Nuclear Receptor Constitutive Androstane Receptor Research Articles

RF03 | PMON287 Examining the role of Constitutive Androstane Receptor during Imidacloprid exposure

Abstract Imidacloprid (IMI) is a neonicotinoid pesticide used for pest control and seed treatments. It mimics nicotine and selectively targets insect nicotinic acetylcholine receptors to induce paralysis and death. Popularly used worldwide, IMI residue is readily detectable in crops, the environment, and human samples. Gastrointestinal symptoms in patients as well as defective gut barrier and hepatoxicity in animal models have been reported subsequent to IMI exposure. However, the mechanism underlying IMI toxicity in mammals is unclear. Pesticide exposure frequently activates xenobiotic nuclear receptors, like constitutive androstane receptor (CAR), to induce detoxification phase I and phase II genes. We found that IMI exposure in wild-type (WT) mice induced hepatic expression of Cyp2b10, a bona fide target of CAR. This data led us to investigate the role of CAR in alleviating IMI toxicity. We dosed wild-type (WT) and Car-/- mice with 50mg/kg BW IMI, twice daily for 21 days and collected serum, liver, and intestine tissues. The absence of CAR resulted in a three-fold increased accumulation of IMI within the liver as well as a 6% increase in body weight. Intriguingly, IMI exposure resulted in hepatic inflammatory pockets in WT mice but did not induce serum liver injury markers (ALT and AST) in either WT or Car-/- mice. We quantified the expression of genes involved in detoxification and oxidative stress within the liver and ileum using qRT-PCR. We found Car-/-mice had significantly blunted expression of Cyp-P450 enzymes in the ileum. To compare gut function, we measured organ coefficient and examined ileal histology using Alcian Blue/PAS staining. We found the absence of CAR altered small intestine (SI) morphology independent of IMI exposure suggesting a basal role of CAR in maintaining overall SI morphology. Car-/-mice also exhibited increased SI weight and length and shorter villi height. AB-PAS staining showed IMI reduced ileal mucin production in WT mice but not Car-/- mice. To further examine CAR's role in the xenobiotic metabolism in the ileum, we will compare microbial diversity and bile acid pools with and without IMI exposure. Our results suggest IMI is not overtly toxic, but the absence of xenobiotic nuclear receptor CAR allows increased accumulation of IMI in the liver and disrupts the structure and Cyp gene expression in the intestine. Presentation: Saturday, June 11, 2022 1:18 p.m. - 1:23 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Deletion of Constitutive Androstane Receptor Led to Intestinal Alterations and Increased Imidacloprid in Murine Liver.

Imidacloprid (IMI) is the most frequently detected neonicotinoid pesticide in the environment. Despite typically low toxicity in vertebrates, IMI exposure is associated with liver and gastrointestinal toxicity. The mechanism underlying IMI toxicity in mammals is unclear. Pesticide exposure frequently activates xenobiotic nuclear receptors, such as the constitutive androstane receptor (CAR), to induce detoxification phase I and phase II genes. This study examined the role of CAR in mediating IMI off-target toxicity. Female Car-/- and wild-type (WT) mice were orally administered imidacloprid (50 mg/kg, twice daily) for 21 days, following which serum, liver, and intestinal tissues were collected. Liver tissue analysis indicated mild inflammation and induction of detoxification gene Cyp2b10 in IMI-exposed WT mice. The absence of CAR increased hepatic IMI accumulation. Microbiome analysis of ileal samples revealed IMI altered microbial diversity in a genotype-specific manner, with increased α-diversity in Car-/- mice while decreased α-diversity in WT mice. We observed Car-/- mice exhibit intestinal alterations with decreased CYP-P450 expression, blunted villi height, and increased small intestine length and weight independent of IMI exposure. Our results suggest that IMI is not overtly toxic. However, the absence of xenobiotic nuclear receptor CAR allows increased accumulation of IMI in the liver and disrupts the villi structure and Cyp gene expression in the intestine.

