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1.
Drug Metab Dispos ; 2023 Oct 13.
Article in English | MEDLINE | ID: mdl-37833076

ABSTRACT

Scientists from Canadian institutions have a rich history of making interesting and important contributions to the journal Drug Metabolism and Disposition (DMD) over the past 51 years. A goal of this minireview is to highlight these contributions and pay tribute to many of the scientists at Canadian institutions that have aided in the evolution of the discipline through their DMD publications. We conducted a geographical and research sectoral analysis of the temporal trends of DMD publications originating from Canadian sources. The fraction of total DMD papers of Canadian origin achieved a peak during the 1990s and since that time, this metric has displayed a pronounced and steady decline to the present situation, where the country needs to be concerned about its potentially vulnerable global status within the realm of drug metabolism and disposition science. Stronger and timely investment by Canadian academic institutions in drug metabolism and disposition science may help to restore the nation's research excellence in this discipline and ensure a more robust pipeline of appropriately trained scientists to take on careers in academia, industry, and government. Significance Statement The substantial contributions made by scientists at Canadian institutions to the journal Drug Metabolism and Disposition (DMD) are highlighted and celebrated in this minireview. Analysis of temporal trends in the fraction of total DMD papers of Canadian origin paints a concerning picture of Canada's current global status in the realm of drug metabolism and disposition science. Further investment in this discipline at Canadian universities may be needed.

2.
Drug Metab Dispos ; 51(6): 657-671, 2023 06.
Article in English | MEDLINE | ID: mdl-36653119

ABSTRACT

The induction of multiple drug-metabolizing enzymes by halogenated and polycyclic aromatic hydrocarbon toxicants is mediated by the aryl hydrocarbon receptor (AHR). This fascinating receptor also has natural dietary and endogenous ligands, and much is now appreciated about the AHR's developmental and physiologic roles, as well as its importance in cancer and other diseases. The past several years has witnessed increasing emphasis on understanding the multifaceted roles of the AHR in the immune system. Most would agree that the "discovery" of the AHR occurred in 1976, with the report of specific binding of a high affinity radioligand in mouse liver, just three years after the launch of the journal Drug Metabolism and Disposition (DMD) in 1973. Over the ensuing 50 years, the AHR and DMD have led parallel and often intersecting lives. The overall goal of this mini-review is to provide a decade-by-decade overview of major historical landmark discoveries in the AHR field and to highlight the numerous contributions made by publications appearing in the pages of DMD. It is hoped that this historical tour might inspire current and future research in the AHR field. SIGNIFICANCE STATEMENT: With the launch of Drug Metabolism and Disposition (DMD) in 1973 and the discovery of the aryl hydrocarbon receptor (AHR) in 1976, the journal and the receptor have led parallel and often intersecting lives over the past 50 years. Tracing the history of the AHR can reveal how knowledge in the field has evolved to the present and highlight the important contributions made by discoveries reported in DMD. This may inspire additional DMD papers reporting future AHR landmark discoveries.


Subject(s)
Neoplasms , Polycyclic Aromatic Hydrocarbons , Animals , Mice , Receptors, Aryl Hydrocarbon/metabolism , Ligands
3.
Can J Physiol Pharmacol ; 98(5): 267-274, 2020 May.
Article in English | MEDLINE | ID: mdl-31825663

ABSTRACT

Expression of NADPH - cytochrome P450 oxidoreductase (POR), electron donor for microsomal P450s, is induced in rat liver by dexamethasone (DEX), an activator of the glucocorticoid receptor (GR) and the pregnane X receptor (PXR). DEX induction of POR in rat liver is primarily PXR-mediated, although GR may contribute to mRNA effects. We examined the role of GR and PXR in the DEX induction of POR mRNA and protein in the H4IIE rat hepatoma cell line. The DEX EC50 for a PXR target, CYP3A23, exceeded that for the GR targets tyrosine aminotransferase and PXR as well as POR itself. POR protein levels were induced 3- and 4-fold, respectively, by DEX concentrations activating GR selectively (100 nM) or both GR and PXR (10 µM). POR was induced by triamcinolone acetonide, a selective GR agonist, but not pregnenolone-16α-carbonitrile, a selective PXR agonist. POR induction was blocked by the GR antagonist RU486 but minimally influenced by the PXR antagonist FLB-12. The half-life for POR mRNA was prolonged by DEX at both 100 nM and 10 µM. GR is more important in DEX-induced POR expression in H4IIE cells compared to rat liver in vivo, calling into question the suitability of this cell model for mechanistic studies.


