but this drug modulates peripubertal androgen imprinting of CYP2C11, CYP3A2, and steroid 5α-reductase in adult female rats.
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Peripubertal androgen imprinting of rat hepatic Cytochrome P450 2C11 and steroid 5 alpha-reductase: pretranslational regulation and impact on microsomal drug activation.
The Journal of pharmacology and experimental therapeutics, 1996Co-Authors: Thomas K. H. Chang, Gail D. BellwardAbstract:To characterize the dose response and time course of peripubertal testosterone imprinting of rat hepatic CYP2C11 and steroid 5 alpha-reductase and to gain further insights into the mechanism and consequences of peripubertal androgen imprinting of these enzymes, prepubertally gonadectomized female rats were injected s.c. with testosterone enanthate (5 mumol/kg/day) on days 35 to 49 (peripubertal period) or days 81 to 89 (adulthood) and then sacrificed on day 90. Androgen treatment during the peripubertal or adult period increased hepatic microsomal testosterone 2 alpha-hydroxylase activity by 4- to 5-fold and decreased steroid 5 alpha-reductase activity by 30 to 50%. By comparison, androgen administration during both periods completely masculinized these two enzyme activities. Whereas shortening the duration of treatment to 5 days during the peripubertal and adult periods resulted in only a partial masculinization of these activities, reducing the dosage of testosterone enanthate from 5 mumol/kg/day to 2.5 mumol/kg/day during both the peripubertal (15 days) and adult periods (9 days) still fully masculinized testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities. Northern blot analysis showed that peripubertal and adult testosterone treatment of female rats increased hepatic CYP2C11 mRNA levels, decreased steroid 5 alpha-reductase mRNA levels and did not change CYP2C6 mRNA levels. Enhanced cyclophosphamide 4-hydroxylation and ifosfamide 4-hydroxylation was found in liver microsomes isolated from adult female rats exposed to testosterone during puberty and adult life. In contrast to once daily subcutaneous injections, continuous testosterone release via subcutaneous implant was ineffective in producing the long-term changes in testosterone 2 alpha-hydroxylase and steroid 5 alpha-reductase activities. Overall, the present study establishes that peripubertal androgen imprinting of CYP2C11 and steroid 5 alpha-reductase can be achieved after daily subcutaneous testosterone administration. This occurs by a pretranslational mechanism(s), which lead to long-lasting effects on microsomal drug activation.
