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Lau-Corona D, Ma H, Vergato C, Sarmento-Cabral A, del Rio-Moreno M, Kineman RD, Waxman DJ. Constitutively Active STAT5b Feminizes Mouse Liver Gene Expression. Endocrinology 2022; 163:bqac046. [PMID: 35396838 PMCID: PMC9070516 DOI: 10.1210/endocr/bqac046] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Indexed: 11/19/2022]
Abstract
STAT5 is an essential transcriptional regulator of the sex-biased actions of GH in the liver. Delivery of constitutively active STAT5 (STAT5CA) to male mouse liver using an engineered adeno-associated virus with high tropism for the liver is shown to induce widespread feminization of the liver, with extensive induction of female-biased genes and repression of male-biased genes, largely mimicking results obtained when male mice are given GH as a continuous infusion. Many of the STAT5CA-responding genes were associated with nearby (< 50 kb) sites of STAT5 binding to liver chromatin, supporting the proposed direct role of persistently active STAT5 in continuous GH-induced liver feminization. The feminizing effects of STAT5CA were dose-dependent; moreover, at higher levels, STAT5CA overexpression resulted in some histopathology, including hepatocyte hyperplasia, and increased karyomegaly and multinuclear hepatocytes. These findings establish that the persistent activation of STAT5 by GH that characterizes female liver is by itself sufficient to account for the sex-dependent expression of a majority of hepatic sex-biased genes. Moreover, histological changes seen when STAT5CA is overexpressed highlight the importance of carefully evaluating such effects before considering STAT5 derivatives for therapeutic use in treating liver disease.
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Affiliation(s)
- Dana Lau-Corona
- Department of Biology and Bioinformatics Program, Boston University, Boston, MA 02215, USA
| | - Hong Ma
- Department of Biology and Bioinformatics Program, Boston University, Boston, MA 02215, USA
| | - Cameron Vergato
- Department of Biology and Bioinformatics Program, Boston University, Boston, MA 02215, USA
| | - Andre Sarmento-Cabral
- Department of Medicine, Section of Endocrinology, Diabetes, and Metabolism, University of Illinois at Chicago and Research and Development Division, Jesse Brown VA Medical Center, Chicago, IL 60612, USA
| | - Mercedes del Rio-Moreno
- Department of Medicine, Section of Endocrinology, Diabetes, and Metabolism, University of Illinois at Chicago and Research and Development Division, Jesse Brown VA Medical Center, Chicago, IL 60612, USA
| | - Rhonda D Kineman
- Department of Medicine, Section of Endocrinology, Diabetes, and Metabolism, University of Illinois at Chicago and Research and Development Division, Jesse Brown VA Medical Center, Chicago, IL 60612, USA
| | - David J Waxman
- Department of Biology and Bioinformatics Program, Boston University, Boston, MA 02215, USA
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Banerjee S, Hayes AM, Shapiro BH. Early expression of requisite developmental growth hormone imprinted cytochromes P450 and dependent transcription factors. Endocr Connect 2021; 10:1167-1179. [PMID: 34424855 PMCID: PMC8494408 DOI: 10.1530/ec-21-0143] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2021] [Accepted: 08/20/2021] [Indexed: 11/22/2022]
Abstract
The sexually dimorphic expression of cytochromes P450 (CYP) drug metabolizing enzymes has been reported in all species examined. These sex differences are initially expressed during puberty and are solely regulated by sex differences in the circulating growth hormone (GH) profiles. Once established, however, the different male- and female-dependent CYP isoforms are permanent and immutable, suggesting that adult CYP expression requires imprinting. Since the hormone that regulates an adult function is likely the same hormone that imprints the function, we selectively blocked GH secretion in some newborn male rats while others also received a concurrent physiologic replacement of rat GH. Rats were subsequently challenged, peripubertally, with either a masculine-like episodic GH regimen or the GH vehicle alone. The results demonstrate that episodic GH regulation of male-specific CYP2C11 and CYP3A2, as well as female-predominant CYP2C6, are dependent on developmental GH imprinting. Moreover, the induction and/or activation of major components in the signal transduction pathway regulating the expression of the principal CYP2C11 isoform is obligatorily dependent on perinatal GH imprinting without which CYP2C11 and drug metabolism would be permanently and profoundly suppressed. Since there are additional adult metabolic functions also regulated by GH, pediatric drug therapy that is known to disrupt GH secretion could unintentionally impair adult health.
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Affiliation(s)
- Sarmistha Banerjee
- Department of Biomedical Sciences, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Allison M Hayes
- Department of Biomedical Sciences, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Bernard H Shapiro
- Department of Biomedical Sciences, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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Banerjee S, Das RK, Shapiro BH. Feminization imprinted by developmental growth hormone. Mol Cell Endocrinol 2019; 479:27-38. [PMID: 30170181 PMCID: PMC6263729 DOI: 10.1016/j.mce.2018.08.011] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/12/2018] [Revised: 08/27/2018] [Accepted: 08/27/2018] [Indexed: 10/28/2022]
Abstract
Previously, we identified early developmental exposure to growth hormone (GH) as the requisite organizer responsible for programming the masculinization of the hepatic cytochromes P450 (CYP)-dependent drug metabolizing enzymes (Das et al., 2014, 2017). In spite of the generally held dogma that mammalian feminization requires no hormonal imprinting, numerous reports that the sex-dependent regulation and expression of hepatic CYPs in females are permanent and irreversible would suggest otherwise. Consequently, we selectively blocked GH secretion in a cohort of newborn female rats, some of whom received concurrent GH replacement or GH releasing factor. As adults, the feminine circulating GH profile was restored in the treated animals. Two categories of CYPs were measured. The principal and basically female specific CYP2C12 and CYP2C7; both completely and solely dependent on the adult feminine continuous GH profile for expression, and the female predominant CYP2C6 and CYP2E1 whose expression is maximum in the absence of plasma GH, suppressed by the feminine GH profile but more so by the masculine episodic GH profile. Our findings indicate that early developmental exposure to GH imprints the inchoate CYP2C12 and CYP2C7 in the differentiating liver to be solely dependent on the feminine GH profile for expression in the adult female. In contrast, adult expression of CYP2C6 and CYP2E1 in the female rat appears to require no GH imprinting.
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Affiliation(s)
- Sarmistha Banerjee
- Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Rajat K Das
- Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA
| | - Bernard H Shapiro
- Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA.
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Das RK, Banerjee S, Shapiro BH. Growth hormone: a newly identified developmental organizer. J Endocrinol 2017; 232:377-389. [PMID: 27980003 PMCID: PMC5241097 DOI: 10.1530/joe-16-0471] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/06/2016] [Accepted: 12/15/2016] [Indexed: 01/23/2023]
Abstract
The sexually dimorphic expression of cytochromes P450 (CYP) drug-metabolizing enzymes has been reported in all species examined. These sex differences are only expressed during adulthood and are solely regulated by sex differences in circulating growth hormone (GH) profiles. Once established, however, the different male- and female-dependent CYP isoform profiles are permanent and immutable, suggesting that adult CYP expression requires imprinting. As the hormone that regulates an adult function is likely the same hormone that imprints the function, we selectively blocked GH secretion in some newborn male rats, whereas others received concurrent physiologic replacement of rat GH. The results demonstrate that adult male GH activation of the signal transduction pathway regulating expression of the principal CYP2C11 isoform is obligatorily dependent on perinatal GH imprinting, without which CYP2C11 and drug metabolism would be permanently and profoundly suppressed. As there are other adult metabolic functions also regulated by GH, pediatric drug therapy known to disrupt GH secretion could unintentionally impair adult health.
