PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides

OCP are xenobiotics which display various toxic effects on animal and human health. One of their effects is to bind and activate estrogen receptor alpha (ERalpha). We have previously studied the down-regulation of induced CYP1A1 (cytochrome P450) expression by this class of molecules in mammary carcinoma cells and shown the importance of ERalpha in this process. However, an alternative mechanism was suggested by those experiments in hepatoma cells. In this study, we have performed Northern blot and transient transfection assays in various cell lines and shown that OCP activate human pregnane X receptor (PXR) and subsequent CYP3A4 mRNA expression. This effect is mediated by the distal xenobiotic responsive element modulator of the promoter. The induction of CYP3A4 by OCP was dose-dependent within the 1-10 microM range. The data suggest that chronic exposure to OCP could alter a major metabolite pathway in human liver and putatively modify the pharmacokinetics of drugs and pollutants.     

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies.     

Modulation of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) by 6-arylpyrrolo[2,1-d][1,5]benzothiazepine derivatives, ligands of peripheral benzodiazepine receptor (PBR)

Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate xenobiotic sensing and metabolism through interactions with multiple exogenous and endogenous chemicals. Compounds that activate CAR are often ligands of PXR; attention is therefore given to discovery of new, receptor-specific chemical entities that may be exploited for therapeutic and basic research purposes. Recently, ligands of the peripheral benzodiazepine receptor (PBR), PK11195 and FGIN-1-27, were shown to modulate both CAR and PXR. PBR is a mitochondrial transport protein responsible for multiple regulatory functions, including heme biosynthesis, a major component in cytochrome P450 (CYP) enzymes. To investigate possible new roles for PBR involvement in metabolic regulation, expression of the CAR and PXR target genes, CYP2B6 and CYP3A4, was measured in human hepatocytes following treatment with a targeted PBR ligand set. Luciferase reporter assays with transiently expressed wild-type CAR (CAR1), splice variant CAR3, or PXR in HuH-7 cells were used to further study activation of these receptors. Four structurally related PBR ligands (benzothiazepines) differentially modulate CAR1, CAR3 and PXR activity. Benzothiazepine NF49 is an agonist ligand of CAR3, a partial agonist of PXR, exhibits greater inverse agonist activity on CAR1 than does PK11195, and is a new tool for studying these closely related nuclear receptors.     

Repression of PXR-mediated induction of hepatic CYP3A gene expression by protein kinase C

Pregnane X receptor (PXR, NR1I2) regulates the inducible expression of the 3A sub-family of cytochrome P450 genes (CYP3A). CYP3A enzymes are responsible for the oxidative metabolism of a wide array of endobiotic and xenobiotic compounds. Hepatic CYP3A gene expression is rapidly down-regulated during inflammation and sepsis. There are twelve protein kinase C (PKC) isoforms, classified into three subfamilies according to the structure of the N-terminal regulatory domain and their sensitivity to calcium and diacylglycerol. It is now well accepted that cytokine stimulation of hepatocytes increases intracellular PKC activity during inflammation and sepsis. We show here that protein kinase C alpha (PKCα) and phorbol ester-dependent PKC signaling dramatically repressed PXR activity in both, cell-based reporter gene assays and in hepatocytes. Moreover, treatment with the protein phosphatase PP1/PP2A inhibitor okadaic acid (OA) totally abolished PXR activity in reporter gene assays and in cultured hepatocytes. In mammalian two-hybrid assays, treatment with phorbol 12-myristate 13-acetate (PMA) increased the strength of interaction between PXR and the nuclear receptor co-repressor protein (NCoR). Treatment with PMA also abolished the ligand-dependent interaction between PXR and the steroid receptor co-activator 1 protein (SRC1). Our findings suggest that activation of the protein kinase C signaling pathway represses PXR activity through alterations in PXR-protein co-factor complexes, possibly through direct alterations in the phosphorylation status of one or all of these proteins. In addition, our data potentially provide important insights into the molecular mechanism of the repression of hepatic CYP3A gene expression that occurs during the inflammatory response.     

The role of hepatocyte RXR alpha in xenobiotic-sensing nuclear receptor-mediated pathways.

Nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) cross talk and serve as xenobiotic sensors to form a safety net against the toxic effects of harmful substances. Retinoid x receptor alpha (RXRalpha) dimerizes with CAR and PXR. In order to analyze the role of RXRalpha in these xeno-sensor-mediated pathways, hepatocyte RXRalpha-deficient mice were challenged by CAR and PXR ligands including androstanol, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), and pregnenolone 16alpha-carbonitrile (PCN). We demonstrate that hepatocyte RXRalpha deficiency prevents TCPOBOP-induced hepatomegaly and morphological changes. We also show that in vivo the cytochrome P450 (CYP) genes including CYP2A5, CYP2B10, CYP3A1, but not CYP2E1 and CYP2D6, are the RXRalpha target genes. Androstanol, TCPOBOP, and PCN can differentially regulate the expression of these CYP450 genes. In addition, the most active peroxisome proliferator activated receptor (PPARalpha) ligand, Wy14,643, also regulates some of the xeno-sensor target genes such as CYP2A5 and CYP2B10 in vivo. Thus, the ligands of different nuclear receptors can regulate common CYP450 genes and hepatocyte RXRalpha is essential for xenobiotic metabolism in vivo.     

Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane transporters through human pregnane X receptor

BACKGROUND AND PURPOSE

Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR).

EXPERIMENTAL APPROACH

Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation.

KEY RESULTS

All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors.

CONCLUSIONS AND IMPLICATIONS

Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.     

Endocrine disruptors induce cytochrome P450 by affecting transcriptional regulation via pregnane X receptor

Pregnane X receptor (PXR) is a nuclear receptor that regulates the expression of genes for cytochrome P450 3A (CYP3A), multidrug resistance 1 (MDR1), and organic anion-transporting peptide 2 (OATP2). These genes control the metabolism (CYP3A subfamily) and aspects of the pharmacokinetics (MDR1 and OATP2) of both endogenous and xenobiotic compounds. Since PXR is important in understanding the actions of endocrine disruptors (EDs), we determined the ability of suspected EDs to interact with PXR. In our study, 7 of 54 xenobiotics compounds interacted with PXR, including methoxychlor and benzophenone. All of the chemicals activated PXR in vitro and induced CYP3A mRNA in the male rat liver. In addition, CYP2C11 was also induced by some PXR agonists and converted methoxychlor into xenoestrogen. These findings suggest that some EDs affect sex hormone receptor indirectly by induction of metabolic enzyme via PXR, to produce rapidly higher concentrations of effective metabolites, leading to disturbance of the endocrine system.     

Structural insights into the promiscuity and function of the human pregnane X receptor

The pregnane X receptor (PXR) is a promiscuous nuclear receptor that responds to a wide variety of drugs, xenobiotics and endogenous compounds, and plays a critical role in mediating drug-drug interactions in humans. PXR is the master regulator of the expression of the CYP3A4 gene, which encodes for the most abundant and promiscuous drug-metabolizing enzyme in humans. PXR also regulates the expression of other genes involved in xenobiotic metabolism, including CYP2C8, CYP2C9, CYP2B6, GSTA2 and MDR1, as well as genes critical to bile acid metabolism. While PXR functions as a xenobiotic sensor in numerous vertebrates, its relatively low sequence conservation across species causes the PXRs from different organisms to respond to distinct subsets of xenobiotics. Thus, PXR promiscuity is directed and not random. The recent determination of crystal structures of the ligand binding domain of human PXR has provided the first detailed molecular view of this promiscuous receptor, and has advanced our understanding of its varied biological functions. We review the evidence establishing the binding promiscuity of PXR and its directed specificity in different species, and analyze the structural determinants of these characteristics. In addition, we examine the relationship between the interaction of PXR with ligands and the manner in which CYP3A4 is thought to bind to substrate molecules. The accumulating structural and functional data on PXR may facilitate the development of improved methods for in vitro, in vivo and in silico screening for PXR activation.     

Nuclear pregnane x receptor and constitutive androstane receptor regulate overlapping but distinct sets of genes involved in xenobiotic detoxification

The nuclear pregnane X receptor (PXR) and constitutive androstane receptor (CAR) play central roles in protecting the body against environmental chemicals (xenobiotics). PXR and CAR are activated by a wide range of xenobiotics and regulate cytochrome P450 and other genes whose products are involved in the detoxification of these chemicals. In this report, we have used receptor-selective agonists together with receptor-null mice to identify PXR and CAR target genes in the liver and small intestine. Our results demonstrate that PXR and CAR regulate overlapping but distinct sets of genes involved in all phases of xenobiotic metabolism, including oxidative metabolism, conjugation, and transport. Among the murine genes regulated by PXR were those encoding PXR and CAR. We provide evidence that PXR regulates a similar program of genes involved in xenobiotic metabolism in human liver. Among the genes regulated by PXR in primary human hepatocytes were the aryl hydrocarbon receptor and its target genes CYP1A1 and CYP1A2. These findings underscore the importance of these two nuclear receptors in defending the body against a broad array of potentially harmful xenobiotics.     

Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 µM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 µM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 µM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 µM and 10 µM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.     

Characterization of human cytochrome P450 induction by pesticides

Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR.