Pregnenolone 16α-carbonitrile Research Articles

Organic anion transporting polypeptide 2-mediated uptake of paclitaxel and 2′-ethylcarbonate-linked paclitaxel in freshly isolated rat hepatocytes

Abstract Objectives The P-glycoprotein (P-gp) efflux pump plays an important role in paclitaxel detoxification. However, hepatic uptake of paclitaxel mediated by a solute-linked carrier transporter family is still poorly understood in animals and humans. Freshly isolated hepatocyte suspensions are a well established in-vitro model for studying drug transport and xenobiotic metabolism. Therefore, the hepatic uptake of paclitaxel and its P-gp-insensitive prodrug, 2′-ethylcarbonate-linked paclitaxel (TAX-2′-Et), has been characterized using freshly isolated and pregnenolone-16-α-carbonitrile (PCN)-treated hepatocytes in rats. Methods Paclitaxel and TAX-2′-Et were incubated with rat hepatocyte suspensions in the presence or absence of inhibitors. Key findings Paclitaxel and TAX-2′-Et showed concentration-dependent uptake in rat hepatocytes. The intrinsic transport capacity was two-fold higher for paclitaxel uptake than for TAX-2′-Et uptake. Rifampicin (a potent inhibitor of organic anion transporting polypeptide (Oatp) 2), but not indometacin (a representative inhibitor of organic anion transporter (Oat) 2 and Oatp1) treatment, significantly inhibited the uptake of paclitaxel and TAX-2′-Et. We characterized the rifampicin-sensitive uptake of paclitaxel and TAX-2′-Et using rat hepatocytes treated with PCN, which dramatically enhances hepatic Oatp2 protein levels. PCN-treated hepatocytes displayed a 1.6-fold greater uptake of paclitaxel and TAX-2′-Et than the vehicle-treated hepatocytes. The uptake of the two compounds was significantly reduced by rifampicin but not by indometacin treatment. These findings demonstrated that the rat Oatp2, but not Oatp1 orOat2, was a candidate transporter for the hepatic uptakeofpaclitaxel and TAX-2′-Et. Conclusions The findings have provided an important step towards identifying a key transporter in hepatic detoxification of paclitaxel and TAX-2′-Et in small animals.

Abstract: 1482 NUCLEAR HORMONE RECEPTORS AS MEDIATORS OF DIETARY CONSTITUENT-INDUCED METABOLIC CHANGES

Introduction: To determine the feasibility of using dominant negative nuclear receptors to dissect the regulation of inducible gene expression in primary cultured hepatocytes, a series of dominant negative nuclear receptor expression plasmids were designed with truncated AF-2 subdomains. Methods: Plasmids expressing dominant negative or wild-type constitutive androstane receptor (CAR), pregnane X receptor (PXR), farnesoid X receptor (FXR), liver X receptor (LXR), or peroxisome proliferator-activated receptor α (PPARα) were transiently cotransfected into primary cultured rat hepatocytes, together with an appropriate reporter plasmid. Results: Treatment with prototypic inducers, 10−4 M phenobarbital (CAR activator), 10−5 M pregnenolone 16α-carbonitrile (PXR activator), 3×10−5 M chenodeoxycholate (FXR activator), or 10−4 M ciprofibrate (PPARα activator), significantly activated expression from the corresponding reporter plasmid. Treatment with 22(R)-hydroxycholesterol (LXR activator) only weakly activated the LXR-responsive reporter, while pregnenolone 16α-carbonitrile treatment significantly activated this reporter. Cotransfection with wild-type LXRα strongly enhanced 22(R)-hydroxycholesterol-inducible expression from the LXR-responsive reporter. Cotransfection of hepatocyte cultures with each of the dominant negative nuclear receptor plasmids significantly inhibited inducible expression of the corresponding reporter while, with one exception (LXRα), cotransfection with the wild-type receptor moderately enhanced or had little effect on reporter expression. When each dominant negative nuclear receptor was cross-examined against all inducer–reporter pairs, effects on multiple inducer–reporter pairs were frequently observed. However, in general, only cotransfection with the appropriate dominant negative inhibited inducible reporter expression to a greater extent than did cotransfection with the corresponding wild-type receptor. Discussion: We suggest that the application of dominant negative nuclear receptors has utility in transient transfection studies aimed at discerning the regulatory role of individual nuclear receptor transcription factors in inducible hepatic gene expression, provided that appropriate controls are employed.

Low affinity glucocorticoid binding site ligands as potential anti-fibrogenics

BackgroundPregnane X receptor (PXR) agonists inhibit liver fibrosis. However, the rodent PXR activator pregnenolone 16α carbonitrile (PCN) blocks, in vitro, hepatic stellate cell-to-myofibroblast trans-differentiation and proliferation in cells from mice with a disrupted PXR gene, suggesting there is an additional anti-fibrogenic drug target for PCN. The role of the low affinity glucocorticoid binding site (LAGS) – which may be identical or associated with the progesterone receptor membrane component 1 (PGRMC1) – in mediating this anti-fibrogenic effect has been examined, since binding of dexamethasone to the LAGS in liver microsomal membranes has previously been shown to be inhibited by PCN.ResultsQuiescent rat and human hepatic stellate cells (HSC) were isolated from livers and cultured to generate liver myofibroblasts. HSC and myofibroblasts expressed PGRMC1 as determined by RT-PCR and Western blotting. Quiescent rat HSC also expressed the truncated HC5 variant of rPGRMC1. Rat PGRMC1 was cloned and expression in COS-7 cells gave rise to specific binding of radiolabelled dexamethasone in cell extracts that was inhibited by PCN, suggesting that PGRMC1 may be identical to LAGS or activates LAGS binding activity. Liver microsomes were used to screen a range of structurally related compounds for their ability to inhibit radiolabelled dexamethasone binding to rat LAGS. These compounds were also screened for their ability to activate rat and human PXR and to inhibit rat HSC-to-myofibroblast trans-differentiation/proliferation. A compound (4 androstene-3-one 17β-carboxylic acid methyl ester) was identified which bound rat LAGS with high affinity and inhibited both rat and human HSC trans-differentiation/proliferation to fibrogenic myofibroblasts without showing evidence of rat or human PXR agonism. However, despite potent anti-fibrogenic effects in vitro, this compound did not modulate liver fibrosis severity in a rat model of liver fibrosis. Immunohistochemical analysis showed that rat liver myofibroblasts in vivo did not express rPGRMC1.ConclusionLAGS ligands inhibit HSC trans-differentiation and proliferation in vitro but show little efficacy in inhibiting liver fibrosis, in vivo. The reason(s) for this disparity is/are likely associated with an altered myofibroblast phenotype, in vitro, with expression of rPGMRC1 in vitro but not in vivo. These data emphasize the limitations of in vitro-derived myofibroblasts for predicting their activity in vivo, in studies of fibrogenesis. The data also demonstrate that the anti-fibrogenic effects of PCN in vivo are likely mediated entirely via the PXR.

Effects of pregnenolone-16α-carbonitrile on vitamin E status and protein levels of antioxidant enzymes in male rats fed a vitamin E-supplemented diet

The aim of this study was to evaluate the effects of the administration of pregnenolone-16α-carbonitrile (PCN), an inducer of the cytochrome P450 3A gene in rats, on vitamin E status and antioxidant enzyme protein levels in rats fed a vitamin E-supplemented diet. Two groups of male Wistar rats were fed for 3 weeks with a basal diet containing 50 ppm of α-tocopherol or the same diet containing 10 times more α-tocopherol. In the final 3 days, each group was divided into two subgroups which were given a single daily intraperitoneal injection of PCN at 75 mg/kg (groups PCN and PCN+VE) or DMSO (groups DS and DS+VE). PCN treatment alone significantly reduced the α-tocopherol content of the liver and plasma and this effect was prevented by supplementation with 10-fold more α-tocopherol. α-Tocopherol levels in the kidneys, lung, heart, and testes were significantly higher in both vitamin E-supplemented groups than in the control groups. TBARS levels in the liver and lung were significantly increased in both PCN-treated groups, as shown by two-way ANOVA analysis. PCN also caused a significant reduction in protein levels of catalase and glutathione peroxidase (GPx) in both groups. Dietary vitamin E supplementation caused a decrease in liver protein levels of GPx and superoxide dismutase, but not catalase, in both groups and protected against PCN-induced lipid peroxidation, which was caused by CYP3A induction and a reduction in antioxidant enzyme levels.