Subject(s)
Carcinoma, Hepatocellular/pathology , Dexamethasone/pharmacology , Liver Neoplasms/pathology , NADPH-Ferrihemoprotein Reductase/biosynthesis , Animals , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Induction/drug effects , NADPH-Ferrihemoprotein Reductase/genetics , Pregnane X Receptor/metabolism , RNA, Messenger/genetics , Rats , Receptors, Glucocorticoid/metabolism
4.
Drug Metab Dispos ; 47(3): 279-282, 2019 03.
Article in English | MEDLINE | ID: mdl-30573465

ABSTRACT

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that activate the aryl hydrocarbon receptor, thereby triggering a range of biologic responses, exemplified by the induction of CYP1A1 PAHs can also regulate the expression of members of the CYP3A subfamily, with reports of mainly suppressive effects on mouse hepatic Cyp3a11 expression, but paradoxically both inductive and suppressive effects on human hepatic CYP3A4 expression. Understanding the regulation of CYP3A4 expression by PAHs is important because of the widespread exposure of humans to these chemicals and the central role of the CYP3A4 enzyme in the metabolism of clinically important drugs and endogenous substances. The present study used 3-methylcholanthrene (MC) as a model PAH to characterize the in vivo regulation of CYP3A4 expression and activity in humanized pregnane X receptor-constitutive androstane receptor-CYP3A4/3A7 mice. Adult mice were treated by intraperitoneal injection with MC (80 mg/kg), or corn oil vehicle, and euthanized 24 or 72 hours later. As a positive control response, pronounced induction of hepatic Cyp1a1 by MC was confirmed at both time points in males and females at the mRNA, protein, and catalytic activity levels. Basal hepatic CYP3A4 expression and activity were significantly higher in female versus male mice. MC treatment suppressed hepatic CYP3A4 in female mice at 72 hours postdosing at the mRNA, protein, and catalytic activity levels. A similar response was observed in male mice, although the suppression of CYP3A4 protein levels did not achieve statistical significance. This mouse model will facilitate further studies of the mechanisms and consequences of CYP3A4 suppression by PAHs.


Subject(s)
Cytochrome P-450 CYP3A/metabolism , Environmental Pollutants/toxicity , Gene Expression Regulation/drug effects , Methylcholanthrene/toxicity , Animals , Constitutive Androstane Receptor , Cytochrome P-450 CYP3A/genetics , Environmental Pollutants/administration & dosage , Female , Injections, Intraperitoneal , Liver/drug effects , Liver/metabolism , Male , Methylcholanthrene/administration & dosage , Mice , Mice, Transgenic , Models, Animal , Pregnane X Receptor/genetics , Pregnane X Receptor/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Sex Factors
5.
Can J Physiol Pharmacol ; 95(6): 768-771, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28384415

ABSTRACT

The marked induction of cytochromes P450 such as CYP1A1 caused by polycyclic aromatic hydrocarbons (PAHs) like 3-methylcholanthrene (MC) is often accompanied by suppression of other hepatic P450s. The molecular mechanisms, functional consequences, and human relevance of P450 downregulation by PAHs are poorly understood. MC suppresses mRNA levels for CYP2C8, an important human P450, in cultured human hepatocytes. To avoid hepatocyte lot-to-lot variability, we assessed CYP2C8 regulation by MC in HepaRG cells, a terminally differentiated human hepatocellular carcinoma cell line that maintains high P450 expression. MC strongly induced CYP1A1 mRNA levels and markedly downregulated CYP2C8 mRNA levels in HepaRG cells. Although MC also suppressed CYP2C8 mRNA levels in the HepG2 human hepatocellular carcinoma cell line, basal CYP2C8 expression was extremely low. HepaRG cells appear to be an appropriate model system for studying the mechanisms and functional consequences of CYP2C8 downregulation by PAHs.


Subject(s)
Carcinoma, Hepatocellular/pathology , Cytochrome P-450 CYP2C8/genetics , Down-Regulation/drug effects , Liver Neoplasms/pathology , Methylcholanthrene/pharmacology , Hep G2 Cells , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism
6.
Drug Metab Dispos ; 45(2): 118-129, 2017 02.
Article in English | MEDLINE | ID: mdl-27856527