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Affiliation(s)
| | | | - Bernard H Shapiro
- Department of Biomedical SciencesUniversity of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania, USA
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5
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Schmölz L, Birringer M, Lorkowski S, Wallert M. Complexity of vitamin E metabolism. World J Biol Chem 2016; 7:14-43. [PMID: 26981194 PMCID: PMC4768118 DOI: 10.4331/wjbc.v7.i1.14] [Citation(s) in RCA: 135] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/24/2015] [Revised: 11/25/2015] [Accepted: 01/19/2016] [Indexed: 02/05/2023] Open
Abstract
Bioavailability of vitamin E is influenced by several factors, most are highlighted in this review. While gender, age and genetic constitution influence vitamin E bioavailability but cannot be modified, life-style and intake of vitamin E can be. Numerous factors must be taken into account however, i.e., when vitamin E is orally administrated, the food matrix may contain competing nutrients. The complex metabolic processes comprise intestinal absorption, vascular transport, hepatic sorting by intracellular binding proteins, such as the significant α-tocopherol-transfer protein, and hepatic metabolism. The coordinated changes involved in the hepatic metabolism of vitamin E provide an effective physiological pathway to protect tissues against the excessive accumulation of, in particular, non-α-tocopherol forms. Metabolism of vitamin E begins with one cycle of CYP4F2/CYP3A4-dependent ω-hydroxylation followed by five cycles of subsequent β-oxidation, and forms the water-soluble end-product carboxyethylhydroxychroman. All known hepatic metabolites can be conjugated and are excreted, depending on the length of their side-chain, either via urine or feces. The physiological handling of vitamin E underlies kinetics which vary between the different vitamin E forms. Here, saturation of the side-chain and also substitution of the chromanol ring system are important. Most of the metabolic reactions and processes that are involved with vitamin E are also shared by other fat soluble vitamins. Influencing interactions with other nutrients such as vitamin K or pharmaceuticals are also covered by this review. All these processes modulate the formation of vitamin E metabolites and their concentrations in tissues and body fluids. Differences in metabolism might be responsible for the discrepancies that have been observed in studies performed in vivo and in vitro using vitamin E as a supplement or nutrient. To evaluate individual vitamin E status, the analytical procedures used for detecting and quantifying vitamin E and its metabolites are crucial. The latest methods in analytics are presented.
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Harkitis P, Daskalopoulos EP, Malliou F, Lang MA, Marselos M, Fotopoulos A, Albucharali G, Konstandi M. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver. PLoS One 2015; 10:e0128708. [PMID: 26466350 PMCID: PMC4605514 DOI: 10.1371/journal.pone.0128708] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2014] [Accepted: 04/29/2015] [Indexed: 12/26/2022] Open
Abstract
Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens.
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Affiliation(s)
- P. Harkitis
- Department of Pharmacology, Faculty of Medicine, University of Ioannina, Ioannina GR-451 10, Greece
| | - E. P. Daskalopoulos
- Department of Pharmacology, Faculty of Medicine, University of Ioannina, Ioannina GR-451 10, Greece
| | - F. Malliou
- Department of Pharmacology, Faculty of Medicine, University of Ioannina, Ioannina GR-451 10, Greece
| | - M. A. Lang
- University of Queensland, National Research Centre for Environmental Toxicology (Entox), 39 Kessels Road, Coopers Plains, QLD 4108, Australia
| | - M. Marselos
- Department of Pharmacology, Faculty of Medicine, University of Ioannina, Ioannina GR-451 10, Greece
| | - A. Fotopoulos
- Department of Nuclear Medicine, Faculty of Medicine, University of Ioannina, Ioannina GR-451 10, Greece
| | - G. Albucharali
- Department of Nuclear Medicine, Faculty of Medicine, University of Ioannina, Ioannina GR-451 10, Greece
| | - M. Konstandi
- Department of Pharmacology, Faculty of Medicine, University of Ioannina, Ioannina GR-451 10, Greece
- * E-mail:
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Banerjee S, Das RK, Giffear KA, Shapiro BH. Permanent uncoupling of male-specific CYP2C11 transcription/translation by perinatal glutamate. Toxicol Appl Pharmacol 2015; 284:79-91. [PMID: 25697375 PMCID: PMC4374021 DOI: 10.1016/j.taap.2015.02.009] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2014] [Revised: 01/12/2015] [Accepted: 02/06/2015] [Indexed: 01/29/2023]
Abstract
Perinatal exposure of rats and mice to the typically reported 4mg/g bd wt dose of monosodium glutamate (MSG) results in a complete block in GH secretion as well as obesity, growth retardation and a profound suppression of several cytochrome P450s, including CYP2C11, the predominant male-specific isoform--all irreversible effects. In contrast, we have found that a lower dose of the food additive, 2mg/g bd wt on alternate days for the first 9days of life results in a transient neonatal depletion of plasma GH, a subsequent permanent overexpression of CYP2C11 as well as subnormal (mini) GH pulse amplitudes in an otherwise normal adult masculine episodic GH profile. The overexpressed CYP2C11 was characterized by a 250% increase in mRNA, but only a 40 to 50% increase in CYP2C11 protein and its catalytic activity. Using freshly isolated hepatocytes as well as primary cultures exposed to the masculine-like episodic GH profile, we observed normal induction, activation, nuclear translocation and binding to the CYP2C11 promoter of the GH-dependent signal transducers required for CYP2C11 transcription. The disproportionately lower expression levels of CYP2C11 protein were associated with dramatically high expression levels of an aberrant, presumably nontranslated CYP2C11 mRNA, a 200% increase in CYP2C11 ubiquitination and a 70-80% decline in miRNAs associated, at normal levels, with a suppression of CYP2C expression. Whereas the GH-responsiveness of CYP2C7 and CYP2C6 as well as albumin was normal in the MSG-derived hepatocytes, the abnormal expression of CYP2C11 was permanent and irreversible.
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Affiliation(s)
- Sarmistha Banerjee
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6009, USA
| | - Rajat Kumar Das
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6009, USA
| | - Kelly A Giffear
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6009, USA
| | - Bernard H Shapiro
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6009, USA.
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Konstandi M, Johnson EO, Lang MA. Consequences of psychophysiological stress on cytochrome P450-catalyzed drug metabolism. Neurosci Biobehav Rev 2014; 45:149-67. [DOI: 10.1016/j.neubiorev.2014.05.011] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2014] [Revised: 04/17/2014] [Accepted: 05/18/2014] [Indexed: 12/11/2022]
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Das RK, Banerjee S, Shapiro BH. Irreversible perinatal imprinting of adult expression of the principal sex-dependent drug-metabolizing enzyme CYP2C11. FASEB J 2014; 28:4111-22. [PMID: 24942648 DOI: 10.1096/fj.13-248864] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2014] [Accepted: 06/02/2014] [Indexed: 01/17/2023]
Abstract
We proposed to determine whether, like other sexual dimorphisms, drug metabolism is permanently imprinted by perinatal hormones, resulting in its irreversible sex-dependent expression. We treated newborn male rats with monosodium glutamate (MSG), a total growth hormone (GH) blocker, and, using cultured hepatocytes, examined expression of adult CYP2C11, the predominant cytochrome-P450 expressed only in males, as well as the signal transduction pathway by which episodic GH solely regulates the isoform's expression. In addition, adolescent hypophysectomized (hypox) male rats served as controls in which GH was eliminated after the critical imprinting period. Whereas renaturalization of the masculine episodic GH profile restored normal male-like levels of CYP2C11, as well as CYP2C12, in hepatocytes from hypox rats, the cells derived from the MSG-treated rats were completely unresponsive. Moreover, GH exposure of hepatocytes from hypox rats resulted in normal induction, activation, nuclear translocation, and binding to the CYP2C11 promoter of the signal transducers mediating GH regulation of CYP2C11 expression, which dramatically contrasted with the complete unresponsiveness of the MSG-derived hepatocytes, also associated with hypermethylation of GH-response elements in the CYP2C11 promoter. Lastly, neonatal MSG treatment had no adverse effect on postnatal and adult testosterone levels. The results demonstrate that the sexually dimorphic expression of CYP2C11 is irreversibly imprinted shortly after birth by a hormone other than the customary testosterone, but likely by GH.