Regulation of Nuclear Receptors PXR and CAR by Small Molecules and Signal Crosstalk: Roles in Drug Metabolism and Beyond.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are ligand-activated transcription factors that regulate the expression of drug metabolizing enzymes and drug transporters. Since their discoveries, they have been studied as important factors for regulating processes related to drug efficacy, drug toxicity, and drug-drug interactions. However, their vast ligand-binding profiles extend into additional spaces, such as endogenously produced chemicals, microbiome metabolites, dietary compounds, and environmental pollutants. Therefore, PXR and CAR can respond to an enormous abundance of stimuli, resulting in significant shifts in metabolic programs and physiologic homeostasis. Naturally, PXR and CAR have been implicated in various diseases related to homeostatic perturbations, such as inflammatory bowel disorders, diabetes, and certain cancers. Recent findings have injected the field with new signaling mechanisms and tools to dissect the complex PXR and CAR biology and have strengthened the potential for future PXR and CAR modulators in the clinic. Here, we describe the historical and ongoing importance of PXR and CAR in drug metabolism pathways and how this history has evolved into new mechanisms that regulate and are regulated by these xenobiotic receptors, with a specific focus on small molecule ligands. To effectively convey the impact of newly emerging research, we have arranged five diverse and representative key recent advances, four specific challenges, and four perspectives on future directions. SIGNIFICANCE STATEMENT: PXR and CAR are key transcription factors that regulate homeostatic detoxification of the liver and intestines. Diverse chemicals bind to these nuclear receptors, triggering their transcriptional tuning of the cellular metabolic response. This minireview revisits the importance of PXR and CAR in pharmaceutical drug responses and highlights recent results with implications beyond drug metabolism.

Comparison of Sweated and Non-Sweated Ethanol Extracts of Salvia miltiorrhiza Bge. (Danshen) Effects on Human and Rat Hepatic UDP-Glucuronosyltransferase and Preclinic Herb-Drug Interaction Potential Evaluation.

The ethanol of Danshen (DEE) preparation has been widely used to treat cardiac-cerebral disease and cancer. Sweating is one of the primary processing methods of Danshen, which greatly influences its quality and pharmacological properties. Sweated and non-sweated DEE preparation combined with various synthetic drugs, add up the possibility of herbal-drug interactions. This study explored the effects of sweated and non-sweated DEE on human and rat hepatic UGT enzyme expression and activity and proposed a potential mechanism. The expression of two processed DEE on rat UGT1A, UGT2B, and nuclear receptors, including pregnane X receptor (PXR), constitutive androstane receptor (CAR), and peroxisome proliferator-activated receptor α (PPARα), were investigated after intragastric administration in rats by Western blot. Enzyme activity of DEE and its active ingredients (Tanshinone I, Cryptotanshinone, and Tanshinone I) on UGT isoenzymes was evaluated by quantifying probe substrate metabolism and metabolite formation in vitro using Ultra Performance Liquid Chromatography. The two processed DEE (5.40 g/kg) improved UGT1A (P<0.01) and UGT2B (P<0.05) protein expression, and the non-sweated DEE (2.70 g/kg) upregulated UGT2B expression protein (P<0.05), compared with the CMCNa group. On day 28, UGT1A protein expression was increased (P<0.05) both in two processed DEE groups meanwhile, the non-sweated DEE significantly enhanced UGT2B protein expression (P<0.05) on day 21, compared with the CMCNa group. The process underlying this mechanism involved the activation of nuclear receptors CAR, PXR, and PPARα. In vitro, sweated DEE (0-80 μg/mL) significantly inhibited the activity of human UGT1A7 (P<0.05) and rat UGT1A1, 1A8, and 1A9 (P<0.05). Non-sweated DEE (0-80 μg/mL) dramatically suppressed the activity of human UGT1A1, 1A3, 1A6, 1A7, 2B4, and 2B15, and rat UGT1A1, 1A3, 1A7, and 1A9 (P<0.05). Tanshinone I (0-1 μM) inhibited the activity of human UGT1A3, 1A6, and 1A7 (P<0.01) and rat UGT1A3, 1A6, 1A7, and 1A8 (P<0.05). Cryptotanshinone (0-1 μM) remarkably inhibited the activity of human UGT1A3 and 1A7 (P<0.05) and rat UGT1A7, 1A8, and 1A9 (P<0.05). Nonetheless, Tanshinone IIA (0-2 μM) is not a potent UGT inhibitor both in humans and rats. Additionally, there existed significant differences between two processed DEE in the expression of PXR, and the activity of human UGT1A1, 1A3, 1A6, and 2B15 and rat UGT1A3, and 2B15 (P<0.05). The effects of two processed DEE on hepatic UGT enzyme expression and activity differed. Accordingly, the combined usage of related UGTs substrates with DEE and its monomer components preparations may call for caution, depending on the drug's exposure-response relationship and dose adjustment. Besides, it is vital to pay attention to the distinction between sweated and non-sweated Danshen in clinic, which influences its pharmacological activity.