Interaction of Abcb6 with Cytochrome P450s

Modification of the enzymatic activity of cytochrome P450 (CYP450) enzymes can alter the metabolism of the drug substrate for that enzyme, and thereby alter the biological activity of the drug leading to potentially harmful drug‐drug interactions. The activity and in some cases the induction of these CYP450 family members is mediated by haem, an essential prosthetic group of these detoxifying enzymes. In the present study we investigated whether the mitochondrial half transporter Abcb6, which regulates cellular haem biosynthesis and cellular haemoprotein pool, plays a role in the expression and/or function of CYP450s. We found that over‐expression of Abcb6 in HepG2 cells lead to an increase in CYP3A4 mRNA expression. In contrast over‐expression of Abcb6 in K562 cells resulted in decreased expression of CYP3A4, CYP2E1 and CYP2D6 mRNA. We tested whether prototypical inducers of CYP450 expression regulated Abcb6 expression and found that both pregnenolone‐16α carbonitrile (PCN) and 1,4‐bis[2‐(3,5‐dichloropyridyloxy) benzene (TCPOBOP), two well known inducers of CYP450, also induced Abcb6 expression both in vitro and in vivo. Cumulatively these findings suggest that Abcb6 might play a role in the metabolism and disposition of drugs and environmental chemicals, and may affect drug clearance and drug toxicity.

Cyclic AMP-dependent Protein Kinase Signaling Modulates Pregnane x Receptor Activity in a Species-specific Manner

Pregnane x receptor is a ligand-activated transcription factor that regulates drug-inducible expression of several key cytochrome P450 enzymes and drug transporter proteins in liver and intestine in a species-specific manner. Activation of this receptor modulates several key biochemical pathways, including gluconeogenesis, beta-oxidation of fatty acids, fatty acid uptake, cholesterol homeostasis, and lipogenesis. It is of current interest to determine whether the interaction between pregnane x receptor and these key biochemical pathways is evolutionarily conserved. We show here that activation of the cyclic AMP-dependent protein kinase signaling pathway synergizes with pregnane x receptor-mediated gene activation in mouse hepatocytes. Conversely, cyclic AMP-dependent protein kinase signaling has a repressive effect upon pregnane x receptor-mediated gene activation in rat and human hepatocytes. We show that the human pregnane x receptor protein can serve as an effective substrate for catalytically active cyclic AMP-dependent protein kinase in vitro. Metabolic labeling of the protein in vivo indicates that human pregnane x receptor exists as a phosphoprotein and that activation of cyclic AMP-dependent protein kinase signaling modulates the phosphorylation status of pregnane x receptor. Activation of cyclic AMP-dependent protein kinase signaling also modulates the interactions of pregnane x receptor with protein cofactors. Our results define the species-specific impact of cyclic AMP-dependent protein kinase signaling on pregnane x receptor and provide a molecular explanation of cyclic AMP-dependent protein kinase-mediated repression of human pregnane x receptor activity. Taken together, our results identify a novel mode of regulation of pregnane x receptor activity and highlight prominent functional differences in the process across species.

Comparison of effects of VDR versus PXR, FXR and GR ligands on the regulation of CYP3A isozymes in rat and human intestine and liver