ABSTRACT

The aryl hydrocarbon receptor (AHR) nuclear translocator (ARNT), as the AHR's heterodimerization partner, and NADPH-cytochrome P450 oxidoreductase (POR), as the key electron donor for all microsomal P450s, are independent and indispensable components in the adaptive and toxic responses to polycyclic aromatic hydrocarbons. Expression of both ARNT and POR in rat liver is induced by dexamethasone (DEX), a synthetic glucocorticoid known to activate both the glucocorticoid receptor (GR) and the pregnane X receptor (PXR). To better understand the role of GR and PXR in the in vivo DEX induction of rat hepatic ARNT and POR at the mRNA and protein levels, we studied the following: 1) the effects of DEX doses that activate GR (≥0.1 mg/kg) or PXR (≥10 mg/kg); 2) responses produced by GR- and PXR-selective agonists; 3) the impact of GR antagonism on DEX's inducing effects; and 4) whether biologic responses to DEX are altered in PXR-knockout rats. Our findings are consistent with a role for GR as a key mediator of the induction of rat hepatic ARNT expression by glucocorticoids; a role for PXR in the modulation of ARNT protein levels could not be excluded. Although GR activation may contribute to POR mRNA induction, regulation of POR expression and function by DEX is primarily PXR-mediated. This work suggests that the hepatic expression and function of ARNT and POR may be modulated by exposure to exogenous PXR activators and/or conditions that alter glucocorticoid levels such as stress, steroidal therapies, and diseases of excess or deficiency.


Subject(s)
Aryl Hydrocarbon Receptor Nuclear Translocator/biosynthesis , Dexamethasone/pharmacology , Microsomes, Liver/drug effects , NADPH-Ferrihemoprotein Reductase/biosynthesis , Receptors, Glucocorticoid/physiology , Receptors, Steroid/physiology , Animals , Dose-Response Relationship, Drug , Enzyme Induction , Gene Knockout Techniques , Male , Microsomes, Liver/metabolism , Pregnane X Receptor , Rats, Inbred F344 , Rats, Sprague-Dawley , Receptors, Glucocorticoid/genetics , Receptors, Steroid/genetics
7.
Can J Physiol Pharmacol ; 91(12): 1154-7, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24289088

ABSTRACT

The aryl hydrocarbon receptor (AHR) plays physiological roles and mediates adaptive and toxic responses to environmental pollutants. Adrenalectomized rats display decreased hepatic AHR protein levels, with no change in mRNA, and selectively impaired induction of cytochrome P450 1B1. This is similar to reported phenotypes for mice with hepatocyte-specific conditional deletion of AHR-interacting protein (AIP), a chaperone protein of the cytoplasmic AHR complex. In this study, we demonstrated that adrenalectomy (ADX) and acute dexamethasone (DEX) treatment do not alter hepatic AIP mRNA or protein levels. Also, hepatic protein levels of the 90 kDa heat shock protein and p23 were not altered by ADX or acute DEX treatment. These results suggest that the loss of rat hepatic AHR protein following ADX cannot be explained by changes in the levels of the receptor's cytoplasmic chaperone proteins.


Subject(s)
Cytoplasm/metabolism , Liver/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Adrenalectomy/methods , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP1B1 , Cytoplasm/genetics , Dexamethasone/pharmacology , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Hepatocytes/metabolism , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Male , Prostaglandin-E Synthases , RNA, Messenger/genetics , Rats , Rats, Inbred F344
8.
Drug Metab Dispos ; 41(10): 1782-6, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23846873

ABSTRACT

The aryl hydrocarbon receptor (AHR)-dependent induction of cytochromes P450 (P450) such as CYP1A1 by 3-methylcholanthrene (MC) and related polycyclic aromatic hydrocarbons is well characterized. We reported previously that MC treatment triggers a pronounced downregulation, particularly at the protein level, of mouse hepatic Cyp3a11, a counterpart of the key human drug-metabolizing enzyme CYP3A4. To determine whether this effect of MC requires hepatic microsomal P450 activity, we studied liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of the NADPH-cytochrome P450 oxidoreductase gene. In vehicle-treated animals, basal levels of CYP3A11 mRNA and CYP3A protein immunoreactivity were elevated by approximately 9-fold in LCN mice compared with wild-type (WT) mice, whereas CYP3A catalytic activity was profoundly compromised in LCN mice. MC treatment caused suppression of CYP3A11 mRNA, CYP3A protein immunoreactivity, and CYP3A catalytic activity in WT mice, and the MC effects at the mRNA and protein levels were maintained in LCN mice. Flavin-containing monooxygenase-3 (Fmo3) induction by MC was suggested previously to occur via an AHR-dependent mechanism requiring conversion of the parent compound to DNA-damaging reactive metabolites; however, hepatic FMO3 mRNA levels were dramatically increased by MC in both WT and LCN mice. MC did not function as a mechanism-based inactivator of CYP3A enzymes in hepatic microsomes prepared from untreated WT mice, under conditions in which 1-aminobenzotriazole caused marked NADPH-dependent loss of total P450 content and CYP3A catalytic activity. These results indicate that MC downregulates mouse hepatic CYP3A protein via a pretranslational mechanism that does not require hepatic microsomal P450-dependent activity.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Down-Regulation/genetics , Hepatocytes/metabolism , Liver/metabolism , Methylcholanthrene/pharmacology , Animals , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Down-Regulation/drug effects , Hepatocytes/drug effects , Hepatocytes/enzymology , Liver/drug effects , Liver/enzymology , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Oxygenases/genetics , Oxygenases/metabolism , RNA, Messenger/genetics , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism
9.
Drug Metab Dispos ; 41(1): 12-23, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23086197