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Affiliation(s)
- Rajat Kumar Das
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania, USA
| | - Sarmistha Banerjee
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania, USA
| | - Bernard H Shapiro
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania, USA
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Age-related changes in mRNA levels of hepatic transporters, cytochrome P450 and UDP-glucuronosyltransferase in female rats. Eur J Drug Metab Pharmacokinet 2014; 40:239-44. [PMID: 24899460 DOI: 10.1007/s13318-014-0208-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2013] [Accepted: 05/22/2014] [Indexed: 10/25/2022]
Abstract
Hepatic transporters and metabolic enzymes affect drug pharmacokinetics. Limited information exists on the alteration in mRNA levels of hepatic transporters and metabolic enzymes with aging. We examined the effects of aging on the mRNA levels of representative hepatic drug transporters and metabolic enzymes by analyzing their levels in 10-, 30- and 50-week-old male and female rats. Levels of mRNA of drug transporters including multidrug resistance protein (Mdr)1a, multidrug resistance-associated protein (Mrp)2, breast cancer resistance protein (Bcrp) and organic anion-transporting polypeptide (Oatp)1a1, and the metabolic enzymes cytochrome P450 (CYP)3A1, CYP3A2 and UDP-glucuronosyltransferase (UGT)1A1 were analyzed using real-time reverse transcriptase polymerase chain reaction. The mRNA levels of transporters in male rats did not decrease with age, while the mRNA levels of Bcrp and Oatp1a1 in female rats decreased with age. The mRNA levels of CYP3A1 and CYP3A2 in male rats were higher than those in female rats. The mRNA levels of metabolic enzymes decreased with age in female but not male rats. In particular, the mRNA levels of UGT1A1 in 10-week-old female rats were higher than those in male rats. mRNA expression of hepatic transporters and metabolic enzymes are more susceptible to aging in female than male rats. The age-related decreases in the mRNA levels of Bcrp, Oatp1a1, CYP3A1 and CYP3A2 in female rats may affect the metabolism and transport of substrates. This study showed that aging affected the mRNA expression of hepatic transporters and metabolic enzymes in rats.
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Konstandi M. Psychophysiological stress: a significant parameter in drug pharmacokinetics. Expert Opin Drug Metab Toxicol 2013; 9:1317-34. [DOI: 10.1517/17425255.2013.816283] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
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Thangavel C, Boopathi E, Shapiro BH. Inherent sex-dependent regulation of human hepatic CYP3A5. Br J Pharmacol 2013; 168:988-1000. [PMID: 22994453 PMCID: PMC3631386 DOI: 10.1111/j.1476-5381.2012.02222.x] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2012] [Revised: 09/05/2012] [Accepted: 09/11/2012] [Indexed: 11/28/2022] Open
Abstract
BACKGROUND AND PURPOSE Expression of hepatic cytochromes P450 (CYP) in all species examined, including humans, is generally sexually dimorphic. We examined the sex-dependent expression of CYP3A5 and the hormone-regulated molecular mechanism(s) responsible for any dimorphism. EXPERIMENTAL APPROACH CYP3A5 levels as well as nuclear translocation and promoter binding of transcription factors regulating CYP3A5 expression were measured in primary hepatocyte cultures derived from men and women exposed to physiological-like levels of growth hormone alone, dexamethasone alone and the combined regimen. KEY RESULTS We observed a dramatic inherent CYP3A5 sexual dimorphism (women > men) with all treatments as a result of a ~2-fold greater level of hormone-induced activation and nuclear accumulation of hepatocyte nuclear factor-4α (HNF-4α), pregnane X receptor (PXR) and retinoic X receptorα (RXRα) in female hepatocytes. Furthermore, PXR : RXRα exhibited significantly higher DNA binding levels to its specific binding motif on the CYP3A5 promoter in female hepatocytes, inferring a possible explanation for the elevated expression of the isoform in women. Results from experiments using HepG2 cells treated with siRNA-induced knockdown of HNF-4α and/or transfected with luciferase reporter constructs containing the CYP3A5 promoter were in agreement with the basic mechanism observed in primary hepatocytes of both sexes. CONCLUSIONS AND IMPLICATIONS Female-predominant expression of human CYP3A5 is due to an inherent, sex-dependent suboptimal activation of the transcription networks responsible for hormone-induced expression of the isoform in men. Accordingly, in conjunction with previous studies of other human CYPs, men and women are intrinsically unlikely to handle many drugs in the same way; thus, sex should be a requisite component factored into the design of personalized drug therapies.
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Affiliation(s)
- Chellappagounder Thangavel
- Laboratories of Biochemistry, School of Veterinary Medicine, University of PennsylvaniaPhiladelphia, PA, USA
| | - Ettickan Boopathi
- Department of Surgery, School of Medicine, University of PennsylvaniaPhiladelphia, PA, USA
| | - Bernard H Shapiro
- Laboratories of Biochemistry, School of Veterinary Medicine, University of PennsylvaniaPhiladelphia, PA, USA
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Abstract
AIM Identify sex- and hormone-independent housekeeping genes in rat liver by using a commercially available quantitative reverse transcription-polymerase chain reaction array designed to measure the expression of 32 rat housekeeping genes. RESULTS We found that the levels of five of the genes were sexually dimorphic, 22 genes were overexpressed, and one was underexpressed in multi-hormone-deficient hypophysectomized rats of both sexes. Only three genes fulfilled the stability criteria determined by geNorm and NormFinder as suitable housekeeping genes. Normalizing quantitative reverse transcription-polymerase chain reaction data with either of these three genes alone, the geometric means of any two of the genes, or even the geometric mean of all the three genes, produced similar results. In contrast, application of unproven housekeeping genes could lead to erroneous conclusions, having found that insulin-like growth factor 1 messenger RNA levels could be calculated dramatically either as male or as female predominant depending on the choice of housekeeping gene. CONCLUSION It is essential to validate the constancy of housekeeping genes under every experimental condition. (This research protocol was approved by the university's Institutional Animal Care and Use Committee.).
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Affiliation(s)
| | | | - Bernard H. Shapiro
- Corresponding author to whom proofs should be sent at the above address 215-898-1772 [telephone],
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Fu ZD, Csanaky IL, Klaassen CD. Effects of aging on mRNA profiles for drug-metabolizing enzymes and transporters in livers of male and female mice. Drug Metab Dispos 2012; 40:1216-25. [PMID: 22446518 PMCID: PMC3362785 DOI: 10.1124/dmd.111.044461] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2011] [Accepted: 03/23/2012] [Indexed: 11/22/2022] Open
Abstract
Aging is a physiological process characterized by progressive functional decline in various organs over time. To reveal possible molecular mechanisms of altered xenobiotic disposition and toxicity in elderly individuals, age-dependent mRNA profiles for 101 xenobiotic-processing genes (XPGs), including seven uptake transporters, 41 phase I enzymes, 36 phase II enzymes, 10 efflux transporters, and seven transcription factors, were characterized in livers of male and female mice from 3 to 27 months of age. Gender differences across the lifespan (significant at five ages or more) were observed for 52 XPGs, including 15 male-predominant genes (e.g., Oatp1a1, Cyp3a11, Ugt1a6a, Comt, and Bcrp) and 37 female-predominant genes (e.g., Oatp1a4, Cyp2b10, Sult1a1, Ugt1a1, and Mrp3). During aging, the mRNA levels for 44% of the 101 XPGs changed in male mice and 63% changed in female mice. In male mice, mRNA levels for 40 XPGs (e.g., Oatp1a1, Ces2c, Gstm4, Gstp1, and Ces1e) were lower in aged mice (more than 21 months of age), whereas mRNA levels for four XPGs (e.g., Oat2 and Gstm2) were higher in aged mice. In female mice, mRNA levels for 43 XPGs (e.g., Oatp1a1, Cyp1a2, Ces1f, Sult3a1, Gstt2, Comt, Ent1, Fmo3, and Mrp6) were lower in aged mice, whereas mRNA levels for 21 XPGs (e.g., Oatp1a4, Nqo1, Adh7, Sult2a1/2, Gsta1, and Mrp4) were higher in aged mice. In conclusion, 51% of the 101 XPGs exhibited gender differences in liver mRNA levels across the lifespan of mice; the mRNA levels for 40% of the XPGs were lower in aged male mice and 43% were lower in aged female mice.