In this study, we compared the regulation of CYP3A isozymes by the vitamin D receptor (VDR) ligand 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2D 3) against ligands of the pregnane X receptor (PXR), the glucocorticoid receptor (GR) and the farnesoid X receptor (FXR) in precision-cut tissue slices of the rat jejunum, ileum, colon and liver, and human ileum and liver. In the rat, 1,25(OH) 2D 3 strongly induced CYP3A1 mRNA, quantified by qRT-PCR, along the entire length of the intestine, induced CYP3A2 only in ileum but had no effect on CYP3A9. In contrast, the PXR/GR ligand, dexamethasone (DEX), the PXR ligand, pregnenolone-16α carbonitrile (PCN), and the FXR ligand, chenodeoxycholic acid (CDCA), but not the GR ligand, budesonide (BUD), induced CYP3A1 only in the ileum, none of them influenced CYP3A2 expression, and PCN, DEX and BUD but not CDCA induced CYP3A9 in jejunum, ileum and colon. In rat liver, CYP3A1, CYP3A2 and CYP3A9 mRNA expression was unaffected by 1,25(OH) 2D 3, whereas CDCA decreased the mRNA of all CYP3A isozymes; PCN induced CYP3A1 and CYP3A9, BUD induced CYP3A9, and DEX induced all three CYP3A isozymes. In human ileum and liver, 1,25(OH) 2D 3 and DEX induced CYP3A4 expression, whereas CDCA induced CYP3A4 expression in liver only. In conclusion, the regulation of rat CYP3A isozymes by VDR, PXR, FXR and GR ligands differed for different segments of the rat and human intestine and liver, and the changes did not parallel expression levels of the nuclear receptors.

Spence Galbraith

Cryopreserved rat hepatocytes entrapped within an alginate matrix commercially available as Liverbeads™, were evaluated for their relevance as a screening tool for gene induction <i>in vitro</i>, using qRT-PCR. They were...

Effects of Cytochrome P450 Inducers on the Gene Expression of Ocular Xenobiotic Metabolizing Enzymes in Rats

In our current study, we investigated the expression profiles of the cytochromes P450 (CYPs) and transporters of the ocular tissues in Sprague-Dawley (SD) rats. Extensive expression of CYP1A1 in the cornea and CYP2E1 in the iris was observed whereas the expression of CYP2B1 and transporters was mostly ubiquitous throughout the ocular tissues. To further understand the regulation of these genes in ocular tissues, we investigated the effects of CYP inducers on the expression of the CYP and other xenobiotic-metabolizing enzyme genes in the cornea and lens. The administration of β-naphthoflavone (BNF), an agonist for aryl hydrocarbon receptor (AhR), induced CYP1A1 gene expression in the cornea, lens and liver of the rats, although the levels of induction were greatest in the liver. An AhR-sensitive UDP-glucuronosyl transferase (UGT) 1A6 gene was also induced in the cornea and the lens by BNF. Phenobarbital (PB) is a known inducer of the CYP2B genes, the expression of which is mediated by constitutive activated receptor (CAR), but did not induce CYP2B1 in the cornea or lens. This insensitivity to PB may be due to the lack of CAR expression in the ocular tissues as revealed in our present study. Pregnenolone-16α-carbonitrile (PCN) is known to induce CYP3A gene expression in the liver via the activation of pregnane X receptor (PXR). However, although PCN was found to induce the CYP3A1 gene in the rat cornea and liver, it failed to do so in the lens. In addition, another of the PXR-mediated genes, multidrug resistance-associated protein 3 (Mrp3), was not induced by PCN in either ocular region. Since the expression of the PXR gene was not detected in the rat ocular tissues, an unknown mechanism for the inducible regulation of CYP3A1 gene expression by PCN in the cornea is suggested.

Drug metabolizing enzyme induction pathways in experimental non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is a disease that compromises hepatic function and the capacity to metabolize numerous drugs. Aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor-E2 related factor 2 (Nrf2) are xenobiotic activated transcription factors that regulate induction of a number of drug metabolizing enzymes (DMEs). The purpose of the current study was to determine whether experimental NASH alters the xenobiotic activation of these transcription factors and induction of downstream DME targets Cyp1A1, Cyp2B10, Cyp3A11, Cyp4A14 and NAD(P)H:quinone oxidoreductase 1 (Nqo1), respectively. Mice fed normal rodent chow or methionine-choline-deficient (MCD) diet for 8 weeks were then treated with microsomal enzyme inducers beta-naphoflavone (BNF), 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), pregnenolone-16alpha-carbonitrile (PCN), clofibrate (CFB) or oltipraz (OPZ), known activators of AhR, CAR, PXR, PPARalpha and Nrf2, respectively. Results of this study show that (1) Hepatic PXR mRNA levels were significantly increased (1.4-fold) in mice fed MCD diet, while AhR, CAR, PPARalpha and Nrf2 were not affected. (2) The MCD diet did not alter hepatic inducibility of Cyp1A1, Cyp2B10, Cyp3A11 mRNA levels by their respective microsomal inducers. (3) Constitutive levels of Cyp4A14 mRNA were significantly increased in mice fed the MCD diet, yet further induction by clofibrate was not observed. (4) Hepatic Nqo1 mRNA levels were significantly increased by the MCD diet; however, additional induction of Nqo1 was still achievable following treatment with the Nrf2 activator OPZ.