ABSTRACT

This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b(5), squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b(5) are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b(5) on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism.


Subject(s)
Microsomes, Liver/enzymology , NADPH-Ferrihemoprotein Reductase/metabolism , Pharmaceutical Preparations/metabolism , Alleles , Animals , Cytochromes b5/metabolism , Mice , Mice, Knockout , NADPH-Ferrihemoprotein Reductase/genetics , Tumor Cells, Cultured
10.
Drug Metab Dispos ; 41(2): 457-65, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23169610

ABSTRACT

3-Methylcholanthrene (MC) is a readily metabolized aryl hydrocarbon receptor (AHR) agonist. MC disrupts expression of mouse hepatic growth hormone (GH) signaling components and suppresses cytochrome P450 2D9 (Cyp2d9), a male-specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). To determine if these effects of MC depend on hepatic microsomal P450-mediated activity, we examined biologic responses to MC treatment in liver Cpr-null (LCN) mice with hepatocyte-specific conditional deletion of NADPH-cytochrome P450 oxidoreductase (POR). MC caused mild induction of Por and a hepatic inflammatory marker in wild-type mice, whereas MC caused strong induction of AHR target genes, Cyp1a1, Cyp1a2, and Cyp1b1 in wild-type and LCN mice. Two mouse hepatic STAT5b target genes, Cyp2d9 and major urinary protein 2 (Mup2), were suppressed by MC in wild-type mice, and the CYP2D9 mRNA response was maintained in LCN mice. In wild-type mice only, MC decreased hepatic GH receptor (GHR) mRNA but increased GHR protein levels. There was an apparent impairment of STAT5 phosphorylation by MC in wild-type and LCN mice, but large interanimal variation prevented achievement of statistical significance. In vehicle-treated mice, basal levels of MUP2 mRNA, GHR mRNA, GHR protein, and the activation status of extracellular signal-regulated kinase 2 and Akt were influenced by hepatic Por genetic status. These results indicate that the effects of MC on hepatic GH signaling components and target genes are complex, involving aspects that are both dependent and independent of hepatic microsomal P450-mediated activity.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Growth Hormone/metabolism , Liver/drug effects , Methylcholanthrene/pharmacology , Signal Transduction/drug effects , Animals , Basic Helix-Loop-Helix Transcription Factors/agonists , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cytochrome P-450 Enzyme System/genetics , Gene Expression Regulation, Enzymologic , Inflammation Mediators/metabolism , Isoenzymes , Janus Kinase 2/drug effects , Janus Kinase 2/metabolism , Liver/enzymology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , NADPH-Ferrihemoprotein Reductase/deficiency , NADPH-Ferrihemoprotein Reductase/genetics , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/agonists , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Somatotropin/drug effects , Receptors, Somatotropin/genetics , Receptors, Somatotropin/metabolism , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Signal Transduction/genetics , Time Factors
11.
Mol Pharmacol ; 83(2): 377-88, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23150485