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Affiliation(s)
- Zidong Donna Fu
- Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417, USA
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Thangavel C, Boopathi E, Shapiro BH. Intrinsic sexually dimorphic expression of the principal human CYP3A4 correlated with suboptimal activation of GH/glucocorticoid-dependent transcriptional pathways in men. Endocrinology 2011; 152:4813-24. [PMID: 21952236 PMCID: PMC3230058 DOI: 10.1210/en.2011-1274] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
Cytochrome P450 (CYP)3A4 is the principal and most abundant human isoform of CYP responsible for the metabolism of more than 50% of all consumed drugs and innumerable endogenous compounds. Expression of CYP3A4 is sexually dimorphic and regulated by the combined actions of GH and glucocorticoids. In the case of the rat, nearly all of the CYPs are "intrinsically" or "inherently" sexually dimorphic, meaning that the expressed sex differences are permanent and irreversible. Using primary hepatocyte cultures derived from men and women exposed to physiologic-like levels of continuous GH (the feminine circulating profile) alone, dexamethasone alone, and the combined regimen, we observed a dramatic inherent CYP3A4 sexual dimorphism (women more than men) with all treatments. The molecular basis for this intrinsic sexually dimorphic expression of CYP3A4 appears to be due, at least in part, to a greater level of hormone-dependent activation and nuclear translocation of both hepatocyte nuclear factor-4α (HNF-4α) and pregnane X receptor in female hepatocytes. Furthermore, these transcription factors exhibited significantly higher DNA binding levels to their specific motifs on the CYP3A4 promoter in female hepatocytes, inferring a possible explanation for the elevated expression of CYP3A4 in women. Accordingly, experiments using HepG2 cells treated with small inhibitory RNA-induced knockdown of HNF-4α and/or transfected with luciferase reporter constructs containing a CYP3A4 promoter lacking HNF-4α-binding motifs demonstrated that GH, to a greater extent dexamethasone, and to the greatest extent the combine hormone regimen, stimulated HNF-4α and pregnane X receptor promoter transactivation, signifying enhanced transcription of CYP3A4 and, thus, identifying a molecular mechanism contributing to the intrinsic sexual dimorphic expression of human CYP3A4.
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Affiliation(s)
- Chellappagounder Thangavel
- Laboratories of Biochemistry, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, Pennsylvania 19104-6009, USA
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Sharma MR, Dworakowski W, Shapiro BH. Intrasplenic transplantation of isolated adult rat hepatocytes: sex-reversal and/or suppression of the major constituent isoforms of cytochrome P450. Toxicol Pathol 2011; 40:83-92. [PMID: 22083583 DOI: 10.1177/0192623311425061] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Adult male and female rat hepatocytes were individually transplanted into the spleens of adult male and female rats. The recipients were euthanized at either eight, sixteen, thirty, or forty-five weeks following transplantation, at which time hepatic and splenic levels of liver-specific rat albumin mRNA as well as sex-dependent transcript levels of CYP2C11, -2C12, -2C7, -2A1, and -3A2-which accounts for > 60% of the total concentration of hepatic constituent cytochrome P450-were determined. Whereas the pre-infused hepatocytes expressed their expected cytochrome P450 sexual dimorphisms (female-specific CYP2C12, male-specific CYP3A2, and female-predominant CYP2A1), their post-transplantational competence now reflected the sexual dimorphisms of the recipient (as observed in the host's liver), which supports the concept that the sex-dependent growth hormone circulating profiles are the determinants regulating the expression levels of hepatic cytochrome P450. Also expressed at normal concentrations in the pre-infused hepatocytes, male-specific CYP2C11 and female-predominant CYP2C7 were inexplicably undetectable in the spleens of both recipient males and females, regardless of the sex of the donor hepatocytes, almost one year after transplantation.
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Affiliation(s)
- Meena R Sharma
- Laboratories of Biochemistry, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, Pennsylvania 19104-6048, USA
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Sun D, Jiang H, Wu H, Yang Y, Kaley G, Huang A. A novel vascular EET synthase: role of CYP2C7. Am J Physiol Regul Integr Comp Physiol 2011; 301:R1723-30. [PMID: 21940400 DOI: 10.1152/ajpregu.00382.2011] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
We demonstrated previously that cytochrome P-450 (CYP) 2C29 is the epoxyeicosatrienoic acid (EET) synthase responsible for the EET-mediated flow/shear stress-induced dilation of vessels of female nitric oxide (NO)-deficient mice (Sun D, Yang YM, Jiang H, Wu H, Ojami C, Kaley G, Huang A. Am J Physiol Regul Integr Comp Physiol 298: R862-R869, 2010). In the present study, we aimed to identify which specific CYP isoform(s) is the source of the synthesis and release of EETs in response to stimulation by shear stress in vessels of rats. Cannulated mesenteric arteries isolated from both sexes of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats were perfused with 2 and 10 dyn/cm(2) shear stress, followed by collection of the perfusate to determine EET concentrations and isoforms. Shear stress stimulated release of EETs in the perfusate of female (but not male) NO-deficient vessels, associated with an EET-mediated vasodilation, in which 11,12- and 14,15-EET contributed predominantly to the responses. Rat CYP cDNA array screened a total of 32 CYP genes of mesenteric arteries, indicating a significant upregulation of CYP2C7 in female L-NAME-treated rats. Endothelial RNA and protein were extracted from intact single vessels. Expression of CYP2C7 mRNA and protein in pooled extractions of endothelial lysate was identified by PCR and Western blot analyses. Transfection of the vessels with CYP2C7 short interfering RNA eliminated the release of EETs, consequently abolishing the EET-mediated flow-induced dilation; these responses, however, were maintained in vessels transfected with nonsilencing short interfering RNA. Knockdown of endothelial CYP2C7 was confirmed by PCR and Western blot analyses. In conclusion, CYP2C7 is an endothelial EET synthase in the female rat vasculature, by which, in NO deficiency, shear stress stimulates the release of EETs to initiate vasodilation.
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Affiliation(s)
- Dong Sun
- Department of Physiology, New York Medical College, Valhalla, New York 10595, USA
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Nagahori H, Matsunaga H, Tomigahara Y, Isobe N, Kaneko H. Metabolism of 2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3-[5-(trifluoromethyl)-2-pyridyloxy]propyl ether (pyridalyl) in rats after repeated oral administration and a simple physiologically based pharmacokinetic modeling in brown and white adipose tissues. Drug Metab Dispos 2010; 38:824-32. [PMID: 20164113 DOI: 10.1124/dmd.109.031914] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/13/2025] Open
Abstract
Male and female Sprague-Dawley rats received repeated oral administration of 14C-2,6-dichloro-4-(3,3-dichloroallyloxy)phenyl 3- [5-(trifluoromethyl)-2-pyridyloxy]propyl ether (14C-pyridalyl) at 5 mg/kg/day for 14 consecutive days, and 14C excretion, 14C concentration in tissues, and the metabolic fate were determined. Most 14C was excreted into feces. The 14C concentrations in the blood and tissues attained steady-state levels at days 6 to 10, whereas those in white adipose tissues increased until day 14. Tissue 14C concentrations were highest in brown and white adipose tissue (38.37-57.50 ppm) but were 5.60 ppm or less in all the other tissues. Total 14C residues in blood and tissues on the 27th day after the first administration accounted for 2.6 to 3.2% of the total dose. A major fecal metabolite resulted from O-dealkylation. Analysis of metabolites in tissues revealed that the majority of 14C in perirenal adipose tissue and lungs was pyridalyl, accounting for greater than 90 and 60%, respectively, of the total, whereas a major metabolite in whole blood, kidneys, and liver was a dehalogenated metabolite. The experimental data were simulated with simple physiologically based pharmacokinetics using four-compartment models with assumption of lymphatic absorption and membrane permeability in adipose tissues. The different kinetics in brown and white adipose tissues was reasonably predicted in this model, with large distribution volume in adipose tissues and high hepatic clearance in liver. Sex-related difference of pyridalyl concentration in liver was considered to be a result of different unbound fraction times the hepatic intrinsic clearance (f x CL(int)) of 1.8 and 12 l/h for male and female, respectively.
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Affiliation(s)
- Hirohisa Nagahori
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 1-98, 3-Chome, Kasugade-Naka, Konohana-Ku, Osaka 554-8558, Japan.