Coordinated Nuclear Receptor Regulation of the Efflux Transporter, Mrp2, and the Phase-Ii Metabolizing Enzyme, GSTπ, at the Blood—Brain Barrier

Xenobiotic efflux pumps at the blood-brain barrier are critical modulators of central nervous system pharmacotherapy. We previously found expression of the ligand-activated nuclear receptor, pregnane X receptor (PXR), in rat brain capillaries, and showed increased expression and transport activity of the drug efflux transporter, P-glycoprotein, in capillaries exposed to PXR ligands (pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone) in vitro and in vivo. Here, we show increased protein expression and transport activity of another efflux pump, multidrug resistance-associated protein isoform 2 (Mrp2), in rat brain capillaries after in vitro and in vivo exposure to PCN and dexamethasone. The phase-II drug-metabolizing enzyme, glutathione S-transferase-pi (GSTpi), was found to be expressed in brain capillaries, where it colocalized to a large extent with Mrp2 at the endothelial cell luminal plasma membrane. Like Mrp2, GSTpi protein expression increased with PXR activation. Colocalization and coordinated upregulation suggest functional coupling of the metabolizing enzyme and efflux transporter. These findings indicate that, as in hepatocytes, brain capillaries possess a regulatory network consisting of nuclear receptors, metabolizing enzymes, and efflux transporters, which modulate blood-brain barrier function.

Comparison of the induction profile for drug disposition proteins by typical nuclear receptor activators in human hepatic and intestinal cells

Certain nuclear receptors (NRs) such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR) mediate induction of some cytochrome P450 enzymes and ABC transporters but conflicting reports exist. The purpose of this study was to assess the reasons for these discrepancies and use a standardized approach to compare activators of NRs. Dexamethasone, pregnenolone 16alpha-carbonitrile, rifampicin, phenobarbital and chenodeoxycholic acid were incubated with HepG2, Caco-2 and cryopreserved human hepatocytes prior to analysis of mRNA and protein for CYP2B6, CYP3A4, CYP3A5, ABCB1, ABCC1, ABCC2, PXR, CAR and FXR. Dexamethasone significantly up-regulated PXR, CYP3A4 and ABCB1 expression in HepG2 and Caco-2 cells. As a result, including dexamethasone as a media supplement masked the induction of these genes by pregnenolone 16alpha-carbonitrile, which may explain discrepancies between previous reports. In the absence of dexamethasone, significant activator-dependent induction was observed in all cell types. Significant correlations were observed between the fold increase in mRNA and in protein, which were, for most instances, logarithmic. Changes in mRNA expression were greater in cell lines than primary cells but for most transcripts correlations were observed between fold increases in HepG2 and hepatocytes. Clearly, no in vitro system can imitate the physiology of a hepatocyte or intestinal cell within an intact organ in vivo, but these data explain some of the discrepancies reported between laboratories and have important implications for study design.