Understanding the physiological functions of the host xenobiotic-sensing nuclear receptors PXR and CAR on the gut microbiome using genetically modified mice

Pharmacological activation of the xenobiotic-sensing nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) is well-known to increase drug metabolism and reduce inflammation. Little is known regarding their physiological functions on the gut microbiome. In this study, we discovered bivalent hormetic functions of PXR/CAR modulating the richness of the gut microbiome using genetically engineered mice. The absence of PXR or CAR increased microbial richness, and absence of both receptors synergistically increased microbial richness. PXR and CAR deficiency increased the pro-inflammatory bacteria Helicobacteraceae and Helicobacter. Deficiency in both PXR and CAR increased the relative abundance of Lactobacillus, which has bile salt hydrolase activity, corresponding to decreased primary taurine-conjugated bile acids (BAs) in feces, which may lead to higher internal burden of taurine and unconjugated BAs, both of which are linked to inflammation, oxidative stress, and cytotoxicity. The basal effect of PXR/CAR on the gut microbiome was distinct from pharmacological and toxicological activation of these receptors. Common PXR/CAR-targeted bacteria were identified, the majority of which were suppressed by these receptors. hPXR-TG mice had a distinct microbial profile as compared to wild-type mice. This study is the first to unveil the basal functions of PXR and CAR on the gut microbiome.

Targeting Xenobiotic Nuclear Receptors PXR and CAR to Prevent Cobicistat Hepatotoxicity.

Liver-related diseases including drug-induced liver injury are becoming increasingly prominent in AIDS patients. Cobicistat (COBI) is the backbone of multiple regimens for antiretroviral therapy. The current work investigated the mechanisms of adverse drug-drug interactions associated with COBI that lead to liver damage. For individuals co-infected with HIV and tuberculosis (TB), the World Health Organization recommends the initiation of TB treatment followed by antiretroviral therapy. Rifampicin (RIF), a first line anti-TB drug, is a human specific activator of pregnane X receptor (PXR). Using PXR-humanized mice, we found that RIF-mediated PXR activation potentiates COBI hepatotoxicity. In contrast, rifabutin, a PXR-neutral analog of RIF, has no impact on COBI hepatotoxicity. Because of the crosstalk between PXR and the constitutive androstane receptor (CAR), the role of CAR in COBI hepatotoxicity was also investigated. Similar to PXR, ligand-dependent activation of CAR also potentiates COBI hepatotoxicity. Our further studies illustrated that PXR and CAR modulate COBI hepatotoxicity through the CYP3A4-dependent pathways. In summary, the current work determined PXR and CAR as key modulators of COBI hepatotoxicity. Given the fact that many prescription drugs and herbal supplements can activate PXR and CAR, these two receptors should be considered as targets to prevent COBI hepatotoxicity in the clinic.

The Connection of Azole Fungicides with Xeno-Sensing Nuclear Receptors, Drug Metabolism and Hepatotoxicity.

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role of nuclear receptors such as the constitutive androstane receptor (CAR), the pregnane-X-receptor (PXR), or the aryl hydrocarbon receptor (AHR), within the molecular pathways leading to hepatotoxicity of these compounds. Nuclear receptor-mediated hepatic effects may comprise rather adaptive changes such as the induction of drug-metabolizing enzymes, to hepatocellular hypertrophy, histopathologically detectable fatty acid changes, proliferation of hepatocytes, and the promotion of liver tumors. Here, we present a comprehensive review of the current knowledge of the interaction of major agricultural azole-class fungicides with the three nuclear receptors CAR, PXR, and AHR in vivo and in vitro. Nuclear receptor activation profiles of the azoles are presented and related to histopathological findings from classic toxicity studies. Important issues such as species differences and multi-receptor agonism and the consequences for data interpretation and risk assessment are discussed.

Influence of signaling kinases on functional dynamics of nuclear receptor CAR.