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Wauthier V, Sugathan A, Meyer RD, Dombkowski AA, Waxman DJ. Intrinsic sex differences in the early growth hormone responsiveness of sex-specific genes in mouse liver. Mol Endocrinol 2010; 24:667-78. [PMID: 20150183 PMCID: PMC2840812 DOI: 10.1210/me.2009-0454] [Citation(s) in RCA: 66] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2009] [Accepted: 12/30/2009] [Indexed: 11/19/2022] Open
Abstract
Sex differences in liver gene expression are dictated by sex differences in circulating GH profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that could contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex differences characterize hepatic responses to plasma GH stimulation. Global RNA expression analysis identified two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class I) and genes subject to negative regulation by pituitary hormones (class II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90 min of GH pulse treatment at a physiological dose were identified as putative direct targets of GH action (early response genes). Intrinsic sex differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were induced by GH within 30 min in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor myocyte enhancer factor 2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex differences in predisposition to liver cancer or other hepatic patho-physiologies.
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Affiliation(s)
- Valerie Wauthier
- Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, Massachusetts 02215, USA
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20
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Abstract
Abstract
We report here a novel observation that zolmitriptan induced CYP3A2 in male but not female rats. As part of our research programme to evaluate sex differences in the response to zolmitriptan, we studied the effects of zolmitriptan on CYP3A activity, protein and gene expression in male and female rats. Zolmitriptan was found to induce CYP3A activity, measured as testosterone and diazepam metabolism in-vitro, as well as midazolam pharmacokinetics in-vivo, in male but not female rats. The sex difference in response to zolmitriptan was further evaluated by analysis of CYP3A1/2 mRNA levels using real-time PCR, and CYP3A1/2 protein levels using immunoblotting. Zolmitriptan preferentially induced CYP3A2 in male but not female rats. No obvious effects on CYP3A1 were observed at any dose in either sex. Thus, we concluded that the observed sex-dependent induction of CYP3A by zolmitriptan was largely due to induction of CYP3A2 in male rats.
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Affiliation(s)
- Lushan Yu
- Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Sijie Lu
- Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Naping Zhao
- Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Shaoqing Ni
- Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Tongwei Yao
- Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
| | - Su Zeng
- Department of Pharmaceutical Analysis and Drug Metabolism, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China
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Waxman DJ, Holloway MG. Sex differences in the expression of hepatic drug metabolizing enzymes. Mol Pharmacol 2009; 76:215-28. [PMID: 19483103 PMCID: PMC2713118 DOI: 10.1124/mol.109.056705] [Citation(s) in RCA: 535] [Impact Index Per Article: 33.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2009] [Accepted: 05/29/2009] [Indexed: 12/26/2022] Open
Abstract
Sex differences in pharmacokinetics and pharmacodynamics characterize many drugs and contribute to individual differences in drug efficacy and toxicity. Sex-based differences in drug metabolism are the primary cause of sex-dependent pharmacokinetics and reflect underlying sex differences in the expression of hepatic enzymes active in the metabolism of drugs, steroids, fatty acids and environmental chemicals, including cytochromes P450 (P450s), sulfotransferases, glutathione transferases, and UDP-glucuronosyltransferases. Studies in the rat and mouse liver models have identified more than 1000 genes whose expression is sex-dependent; together, these genes impart substantial sexual dimorphism to liver metabolic function and pathophysiology. Sex differences in drug metabolism and pharmacokinetics also occur in humans and are due in part to the female-predominant expression of CYP3A4, the most important P450 catalyst of drug metabolism in human liver. The sexually dimorphic expression of P450s and other liver-expressed genes is regulated by the temporal pattern of plasma growth hormone (GH) release by the pituitary gland, which shows significant sex differences. These differences are most pronounced in rats and mice, where plasma GH profiles are highly pulsatile (intermittent) in male animals versus more frequent (nearly continuous) in female animals. This review discusses key features of the cell signaling and molecular regulatory mechanisms by which these sex-dependent plasma GH patterns impart sex specificity to the liver. Moreover, the essential role proposed for the GH-activated transcription factor signal transducer and activator of transcription (STAT) 5b, and for hepatic nuclear factor (HNF) 4alpha, as mediators of the sex-dependent effects of GH on the liver, is evaluated. Together, these studies of the cellular, molecular, and gene regulatory mechanisms that underlie sex-based differences in liver gene expression have provided novel insights into the physiological regulation of both xenobiotic and endobiotic metabolism.
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Affiliation(s)
- David J Waxman
- Division of Cell and Molecular Biology, Department of Biology, Boston University, Boston, MA 02215, USA.
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Abstract
Metabolism of steroids and drugs in rodents is sexually differentiated. The reason for this turned out to be the sexually differentiated growth hormone (GH) secretory pattern regulating the expression of a number of hepatic cytochrome P-450 genes. Although not fully resolved, it is clear that several signaling pathways and transcription factors are involved in mediating the effects of GH. It may be argued that such a well-controlled physiological system should have an important biological role and we speculate that the demands of a robust hepatic steroid metabolism during pregnancy has led to the development of this sexually differentiated hypothalamo-pituitary-liver axis.
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Affiliation(s)
- Agneta Mode
- Department of Medical Nutrition, Karolinska Institute, Novum, Huddinge, Sweden.
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Thangavel C, Shapiro BH. Inherent sexually dimorphic expression of hepatic CYP2C12 correlated with repressed activation of growth hormone-regulated signal transduction in male rats. Drug Metab Dispos 2008; 36:1884-95. [PMID: 18559485 PMCID: PMC2656384 DOI: 10.1124/dmd.108.021451] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Because of its myriad physiologic functions, it is not surprising that the actions of growth hormone (GH) are mediated by recruiting/activating dozens of signaling molecules involved in numerous transduction pathways. The particular signal transduction pathway activated by the hormone is determined by the affected target cell, the sexually dimorphic secretory GH profile (masculine episodic or feminine continuous) to which the cell is exposed, and the individual's sex. In this regard, expression of female-specific CYP2C12, the most abundant cytochrome P450 in female rat liver, is solely regulated by the feminine GH profile. Sex is a modulating factor in this response in that males are considerably less responsive than females to the CYP2C12-induction effects of continuous GH. Using primary hepatocytes derived from male and female hypophysectomized rats, we have identified several factors in a transduction pathway activated by the feminine GH regime and associated with the induction of hepatic CYP2C12. Elements in the proposed pathway, in their likely order of activation, are the growth hormone receptor, extracellular signal-regulated kinases, the cAMP-response element-binding protein, and hepatocyte nuclear factors 4alpha and 6, which subsequently bind and activate the CYP2C12 promoter. Recruitment and/or activation levels of all of the component factors in the pathway were highly suppressed in male hepatocytes, possibly explaining the dramatically lower induction levels of CYP2C12 in males exposed to the same continuous GH profile as females.
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Wauthier V, Waxman DJ. Sex-specific early growth hormone response genes in rat liver. Mol Endocrinol 2008; 22:1962-74. [PMID: 18483176 PMCID: PMC2725767 DOI: 10.1210/me.2007-0549] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2007] [Accepted: 05/09/2008] [Indexed: 11/19/2022] Open
Abstract
Pituitary GH-secretory profiles are sex dependent and regulate the sexually dimorphic expression of a large number of genes in the liver. The slow response of many sex-specific liver genes to changes in plasma GH status suggests that GH acts in the liver via both direct and indirect mechanisms organized in a hierarchical regulatory network. Presently, genome-wide liver transcription profiling was conducted to elucidate the global impact of pituitary hormone ablation on the sex specificity of rat liver gene expression and to identify sex-specific genes that respond rapidly to GH as candidates for direct targets of GH action. Hypophysectomy abolished the sex specificity of approximately 90% of 1032 sex-dependent genes, consistent with the dominant role of pituitary GH in regulating liver sexual dimorphism. Two major classes of sex-specific genes were identified: genes that were down-regulated after hypophysectomy and may be subject to positive GH regulation (461 class I genes), and genes that were up-regulated after hypophysectomy and may be subject to negative GH regulation (224 class II genes). Fifty class I sex-specific genes were induced, and 38 class II sex-specific genes were suppressed within 90 min of a physiological GH pulse, suggesting they are primary GH response genes. A further 71 sex-specific genes responded after a second GH treatment and may correspond to secondary response genes. Twenty four DNA-binding proteins were identified as early GH response genes, of which 15 were induced and nine were suppressed by GH. Five of these 24 genes displayed sex-specific expression, consistent with a hierarchical transcriptional network controlling sex-specific liver gene expression. Class II male-specific genes, such as Cyp2a2 and Cyp2c13, were down-regulated within 30 min of GH pulse treatment, as determined by heterogeneous nuclear RNA analysis, suggesting that transcription of these genes is restricted to the GH-free interpulse period in adult male rat liver. We conclude that GH acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression.