Hepatic Fatty Acid Transporter Cd36 Is a Common Target of LXR, PXR, and PPARγ in Promoting Steatosis

Liver X receptor (LXR) is known to promote hepatic lipogenesis by activating the lipogenic transcriptional factor sterol regulatory element-binding protein (Srebp). Pregnane X receptor (PXR), a previously known "xenobiotic receptor," could mediate a Srebp-independent lipogenic pathway by activating the free fatty acid uptake transporter Cd36. The goal of this study is to investigate further the role of Cd36 in hepatic steatosis. Wild-type, LXR transgenic, PXR transgenic, and Cd36 null mice were used to study the regulation of Cd36 and other hepatic lipogenic genes and the implication of this regulation in hepatic steatosis. Promoter sequences of Cd36 and peroxisome proliferator-activated receptor (PPAR) gamma were cloned, and their respective regulation by LXR and PXR was investigated by combinations of receptor-DNA binding and reporter gene assays. We showed that genetic (transgene) or pharmacologic (ligands) activation of LXR induced Cd36. Promoter analysis established Cd36 as a novel transcription target of LXRalpha. Moreover, the hepatic steatosis induced by LXR agonists was largely abolished in Cd36 null mice. We also showed that PPARgamma, a positive regulator of Cd36, is a transcriptional target of PXR, suggesting that PXR can regulate Cd36 directly or through its activation of PPARgamma. Interestingly, both LXR-mediated Cd36 regulation and PXR-mediated PPARgamma regulation are liver specific. We conclude that Cd36 is a shared target of LXR, PXR, and PPARgamma. The network of CD36 regulation by LXR, PXR, and PPARgamma establishes this free fatty acid transporter as a common target of orphan nuclear receptors in their mediation of lipid homeostasis.

Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6-phosphatase gene

The nuclear PXR (pregnane X receptor) was originally characterized as a key transcription factor that activated hepatic genes encoding drug-metabolizing enzymes. We have now demonstrated that PXR also represses glucagon-activated transcription of the G6Pase (glucose-6-phosphatase) gene by directly binding to CREB [CRE (cAMP-response element)-binding protein]. Adenoviral-mediated expression of human PXR (hPXR) and its activation by rifampicin strongly repressed cAMP-dependent induction of the endogenous G6Pase gene in Huh7 cells. Using the -259 bp G6Pase promoter construct in cell-based transcription assays, repression by hPXR of PKA (cAMP-dependent protein kinase)-mediated promoter activation was delineated to CRE sites. GST (glutathione transferase) pull-down and immunoprecipitation assays were employed to show that PXR binds directly to CREB, while gel-shift assays were used to demonstrate that this binding prevents CREB interaction with the CRE. These results are consistent with the hypothesis that PXR represses the transcription of the G6Pase gene by inhibiting the DNA-binding ability of CREB. In support of this hypothesis, treatment with the mouse PXR activator PCN (pregnenolone 16alpha-carbonitrile) repressed cAMP-dependent induction of the G6Pase gene in primary hepatocytes prepared from wild-type, but not from PXR-knockout, mice, and also in the liver of fasting wild-type, but not PXR-knockout, mice. Moreover, ChIP (chromatin immunoprecipitation) assays were performed to show a decreased CREB binding to the G6Pase promoter in fasting wild-type mice after PCN treatment. Thus drug activation of PXR can repress the transcriptional activity of CREB, down-regulating gluconeogenesis.

Nuclear Pregnane X Receptor Cross-talk with FoxA2 to Mediate Drug-induced Regulation of Lipid Metabolism in Fasting Mouse Liver

Upon drug activation, the nuclear pregnane X receptor (PXR) regulates not only hepatic drug but also energy metabolism. Using Pxr(-/-) mice, we have now investigated the PXR-mediated repression of lipid metabolism in the fasting livers. Treatment with PXR activator pregnenolone 16alpha-carbonitrile (PCN) down-regulated the mRNA levels of carnitine palmitoyltransferase 1A (in beta-oxidation) and mitochondrial 3-hydroxy-3-methylglutarate-CoA synthase 2 (in ketogenesis) in wild-type (Pxr(+/+)) mice only. In contrast, the stearoyl-CoA desaturase 1 (in lipogenesis) mRNA was up-regulated in the PCN-treated Pxr(+/+) mice. Reflecting these up- and down-regulations and consistent with decreased energy metabolism, the levels of hepatic triglycerides and of serum 3-hydroxybutylate were increased and decreased, respectively, in the PCN-treated Pxr(+/+) mice. Using gel shift, glutathione S-transferase pull-down and cell-based reporter assays, we then examined whether PXR could cross-talk with the insulin response forkhead factor FoxA2 to repress the transcription of the Cpt1a and Hmgcs2 genes, because FoxA2 activates these genes in fasting liver. PXR directly bound to FoxA2 and repressed its activation of the Cpt1a and Hmgcs2 promoters. Moreover, ChIP assays showed that PCN treatment attenuated the binding of FoxA2 to these promoters in fasting Pxr(+/+) but not Pxr(-/-) mice. These results are consistent with the conclusion that PCN-activated PXR represses FoxA2-mediated transcription of Ctp1a and Hmgcs2 genes in fasting liver.