ABSTRACT

The multidrug transporter, breast cancer resistance protein, ABCG2, is up-regulated in certain chemoresistant cancer cells and in the mammary gland during lactation. We investigated the role of the lactogenic hormone prolactin (PRL) in the regulation of ABCG2. PRL dose-dependently induced ABCG2 expression in T-47D human breast cancer cells. This induction was significantly reduced by short-interfering RNA-mediated knockdown of Janus kinase 2 (JAK2). Knockdown or pharmacologic inhibition of the down-stream signal transducer and activator of transcription-5 (STAT5) also blunted the induction of ABCG2 by PRL, suggesting a role for the JAK2/STAT5 pathway in PRL-induced ABCG2 expression. Corroborating these findings, we observed PRL-stimulated STAT5 recruitment to a region containing a putative γ-interferon activation sequence (GAS) element at -434 base pairs upstream of the ABCG2 transcription start site. Introduction of a single mutation to the -434 GAS element significantly attenuated PRL-stimulated activity of a luciferase reporter driven by the ABCG2 gene promoter and 5'-flanking region containing the -434 GAS motif. In addition, this GAS element showed strong copy number dependency in its response to PRL treatment. Interestingly, inhibitors against the mitogen-activated protein kinase and phosphoinositide-3-kinase signaling pathways significantly decreased the induction of ABCG2 by PRL without altering STAT5 recruitment to the GAS element. We conclude that the JAK2/STAT5 pathway is required but not sufficient for the induction of ABCG2 by PRL.


Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Neoplasm Proteins/biosynthesis , Prolactin/pharmacology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Breast Neoplasms/genetics , Carcinoma, Ductal, Breast/genetics , Cell Line, Tumor , Drug Resistance, Multiple , Female , Humans , Interferon-gamma/genetics , Interferon-gamma/metabolism , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , MCF-7 Cells , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Mutation/genetics , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Promoter Regions, Genetic/drug effects , RNA, Messenger/genetics , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects
12.
Can J Physiol Pharmacol ; 90(10): 1354-63, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22978700

ABSTRACT

The aryl hydrocarbon receptor (AHR) has physiological roles in the absence of exposure to exogenous ligands, and mediates adaptive and toxic responses to the environmental pollutant 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD). A readily metabolized AHR agonist, 3-methylcholanthrene, disrupts the expression of mouse hepatic growth hormone (GH) signaling components and suppresses cytochrome P450 2D9 (Cyp2d9), a male-specific gene controlled by pulsatile GH via signal transducer and activator of transcription 5b (STAT5b). Using TCDD as an essentially nonmetabolized AHR agonist, and Ahr (-/-) mice as the preferred model to determine the AHR-dependence of biological responses, we now show that 2 mouse hepatic STAT5b target genes, Cyp2d9, and major urinary protein 2 (Mup2), are suppressed by TCDD in an AHR-dependent manner. TCDD also decreased hepatic mRNA levels for GH receptor, Janus kinase 2, and STAT5a/b with AHR-dependence. Without inducing selected hepatic inflammatory markers, TCDD caused AHR-dependent induction of Cyp1a1 and NADPH-cytochrome P450 oxidoreductase (Por) and suppression of Cyp3a11. In vehicle-treated mice, basal mRNA levels for CYP2D9, CYP3A11, POR, serum amyloid protein P, and MUP2 were influenced by Ahr genetic status. We conclude that AHR activation per se leads to dysregulation of hepatic GH signaling components and suppression of some, but not all, STAT5b target genes.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/agonists , Cytochrome P-450 Enzyme System/metabolism , Environmental Pollutants/toxicity , Liver/drug effects , Polychlorinated Dibenzodioxins/toxicity , Receptors, Aryl Hydrocarbon/agonists , Receptors, Somatotropin/metabolism , Signal Transduction/drug effects , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cytochrome P-450 CYP1A1/biosynthesis , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 CYP3A Inhibitors , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/genetics , Cytochrome P450 Family 2 , Down-Regulation/drug effects , Environmental Pollutants/administration & dosage , Enzyme Induction/drug effects , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Liver/immunology , Liver/metabolism , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH-Ferrihemoprotein Reductase/biosynthesis , NADPH-Ferrihemoprotein Reductase/genetics , Polychlorinated Dibenzodioxins/administration & dosage , Proteins/antagonists & inhibitors , Proteins/genetics , Proteins/metabolism , RNA, Messenger/metabolism , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Receptors, Somatotropin/antagonists & inhibitors , Receptors, Somatotropin/genetics , STAT5 Transcription Factor/antagonists & inhibitors , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism
13.
Drug Metab Dispos ; 39(1): 83-91, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20881032