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Affiliation(s)
- Valerie Wauthier
- Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215, USA
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Thangavel C, Dhir RN, Volgin DV, Shapiro BH. Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone. Biochem Pharmacol 2007; 74:1476-84. [PMID: 17868651 PMCID: PMC2701361 DOI: 10.1016/j.bcp.2007.07.035] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2007] [Revised: 07/25/2007] [Accepted: 07/25/2007] [Indexed: 01/22/2023]
Abstract
CYP2C11, the most commonly expressed isoform of cytochrome P450 in male rat liver, was measured in spleen, thymus and bone marrow by quantitative real-time PCR and enhanced Western blotting. CYP2C11 concentrations in the lymphoid tissues were a fraction of that observed in liver, but like the liver, were sexually dimorphic (M>F) with mRNA and protein levels in agreement. Although the response to hypophysectomy varied according to tissue and sex, expression levels of CYP2C11 in all measured tissues remained greater in males. Further differences in CYP2C11 expression between liver and lymphoid tissue were observed following restoration of the circulating masculine growth hormone profile in hypophysectomized rats. In contrast to the liver where the renaturalized growth hormone profile elevated CYP2C11 expression in both sexes, the response was opposite in spleen and thymus with isoform concentrations declining in both sexes. Lastly, the divergent response of CYP2C11 between the liver and immune system was examined in cultured splenocytes exposed to different mitogens. In contrast to the dramatic depletion of CYP2C11 reported in proliferating hepatocytes, mitogen-stimulation resulted in a significant elevation in splenocyte CYP2C11 expression. In summary, we report for the first time that thymus, spleen and bone marrow express, albeit nominal, sex-dependent levels of CYP2C11 (M>F) whose regulation appears to be under some hormonal control, but very different from that of the hepatic isoform.
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Affiliation(s)
- Chellappagounder Thangavel
- Laboratories of Biochemistry, Univeristy of Pennsylvania, School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6048, USA
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Wauthier V, Dubois P, Verbeeck RK, Calderon PB. Induction of CYP2C12 expression in senescent male rats is well correlated to an increase of HNF3beta expression, while the decline of CYP2C11 expression is unlikely due to a decrease of STAT5 activation. Biochem Pharmacol 2006; 73:923-33. [PMID: 17239351 DOI: 10.1016/j.bcp.2006.12.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2006] [Revised: 11/16/2006] [Accepted: 12/04/2006] [Indexed: 11/22/2022]
Abstract
Ageing affects drugs metabolism influencing the therapeutic efficacy and safety of drugs. By using the experimental model of aged male rats, we investigated the influence of ageing on some CYP2C isoforms, the most important CYP450 sub-family in rats. The activity of the male specific CYP2C11 is decreased by 55% in senescent male rats. This correlates with a significant reduction of both protein content (80%) and mRNA (60%) indicating a demasculinization process. The expression of CYP2C12, a female specific isoform, is induced in senescent male rats indicating a feminization process. Neither the activity nor the expression of CYP2C6, a female predominant isoform, is modified in senescent male rats. Thereafter, certain putative GH mediators like some liver enriched transcription factors (LETFs) or STAT5b were investigated. The amount of HNF3beta mRNA, a transcription factor involved in the up-regulation of CYP2C12, has been shown to increase by about three-fold in senescent male rats. With regard to STAT5b, which has been reported to be involved in the male specific regulation of CYP2C11, large amounts of phosphorylated STAT5 were observed in the liver of senescent male rats. These results indicate that while the induction of CYP2C12 during ageing could be due, at least partially, to the enhanced HNF3beta expression, the decline of CYP2C11 is unlikely related to a decrease of STAT5 activation.
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Affiliation(s)
- Valérie Wauthier
- Unité de Pharmacocinétique, Métabolisme, Nutrition, et Toxicologie (PMNT), Département des sciences pharmaceutiques, Université Catholique de Louvain, Brussels, Belgium
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Wiwi CA, Waxman DJ. Role of Hepatocyte Nuclear Factors in Transcriptional Regulation of Male-specific CYP2A2. J Biol Chem 2005; 280:3259-68. [PMID: 15539409 DOI: 10.1074/jbc.m409294200] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Cytochrome P450 2A2 (CYP2A2) is an adult male-specific rat liver steroid hydroxylase whose sex-dependent expression is regulated at the transcriptional level by sexually dimorphic pituitary growth hormone (GH) secretory patterns. In contrast to CYP2C11 and other male-specific, plasma GH pulse-inducible liver genes, CYP2A2 is highly expressed in hypophysectomized rat liver, despite the absence of GH stimulation. CYP2A2 promoter fragments 0.9-6.2 kb long exhibited unusually high basal promoter activity when transfected into the liver cell line HepG2. A further approximately 2.5-fold increase in activity was obtained by cotransfection of hepatocyte nuclear factor (HNF) 3gamma or HNF4alpha. CYP2A2 promoter activity was inhibited approximately 85% by transfection of HNF3beta or HNF6, both of which are more highly expressed in female than male liver and can strongly trans-activate the female-specific CYP2C12 promoter. The male GH pulse-activated transcription factor STAT5b had no effect on CYP2A2 promoter activity, either alone or in combination with HNF3gamma and HNF4alpha, consistent with the GH pulse-independence of CYP2A2 expression. By contrast, STAT5b synergistically enhanced the transcriptional activity of HNF4alpha toward two other male-specific liver target genes, Cyp2d9 and CYP8B1. Furthermore, STAT5b in combination with the HNF4alpha coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha strongly enhanced the transcriptional activity of HNF4alpha toward CYP8B1 but not toward CYP2A2. These findings support the hypothesis that sex-dependent HNFs contribute to the sexually dimorphic expression of CYP2A2 and other liver CYPs and highlight the ability of STAT5b to act in concert with HNF4alpha to regulate select male-specific liver CYP genes.
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Affiliation(s)
- Christopher A Wiwi
- Division of Cell and Molecular Biology, Department of Biology Boston University, Boston, Massachusetts 02215, USA
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Sierra-Santoyo A, Hernández M, Albores A, Cebrián ME. DDT increases hepatic testosterone metabolism in rats. Arch Toxicol 2004; 79:7-12. [PMID: 15372139 DOI: 10.1007/s00204-004-0603-y] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2004] [Accepted: 08/10/2004] [Indexed: 11/25/2022]
Abstract
DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-14C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2alpha-hydroxytestosterone (OHT), and 16alpha-OHT but higher 6beta-OHT whereas treated females produced less 7alpha-OHT and AD but higher 6beta-OHT and 6alpha-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6beta-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6alpha-/15alpha-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6alpha-/16alpha-OHT and 6-dehydrotestosterone/16alpha-OHT ratios followed a similar tendency, with the ratio 6alpha-/16alpha-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorphism in testosterone metabolism and decreased the metabolic differences between male and female rats.
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Anakk S, Kalsotra A, Kikuta Y, Huang W, Zhang J, Staudinger JL, Moore DD, Strobel HW. CAR/PXR provide directives for Cyp3a41 gene regulation differently from Cyp3a11. THE PHARMACOGENOMICS JOURNAL 2004; 4:91-101. [PMID: 14770174 DOI: 10.1038/sj.tpj.6500222] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
This study reports that Cyp3a41 gene contains 13 exons and is localized on the chromosome 5. CYP3A41 is a female-specific isoform that is predominantly expressed in the liver. Estrogen signaling is not responsible for its female specificity. CYP3A41 expression in kidney and brain is observed only in 50% of mice examined. PXR mediates dexamethasone-dependent suppression of CYP3A41. In contrast to CYP3A11, CYP3A41 expression is not induced by pregnenolone-16alpha-carbonitrile (PCN) in wild-type mice, but is significantly suppressed by PCN in PXR(-/-) mice. Phenobarbital and TCPOBOP induce CYP3A11 expression only in the presence of CAR, but have no effect on CYP3A41 expression. Immunoblot and erythromycin demethylase activity analysis reveal robust CYP3A induction after PCN treatment, which is poorly correlated to CYP3A41. These findings suggest a differential role for CAR/PXR in regulating individual CYP3A isoforms by previously characterized CYP3A inducers.