The effect of pregnane X receptor (PXR)‐mediated CYP3A induction on acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is the leading cause of acute liver failure in the U.S. Cytochromes P450 CYP2E1, CYP1A2, and CYP3A4 have all been implicated in formation of the reactive intermediate of APAP, and thus can contribute to APAP hepatotoxicity. The nuclear pregnane X receptor (PXR) plays a critical role in CYP3A regulation and many drugs and dietary compounds are PXR activators. Thus, it is important to determine whether PXR-mediated CYP3A induction is a risk factor for APAP hepatotoxicity. In this study, a double transgenic human PXR and CYP3A4 mice (TgCYP3A4/hPXR) mouse model was generated in a mouse Pxr-null background. In this model, CYP3A4 was strongly induced by rifampicin (RIF), a human-specific PXR ligand, but not by pregnenolone 16α-carbonitrile, a rodent-specific PXR ligand. Consistent with CYP3A4 expression, hepatic CYP3A4 activity increased ~500% in TgCYP3A4/hPXR mice treated with RIF. However, APAP (250 mg/kg, p.o.) hepatotoxicity did not concurrently increase in TgCYP3A4/hPXR mice upon PXR activation and CYP3A4 induction, as revealed by hepatic histological changes and serum ALT levels. Moreover, there were no significant differences in the major APAP urinary metabolites between RIF-pretreated and vehicle-treated groups. In conclusion, these studies suggest that PXR-mediated CYP3A4 induction does not enhance APAP hepatotoxicity and PXR ligands are not risk factors for hepatotoxicity when combined medication with APAP.

Xenobiotic- and vitamin D-responsive induction of the steroid/bile acid-sulfotransferase Sult2A1 in young and old mice: The role of a gene enhancer in the liver chromatin

The xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D 3-activated nuclear receptor VDR regulate steroid and xenobiotic metabolism by inducing the phase I cytochrome P450 monooxygenases, phase II conjugating transferases, and the phase III transporters, which mediate the efflux of water-soluble lipid metabolites from cells. Metabolic stress due to the deviant expression of steroid- and xenobiotic-metabolizing enzymes is known to have severe health consequences including accelerated aging, and increased expression of these enzymes is associated with extended longevity [Gachon, F, Olela, FF, Schaad, O, Descombes, P and Schibler, U, 2006. The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification. 4, 25–36.; McElwee, JJ, Schuster, E, Blanc, E, Thomas, JH and Gems, D, 2004. Shared Transcriptional Signature in Caenorhabditis elegans Dauer Larvae and Long-lived daf-2 Mutants Implicates Detoxification System in Longevity Assurance. J. Biol. Chem., 279, 44533–43.]. Information on the similarities and dissimilarities in drug metabolism between the young and old, as may be uncovered by studying aging regulation of the genes relevant to steroid and xenobiotic metabolism, is likely to have clinical significance. In this report, we examined the VDR- and PXR-mediated gene induction of the phase II sulfotransferase Sult2A1 in the livers of 4-month- and 20-month-old mice. Sult2A1 converts bile acids, steroids and a number of drugs to the corresponding sulfated metabolites, which are readily eliminated from the body due to increased water solubility. In RT-PCR assay, aging did not change the induction of Sult2A1 mRNAs by the hormonally active vitamin D 3 and the catatoxic synthetic steroid PCN (pregnenolone-16α-carbonitrile). Chromatin immunoprecipitation (ChIP) from liver nuclei showed that aging had no effect on the activity of an IR0 enhancer in the Sult2A1 chromatin to recruit VDR, RXR-α (retinoid X receptor) and PXR in mice injected with D 3 or PCN. Thus, mice in late life are as competent as those in early life in responding to the hormonal and xenobiotic signaling for Sult2A1 induction. This is the first report describing the role of aging in the functional response of an enhancer in the liver chromatin to the nuclear receptor-dependent signaling.