ABSTRACT

The aryl hydrocarbon receptor (AHR) is activated by 3-methylcholanthrene (MC), a polycyclic aromatic hydrocarbon, and environmental contaminants, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin. Adrenalectomized (ADX) rats have decreased hepatic AHR protein and lower levels of MC-induced CYP1B1 mRNA. To further characterize the effects of decreased AHR protein and the response to MC in ADX rats, we measured AHR-mediated responses in the liver of sham-operated (SHAM) and ADX rats, 6 and 54 h after MC treatment. CYP1A2 mRNA was suppressed by 46 to 60% 4 days after ADX in vehicle-treated animals. AHR mRNA was induced 4-fold 6 h after MC in SHAM rats, but no induction was observed in ADX rats. The MC-induced 7-ethoxyresorufin O-deethylation (EROD) activity in ADX rats was 35% of the activity in the MC-treated SHAM group at 6 h. At 54 h after treatment, the induction of EROD activity by MC was more pronounced in ADX rats than at 6 h. To assess the overall capacity for hepatic P450-mediated metabolism, we measured NADPH-cytochrome P450 oxidoreductase (POR) activity. POR activity was decreased by 50% after ADX. We have shown that the response to MC in ADX rats is suppressed for some, but not all, AHR-mediated responses and that reduced POR activity after ADX could contribute to a decreased capacity for P450-dependent metabolism. The current study contributes to our understanding of how adrenal-dependent factors modulate the AHR pathway and the response to MC in vivo.


Subject(s)
Liver/drug effects , Methylcholanthrene/pharmacology , Receptors, Aryl Hydrocarbon/metabolism , Actins/genetics , Actins/metabolism , Adrenalectomy , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP1A2/metabolism , Cytochrome P-450 CYP1B1 , Liver/metabolism , Male , NADPH-Ferrihemoprotein Reductase/genetics , NADPH-Ferrihemoprotein Reductase/metabolism , Oxazines/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred F344
14.
Chem Biol Interact ; 182(2-3): 148-58, 2009 Dec 10.
Article in English | MEDLINE | ID: mdl-19615983

ABSTRACT

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene (MC); the prototypical response is induction of drug-metabolizing enzymes. Factors that regulate AHR levels in vivo are poorly understood and it is also not clear how AHR levels affect aromatic hydrocarbon responsiveness. Our interest in pituitary-dependent regulation of AHR levels was prompted by two findings from our laboratory: (1) hypophysectomized rats have reduced hepatic levels of AHR protein; and (2) glucocorticoids increase AHR expression and aromatic hydrocarbon responsiveness in rodent hepatoma cells. To study whether adrenalectomy and glucocorticoids contribute to hormone-dependent regulation of the hepatic AHR pathway, male adrenalectomized (ADX) or SHAM-ADX rats were treated with dexamethasone (DEX) or vehicle. AHR protein was depleted by 50-60% at 4 days after ADX, but was not altered by DEX treatment. To assess whether the observed AHR depletion affected aromatic hydrocarbon responsiveness, the induction of hepatic cytochrome P450 1B1 (CYP1B1) mRNA by MC was measured as an AHR-mediated adaptive response. MC-induced hepatic CYP1B1 mRNA was reduced by 50% in ADX rats relative to SHAM-ADX. Exogenous glucocorticoid treatment (DEX - 1.5mg/kg) induced hepatic AHR nuclear translocator (ARNT) mRNA by up to 9-fold at 3 and 6h after dosing, with no corresponding change in ARNT protein levels. These data demonstrate that: (1) adrenal-dependent factors contribute to the physiological maintenance of hepatic AHR protein levels; (2) the depletion of hepatic AHR protein in ADX rats coincided with a diminished adaptive response to MC; and (3) exogenous glucocorticoid treatment increases hepatic ARNT mRNA levels regardless of adrenal status. This model is useful for studying the mechanisms of AHR and ARNT regulation and for further characterization of the impact of AHR protein depletion on the response to aromatic hydrocarbons in vivo.


Subject(s)
Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Receptors, Aryl Hydrocarbon/metabolism , Adrenalectomy , Animals , Aryl Hydrocarbon Hydroxylases/genetics , Aryl Hydrocarbon Hydroxylases/metabolism , Aryl Hydrocarbon Receptor Nuclear Translocator/genetics , Aryl Hydrocarbon Receptor Nuclear Translocator/metabolism , Cytochrome P-450 CYP1B1 , Dexamethasone/administration & dosage , Gene Expression/drug effects , Gene Products, tat/genetics , Gene Products, tat/metabolism , Glucocorticoids/administration & dosage , Liver/drug effects , Liver/metabolism , Male , RNA, Messenger/metabolism , Rats , Receptors, Aryl Hydrocarbon/genetics , Signal Transduction/drug effects
15.
Drug Metab Dispos ; 36(9): 1803-11, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18524872

ABSTRACT

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 gene's 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.


Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Gene Expression Regulation, Enzymologic/drug effects , Methylcholanthrene/pharmacology , Promoter Regions, Genetic , Steroid 16-alpha-Hydroxylase/genetics , Animals , Cytochrome P450 Family 2 , Genes, Reporter , Male , RNA, Messenger/genetics , Rats , Rats, Inbred F344 , Reverse Transcriptase Polymerase Chain Reaction
16.
Toxicology ; 248(2-3): 104-12, 2008 Jun 27.
Article in English | MEDLINE | ID: mdl-18440119

ABSTRACT

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.


Subject(s)
5' Flanking Region/physiology , Aryl Hydrocarbon Hydroxylases/genetics , Gene Expression Regulation, Enzymologic/drug effects , Methylcholanthrene/toxicity , Polychlorinated Dibenzodioxins/toxicity , Promoter Regions, Genetic/physiology , Steroid 16-alpha-Hydroxylase/genetics , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Benzoflavones/pharmacology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor/drug effects , Cloning, Molecular , Cytochrome P450 Family 2 , Enzyme Induction , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Luciferases/biosynthesis , Luciferases/genetics , Luciferases/metabolism , Mice , Mutagenesis, Site-Directed , RNA, Messenger/metabolism , Rats , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Steroid 16-alpha-Hydroxylase/metabolism
17.
Drug Metab Dispos ; 36(3): 543-51, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18086832

ABSTRACT

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates most biological responses to 2,3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related aromatic hydrocarbons. Although the role of the AHR in control of drug metabolism and endocrine disruption is partly understood, we know little about the regulation of the AHR itself by endocrine factors. Our work with hypophysectomized rats suggested that hepatic AHR protein level is positively regulated by pituitary-dependent factors. A current hypothesis is that adrenal glucocorticoids elevate AHR expression and enhance responsiveness to AHR agonists. Dexamethasone (DEX) at concentrations that activate the glucocorticoid receptor (GR) increased AHR mRNA, protein, and TCDD-binding by approximately 50% in Hepa-1 mouse hepatoma cells. This response was blocked by the GR antagonist 17beta-hydroxy-11beta-[4-dimethylamino phenyl]-17alpha-[1-propynyl]estra-4,9-dien-3-one (RU486), suggesting GR involvement. This small magnitude increase in AHR levels was functionally significant; pretreatment of Hepa-1 cells with DEX caused a 75% increase in the maximum induction of an AHR-activated luciferase reporter plasmid by TCDD. A luciferase reporter under control of the proximal 2.5 kilobases of the mouse Ahr 5'-flanking region and promoter was induced approximately 2.5-fold by DEX when cotransfected with a mouse GR expression plasmid. This is the first demonstration that glucocorticoids increase AHR levels in hepatoma cells via a GR-dependent transcriptional mechanism, suggesting a novel aspect of cross-talk between the AHR and the GR.


Subject(s)
Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Liver Neoplasms, Experimental/metabolism , Receptors, Aryl Hydrocarbon/biosynthesis , Animals , Cell Line, Tumor , Liver Neoplasms, Experimental/genetics , Methylcholanthrene/pharmacology , Mice , Mifepristone/pharmacology , Polychlorinated Dibenzodioxins/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, Aryl Hydrocarbon/genetics , Receptors, Glucocorticoid/metabolism , Signal Transduction , Transfection
18.
Drug Metab Dispos ; 34(9): 1530-8, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16782765

ABSTRACT

3-Methylcholanthrene (MC) activates the aryl hydrocarbon receptor and increases expression of cytochrome P450 (P450) enzymes such as CYP1A1. MC also decreases expression of CYP2C11, the major hepatic P450 in male rats that is regulated by pulsatile growth hormone (GH) secretion via a pathway partially dependent on signal transducer and activator of transcription 5b (STAT5b). If disruption of this GH signaling pathway is important for MC's ability to suppress CYP2C11 transcription, we hypothesize that MC suppresses other male-specific genes (e.g., mouse Cyp2d9) regulated by pulsatile GH with STAT5b dependence. We examined the time course of MC's effects on hepatic P450s and GH signaling components in male C57BL/6 mice. P450 content, heme content, and NADPH P450 oxidoreductase activity were induced 2.3-, 1.8-, and 1.3-fold, respectively, by MC. MC dramatically induced CYP1A1 mRNA, protein, and catalytic activity. MC caused a 42% decrease in CYP2D9 protein, a 28% decrease in CYP2D9 mRNA, and a 27% decrease in testosterone 16alpha-hydroxylation activity. MC caused a pronounced decrease in CYP3A protein; however, there was no apparent change in testosterone 6beta-hydroxylation activity, and changes in mRNA levels for CYP3A forms were relatively small. Expression of GH receptor and major urinary protein 2, a gene regulated by GH with STAT5b dependence, was decreased by MC at the mRNA level. These results show that MC suppresses mouse Cyp2d9, a pulsatile GH- and STAT5b-dependent male-specific gene, via a pretranslational mechanism that may involve disrupted GH signaling. Mouse CYP3A protein levels are dramatically decreased by MC via a mechanism that is not yet understood.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation, Enzymologic , Growth Hormone/metabolism , Liver/drug effects , Methylcholanthrene/pharmacology , Signal Transduction , Animals , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Cytochrome P450 Family 2 , Down-Regulation , Hydroxylation , Liver/enzymology , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver , Proteins/genetics , Proteins/metabolism , RNA, Messenger/metabolism , Receptors, Somatotropin/genetics , Receptors, Somatotropin/metabolism , Sex Factors , Testosterone/metabolism , Time Factors
19.
Biochem Pharmacol ; 72(3): 267-79, 2006 Jul 28.
Article in English | MEDLINE | ID: mdl-16488401

ABSTRACT

The aryl hydrocarbon receptor (AHR) participates in a wide range of critical cellular events in response to endogenous signals or xenobiotic chemicals. Hence, it is important that AHR levels and activity themselves be well controlled in target tissues. The AHR is essentially ubiquitous in its distribution in mammalian tissues. However, levels of the receptor vary widely across different tissues and among different cell types. AHR levels and activity are modulated by exposure to the receptor's own ligands and are influenced by other xenobiotic chemicals. Many different factors impinge on AHR levels and AHR activity. These factors may alter responsiveness of downstream pathways, thereby affecting normal physiologic functions as well as responses to toxic environmental chemicals such as dioxins. Our commentary appraises the current literature on factors that regulate AHR levels/activity and attempts to identify fruitful strategies towards discovery of key pathways by which AHR levels are modulated in response to endogenous signals and in response to xenobiotic chemicals. An extraordinarily large number of agents alter the level or activity of the AHR. We have not yet entered an age of enlightenment sufficient to achieve true understanding of the interplay of mechanisms that regulate AHR expression in space and in time.


Subject(s)
Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Animals , Biological Factors/pharmacology , Gene Expression/drug effects , Humans , Ligands , Polymorphism, Genetic , Receptors, Aryl Hydrocarbon/physiology
20.
Drug Metab Dispos ; 33(8): 1083-96, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16049130

ABSTRACT

Drug-metabolizing enzymes and drug transporters are key determinants of the pharmacokinetics and pharmacodynamics of many antineoplastic agents. Metabolism and transport influence the cytotoxic effects of antineoplastic agents in target tumor cells and normal host tissues. This article summarizes several state-of-the-art approaches to enhancing the effectiveness and safety of cancer therapy based on recent developments in our understanding of antineoplastic drug metabolism and transport. Advances in four interrelated research areas presented at a recent symposium sponsored by the Division for Drug Metabolism of the American Society for Pharmacology and Experimental Therapeutics (Experimental Biology 2004; Washington D.C., April 17-21, 2004) are discussed: 1) interactions of anthracyclines with drug-metabolizing enzymes; 2) use of hypoxia-selective gene-directed enzyme prodrug therapy (GDEPT) in combination with bioreductive prodrugs; 3) synergy between glutathione conjugation and conjugate efflux in conferring resistance to electrophilic toxins; and 4) use of cytochromes P450 as prodrug-activating enzymes in GDEPT strategies. A clear theme emerged from this symposium: drug metabolism and transport processes can be modulated and exploited in ways that may offer distinct therapeutic advantages in the management of patients with cancer.


Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Cytochrome P-450 Enzyme System/metabolism , Genetic Therapy , Liver/metabolism , Neoplasms/drug therapy , Prodrugs/metabolism , Prodrugs/therapeutic use , Animals , Antibiotics, Antineoplastic/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Alkylating/metabolism , Antineoplastic Agents, Alkylating/pharmacology , Biological Transport , Cell Line, Tumor/drug effects , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/therapeutic use , Doxorubicin/metabolism , Drug Resistance, Neoplasm , Drug-Related Side Effects and Adverse Reactions/prevention & control , Genetic Vectors , Glutathione/metabolism , Glutathione Transferase/antagonists & inhibitors , Glutathione Transferase/metabolism , Humans , Liver/enzymology , Neoplasms/metabolism , Oxidation-Reduction , Prodrugs/classification
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