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MESH Headings
- Animals
- Aryl Hydrocarbon Hydroxylases/biosynthesis
- Aryl Hydrocarbon Hydroxylases/genetics
- Constitutive Androstane Receptor
- Cytochrome P-450 CYP3A
- Cytochrome P-450 Enzyme System/biosynthesis
- Cytochrome P-450 Enzyme System/genetics
- Dexamethasone/pharmacology
- Dose-Response Relationship, Drug
- Female
- Gene Expression Regulation, Enzymologic/drug effects
- Gene Expression Regulation, Enzymologic/physiology
- Isoenzymes/biosynthesis
- Isoenzymes/genetics
- Isoenzymes/physiology
- Male
- Membrane Proteins
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Microsomes, Liver/drug effects
- Microsomes, Liver/enzymology
- Oxidoreductases/biosynthesis
- Oxidoreductases/genetics
- Oxidoreductases, N-Demethylating/biosynthesis
- Oxidoreductases, N-Demethylating/genetics
- Pregnane X Receptor
- Receptors, Cytoplasmic and Nuclear/biosynthesis
- Receptors, Cytoplasmic and Nuclear/deficiency
- Receptors, Cytoplasmic and Nuclear/genetics
- Receptors, Cytoplasmic and Nuclear/physiology
- Receptors, Steroid/biosynthesis
- Receptors, Steroid/deficiency
- Receptors, Steroid/genetics
- Receptors, Steroid/physiology
- Transcription Factors/deficiency
- Transcription Factors/genetics
- Transcription Factors/physiology
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Affiliation(s)
- S Anakk
- Department of Biochemistry & Molecular Biology, The University of Texas Medical School of Houston, TX 77225, USA
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30
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Dhir RN, Shapiro BH. Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats. Proc Natl Acad Sci U S A 2003; 100:15224-8. [PMID: 14638941 PMCID: PMC299965 DOI: 10.1073/pnas.2434273100] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Humans as well as other mammals experience an aging-related decline in drug metabolism as well as a diminution in growth hormone secretion. In the case of rats, these events are more pronounced in senescent males, whose expression of male-specific isoforms of cytochrome P450, the major drug-metabolizing enzymes and constituting approximately 60-70% of the total cytochrome P450 in male rat liver, is completely suppressed, whereas female-dependent isoforms are remarkably induced to female-like levels. Overlooked in these independently reported studies is the fact that "signals" inherent in the masculine episodic and female continuous growth hormone profiles regulate expression and/or suppression of the dozen or so sex-dependent cytochrome P450 isoforms in rat liver. Whereas previous studies identified profound reductions in the pulse amplitudes of the masculine growth hormone profile as the cause for the diminished hormone secretion during aging, pulse heights are not recognized by the cytochromes as regulatory signals. Instead, we have shown that just a nominal secretion of growth hormone during the usual growth hormone-devoid interpulse period in the masculine episodic profile can explain the complete repression of male-specific CYP2C11, CYP3A2, and CYP2A2 and induction of female-dependent CYP2C12, CYP2C6, and CYP2A1 observed in senescent male rats.
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Affiliation(s)
- Ravindra N Dhir
- Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce Street, Philadelphia, PA 19104-6048, USA
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31
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Ishizuka M, Yonemoto J, Zaha H, Tohyama C, Sone H. Perinatal exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters sex-dependent expression of hepatic CYP2C11. J Biochem Mol Toxicol 2003; 17:278-85. [PMID: 14595850 DOI: 10.1002/jbt.10090] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
The cytochrome P450 (CYP) isoform CYP2C11 is specifically expressed in the liver of adult male rats, and 5alpha-reductase is specifically expressed in the liver of the adult female rats. The sexually dimorphic expressions of these hepatic enzymes are regulated by the sex-dependent profiles of the circulating growth hormone (GH). However, it is not well known whether hormonal imprinting or activation factors in the neonatal brain influence the sexually dimorphic expression patterns of hepatic enzymes. We therefore examined the effect of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sex-dependent expressions of hepatic enzymes. Pregnant rats were treated with TCDD at a dose of 0, 200, or 800 ng/kg on gestation day 15, exposing the pups to the chemical. Although the expression of CYP2C11 protein in the livers of male pups on postnatal day (PND) 49 was significantly higher than that of the controls, but the 5alpha-reductase activities in the livers of female pups were not altered by exposure to TCDD. Focusing on perinatal periods, testosterone and estrogen levels significantly increased in the brain of male pups on PND 2. The results suggest that the alteration of testosterone and estrogen levels affect hormonal imprinting in the neonatal brain of male pups, and thus induces a change in the level of male-specific hepatic CYP2C11. We conclude that perinatal exposure to TCDD at low doses may change the sexual differentiation of the neonatal brain in male rats.
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Affiliation(s)
- Mayumi Ishizuka
- Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba 305-8506, Japan
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32
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Skálová L, Wsól V, Baliharová V, Král R, Szotáková B, Velík J, Lamka J. Reduction of flobufen in pig hepatocytes: effect of pig breed (domestic, wild) and castration. Chirality 2003; 15:213-9. [PMID: 12582986 DOI: 10.1002/chir.10182] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
Knowledge of the biotransformation processes of veterinary drugs and food supplements in food-producing animals is increasingly important. Residual levels of parent compounds or their metabolites in food of animal origin may differ with the breed, breeding conditions, and gender of animals. The nonsteroidal antiinflammatory drug flobufen, 4-(2',4'-difluorobiphenyl-4-yl)-2-methyl-4-oxobutanoic acid (racemic or its individual enantiomers) was used as a model to evaluate differences in activity, stereoselectivity, and stereospecificity of reductases in primary cultures of hepatocytes from intact male or castrated male domestic pigs (Sus scrofa domestica) or male wild pig (Sus scrofa scrofa). Time-dependent consumption of flobufen enantiomers and formation of dihydroflobufen (DHF) diastereoisomers as their principal metabolites in hepatocytes were measured using chiral HPLC. Flobufen reduction in hepatocytes from all three experimental groups of animals was stereoselective ((+)-R-flobufen was predominantly metabolized) and stereospecific (2R;4S-DHF and 2S;4S-DHF diastereoisomers were preferentially formed). Flobufen reductases activity in male domestic pigs was 30 times higher compared to castrated pigs. Flobufen reductases activity was similar in domestic and wild pigs. The stereospecificity and stereoselectivity of DHF production did not significantly differ with breed or castration of animal. Chiral inversion of flobufen enantiomers was also studied and differences between castrated and intact male pigs were seen.
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Affiliation(s)
- Lenka Skálová
- Department of Biochemical Sciences, Research Centre LN00B125, Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Hradec Králové, Czech Republic.