P53-Independent Induction of Rat Hepatic Mdm2 following Administration of Phenobarbital and Pregnenolone 16α-Carbonitrile

Murine double minute 2 (Mdm2) negatively regulates p53 by mediating its ubiquitination and proteosomal degradation, and Mdm2 is recognized as a proto-oncogene. In the present study, hepatic gene expression patterns induced by phenobarbital (PB; 100 mg/kg) and pregnenolone 16alpha-carbonitrile (PCN, 100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.

Identification of Genes Controlled by the Pregnane X Receptor by Microarray Analysis of mRNAs from Pregnenolone 16 -Carbonitrile-Treated Rats

Mammalian liver contains a pregnane X receptor (PXR, NR1I2), which binds drugs and other xenobiotics, and stimulates (or suppresses) expression of numerous genes involved in the metabolic elimination of foreign compounds and some toxic endogenous substances. In the present study, we used microarray analysis to identify genes whose expression in rat liver was significantly altered by pregnenolone 16alpha-carbonitrile (PCN) treatment. PCN is a synthetic steroid that induces cytochrome P4503A expression and is hepatoprotective by increasing resistance to subsequent stressful insults. Significant induction was seen for 138 genes while expression of 82 genes was significantly repressed. We found induction of genes known to be induced by PCN, such as enzymes involved in drug metabolism and transport. In addition, many genes were differentially expressed whose functions concerned intracellular metabolism, transport of essential small molecules, cell cycle, and redox balance. Our results support the idea that the domain of PXR-controlled gene networks may be even more extensive than currently thought and may extend to functions apart from xenobiotic metabolism.

Role for enhanced faecal excretion of bile acid in hydroxysteroid sulfotransferase-mediated protection against lithocholic acid-induced liver toxicity

The efficient clearance of toxic bile acids such as lithocholic acid (LCA) requires drug-metabolizing enzymes. We therefore assessed the influence of pregnenolone 16α-carbonitrile (PCN) treatment on LCA-induced hepatotoxicity and disposition of LCA metabolites using female farnesoid X receptor (FXR)-null and wild-type mice. Marked decreases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and hepatic tauroLCA (TLCA) concentrations were found in LCA-fed wild-type mice co-treated with PCN. Whereas induction of Cyp3a and hydroxysteroid sulfotransferase (Sult2a) proteins was observed in FXR-null and wild-type mice, clear increases in biliary 3α-sulfated TLCA but not total 6α-hydroxy LCA (taurohyodeoxycholic acid and hyodeoxycholic acid) were only observed in PCN-treated wild-type mice. Biliary 3α-sulfated TLCA output rate was increased 7.2-fold, but accounts for only 4.2% of total bile acid output rate in LCA and PCN-co-treated wild-type mice. Total 3α-sulfated LCA (LCA and TLCA) was, however, the most abundant bile acid component in faeces suggesting that efficient faecal excretion of biliary 3α-sulfated TLCA through escape from enterohepatic circulation. FXR-null mice, which have constitutively high levels of the Sult2a protein, were fed a diet supplemented with 1% LCA and 0.4% dehydroepiandrosterone (DHEA), a typical Sult2a substrate/inhibitor. The faecal total 3α-sulfated bile acid excretion was reduced to 62% of FXR-null mice fed only the LCA diet. Hepatic TLCA concentration and serum AST activity were significantly higher in FXR-null mice fed DHEA and LCA diet than in FXR-null mice fed the LCA diet or DHEA diet. These results suggest that hepatic formation of 3α-sulfated TLCA is a crucial factor for protection against LCA-induced hepatotoxicity.