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Abstract
The hepatotropic viruses, measles, and herpesviruses as well as different drugs were repeatedly shown to act presumably as a trigger in patients with autoimmune hepatitis (AI-H). On the other hand, it is known that viral infections stimulate interferon production, which inactivates the cytochrome P-450 enzymes involved in the metabolism of several endogenous substances and exogenous environmental agents. Moreover, it was reported that several cytokines, including interferons, as well as transforming growth factor beta1 and human hepatocyte growth factor, which are abundantly produced and released in the body during infections, also downregulated expression of major cytochrome P-450 and/or other biotransformation enzymes. It seems that all these factors, in addition to individual immune response and the nature and amount of the neoantigen(s) produced, impair the equilibrium of bioactivation and detoxication pathways, thus leading to the development of AI-H in a genetically predisposed person continually exposed to harmful environmental factor(s). Possible increased/decreased density of lysine residues at position D-related human leukocyte antigen locus (DR)beta71 of the antigen-binding groove may affect the eventual steroid-sparing effect of this critical amino acid at the cellular level. In addition, some food additives, such as monosodium glutamate (MSG) and/or aspartame regularly consumed in excessive amounts, may eventually disturb the delicate balance between a positively charged amino acid residue at position DRbeta71 (lysine or arginine) and a negatively charged amino acid residue at position P4 on the antigenic peptide (glutamic acid or aspartic acid). This may favor formation of a salt bridge between these amino acid residues within the hypervariable region 3 on the alpha-helix of the DRbeta polypeptide and facilitate autoantigen presentation and CD4 T-helper cell activation. MSG and aspartate may also depress serum concentrations of growth hormone, which downregulate the activity of several cytochrome P-450 hepatic and other drug-metabolizing enzymes, thus increasing sensitivity to some environmental agents and possibly influencing efficacy of treatment regimens and final outcome of patients with type 1 AI-H.
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Affiliation(s)
- Joseph Prandota
- Department of Public Health, University Medical School, Wroclaw, Poland.
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Jarukamjorn K, Sakuma T, Nemoto N. Sexual dimorphic expression of mouse hepatic CYP2B: alterations during development or after hypophysectomy. Biochem Pharmacol 2002; 63:2037-41. [PMID: 12093481 DOI: 10.1016/s0006-2952(02)00989-9] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The constitutive expression of CYP2B mRNA in the livers of mice in the prepubertal stage was sex-independent, with CYP2B9 as the principal isoform. During the maturation stage, CYP2B10 was expressed in both sexes, whereas CYP2B9 was diminished markedly in the males, resulting in a sexually dimorphic expression in adult mice. Hypophysectomy eliminated the sexual dimorphism in the mouse CYP2B subfamily by markedly increasing the expression of both CYP2B9 and CYP2B10 in males to levels similar to those in females.
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Affiliation(s)
- Kanokwan Jarukamjorn
- Department of Toxicology, Faculty of Pharmaceutical Sciences, Toyama Medical & Pharmaceutical University, 2630 Sugitani, Toyama, Japan.
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Iatsimirskaia EA, Gregory ML, Anderes KL, Castillo R, Milgram KE, Luthin DR, Pathak VP, Christie LC, Vazir H, Anderson MB, May JM. Effect of testosterone suppression on the pharmacokinetics of a potent gnRH receptor antagonist. Pharm Res 2002; 19:202-8. [PMID: 11883648 DOI: 10.1023/a:1014281018271] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
PURPOSE The expression of cytochrome P450 enzymes (CYPs) in animals and humans is under complex hormonal regulation. Chronic treatment with drugs that alter sex hormone levels such as GnRH receptor agonists or antagonists may affect the expression of hormone-dependent CYPs, and as a result the pharmacokinetics of drugs metabolized by them. METHODS Enzyme kinetic parameters were obtained by incubating AG-045572 (0.1-30 microM) with human or rat liver microsomes, or expressed CYP3A4 and CYP3A5. The pharmacokinetics of AG-045572 (10 mg/kg i.v. or 20 mg/kg p.o.) were studied in intact male, female, castrated male and male rats pretreated with AG-045572 for 4 days. RESULTS AG-045572 is metabolized by CYP3A in both rats and humans. The Km values were similar in male and female human, female rat liver microsomes, and expressed CYP3A4 and CYP3A5 (0.39, 0.27, 0.28, 0.25, and 0.26 microM, respectively). The Km in male rat liver microsomes was 1.5 microM, suggesting that in male and female rats AG-045572 is metabolized by different CYP3A isozymes. The oral bioavailability of AG-045572 in intact male rats was 8%, while in female or castrated male rats it was 24%. Pretreatment of intact male rats with AG-045572 i.m. for 4 days resulted in suppression of testosterone to castrate levels, accompanied by an increase in oral bioavailability of AG-045572 to 27%. In the same experiment, the male-specific pulsatile pattern of growth hormone remained unchanged with slightly elevated baseline levels. CONCLUSIONS The potent GnRH receptor antagonist AG-045572 is metabolized by hormone-dependent CYP3A. As a result, suppression of testosterone by pretreatment with AG-045572 "feminized" its own pharmacokinetics.
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Affiliation(s)
- Eugenia A Iatsimirskaia
- Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development/Agouron Pharmaceuticals, Inc, San Diego, California 92121, USA.
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36
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Yamada A, Yamada M, Fujita Y, Nishigami T, Nakasho K, Uematsu K. Self-augmentation effect of male-specific products on sexually differentiated progesterone metabolism in adult male rat liver microsomes. J Biol Chem 2001; 276:4604-10. [PMID: 10995741 DOI: 10.1074/jbc.m003355200] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
It is well known that several 3-keto-4-ene steroids such as progesterone and testosterone are metabolized in a gender-specific or -predominant manner by adult rat liver microsomes. In the male, these steroids are primarily metabolized into two oxidized (16alpha-hydroxyl and 6beta-hydroxyl) products mainly by the respective, male-specific cytochrome P450 subforms, CYP2C11 and CYP3A2, while they are primarily metabolized into the 5alpha-reduced products by female-predominant 5alpha-reductase in the female. These sexually differentiated enzyme activities are largely regulated at the transcription level under endocrine control. In the present study, we show that unlabeled 16alpha-hydroxyprogesterone and 6beta-hydroxyprogesterone inhibited the 5alpha-reductive [(3)H]progesterone metabolism by adult male rat liver microsomes without significantly inhibiting the CYP2C11 and CYP3A2 activities producing themselves, whereas 3alpha-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnane-3,20-dione not only stimulated the 5alpha-reductive metabolism producing themselves but also inhibited the male-specific oxidative metabolism. This finding compels us to propose a novel hypothesis that adult male rat liver microsomes may possess a self-augmentation system regulated by the male-specific products on sexually differentiated steroid metabolism, besides regulation by gene expressions of the related enzymes.
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Affiliation(s)
- A Yamada
- Second Department of Pathology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan.
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Pampori NA, Shapiro BH. Nominal growth hormone pulses in otherwise normal masculine plasma profiles induce intron retention of overexpressed hepatic CYP2C11 with associated nuclear splicing deficiency. Endocrinology 2000; 141:4100-6. [PMID: 11089541 DOI: 10.1210/endo.141.11.7751] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Restoration of circulating masculine GH profiles at minipulse amplitudes (i.e. approximately 10% of normal) to hypophysectomized male rats and neonatal administration of monosodium glutamate (MSG), producing a similar plasma GH profile, both result in an overexpression (approximately 200-300%) of CYP2C11 messenger RNA (mRNA), the predominant hepatic cytochrome P450 (CYP) drug-metabolizing enzyme in adult male rats. Coincident with the severalfold elevation in transcript level is a modest 10-30% overexpression of CYP2C11 protein and its catalytic activities. Using hepatic tissue from adult, neonatally MSG-treated rats, we have cloned a variant species of CYP2C11 mRNA containing all of the essential elements of a full-length complementary DNA, including initiating codon, termination codon, and polyadenylase tail. In addition, the transcript contains a 742-bp intervening sequence (identical to the complete terminal intron) between the last and penultimate exons, and an intron-specific oligo probe for Northern blotting demonstrates the presence of the variant transcript in liver of MSG-treated rats. Associated with the overexpression and intron retention of the transcript is a 50% reduction in the nuclear splicing capacity of the liver for model precursor CYP2C11 mRNA. It is proposed that this splicing defect may be a consequence of the mini-GH pulses (secreted in otherwise normal masculine plasma profiles) signaling abnormal processing of precursor CYP2C11 mRNA to produce a substantial portion of intron retained, nontranslatable transcript.
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Affiliation(s)
- N A Pampori
- Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6048, USA
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Kawai M, Bandiera SM, Chang TK, Bellward GD. Growth hormone regulation and developmental expression of rat hepatic CYP3A18, CYP3A9, and CYP3A2. Biochem Pharmacol 2000; 59:1277-87. [PMID: 10736428 DOI: 10.1016/s0006-2952(00)00247-1] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022]
Abstract
The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.
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Affiliation(s)
- M Kawai
- Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada
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