Constitutive androstane receptor (CAR) is a xenobiotic nuclear receptor known to regulate genes involved in key physiological processes like drug metabolism, maintenance of energy homeostasis, and cell proliferation. Owing to the diverse regulatory roles played by the receptor, it is critical to understand the precise cellular signals that dictate functional dynamics of CAR. With the objective of exploring the hitherto unknown regulatory pathways modulating CAR, we subjected the CAR protein sequence to a kinase prediction tool and identified several kinases recognizing CAR as a substrate. Using fluorescence live cell imaging and specific inhibitors it was observed that CAR functions under the regulation of mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3) signaling cascade. Additionally, insulin-like growth factor 1 (IGF1)-mediated inhibition of GSK3 also induced nuclear translocation of CAR linking CAR to the Akt signaling pathway. Identification of T38 residue of CAR as the GSK3 target site further substantiated our observations. Taking cues from these findings, we propose a hypothetical model elucidating the GSK3-mediated regulation of CAR dynamics through the involvement of Akt pathway. Further research into this area is expected to provide novel therapeutic targets in disease conditions like type 2 diabetes and hepatocellular carcinoma.

Phenanthrene, but not its isomer anthracene, effectively activates both human and mouse nuclear receptor constitutive androstane receptor (CAR) and induces hepatotoxicity in mice

Polycyclic aromatic hydrocarbons (PAHs) are a class of pervasive global environmental pollutants and adversely affect human health. Among PAHs, phenanthrene and anthracene are isomers consisting of three benzene rings. In the present study, we have made comparisons of constitutive androstane receptor (CAR) activation and toxic effects on the liver between these two isomers. Phenanthrene, but not anthracene, significantly induced promoter activity and gene expression of human drug metabolizing enzyme CYP2B6 in HepG2 cells and human primary hepatocytes, respectively. Phenanthrene, but not anthracene, significantly increased CYP2B10 expression levels and caused hepatotoxicity in mice. Phenanthrene induced the nuclear accumulation of CAR in the liver of wild-type mice, but not CAR−/− mice. Hepatocellular necrosis, elevated expression levels of some CAR-related genes such as CYP2B10, CYP3A11, UGT1A1, SULT2A1 and GSTM3, and lower hepatic glutathione levels were found in phenanthrene-exposed wild-type mice but not CAR−/− mice. Additionally, phenanthrene and anthracene were detected in both raw and grilled lamb samples. The average concentrations of phenanthrene were much higher than those of anthracene in these samples. This study is the first to demonstrate that phenanthrene, but not its isomer anthracene, effectively activates both human and mouse nuclear receptor CAR, and CAR plays a crucial role in phenanthrene-induced mouse hepatotoxicity. Compared with anthracene, K region may be an important electronic structure of phenanthrene for activation of CAR. Dietary consumption of PAHs-contaminated food is an important exposure route for humans. Exposure to phenanthrene may affect human health especially associated with liver.

Response of the nuclear receptors PXR and CAR and their target gene mRNA expression in female piglets exposed to zearalenone

A study was conducted to determine the effects of zearalenone (ZEN) on the mRNA expression of pregnane X receptor (PXR), constitutive and rostane receptor (CAR), and phase I and II enzymes as well as the toxicity in the liver of female weanling piglets. Thirty-two female weanling piglets (Duroc × Landrace × Large white, 12.27 ± 0.30 kg)were divided into four groups (n = 8 piglets/group) that were supplemented with 0 (control), 0.5, 1 or 2 mg/kg ZEN. The trial period lasted for 28 d. The results showed that the ZEN supplementation in the diets (0.5–2 mg/kg) had no effect on growth performance but dose-dependently increased serum aspartate aminotransferase, alanineaminotransferase, alkaline phosphatase, and γ-glutamyltransferase activities (P < 0.05). The ZEN residue in the liver (P < 0.01) was also linearly and dose-dependently increased. Furthermore, the mRNA expression of PXR, CAR, phase I enzymes (i.e., cyp2e1, cyp3a5, cyp2a6, cyp1a1, and cyp1a2), and phase II enzymes (i.e., gsta1, gsta2, ugt1a3) significantly increased linearly in a dose-dependent manner (P < 0.05). However, the spleen relative weight and the glutathione peroxidase activity in the liver (P < 0.05) linearly decreased as the dietary ZEN concentration increased; the mRNA expression of the nuclear receptors PXR and CAR is responsive to ZEN in female piglets, and ZEN increases the mRNA expression of their target genes. This finding shows that the nuclear receptor signaling system plays an important role in the defense against ZEN.

Bile acid detoxifying enzymes limit susceptibility to liver fibrosis in female SHRSP5/Dmcr rats fed with a high-fat-cholesterol diet.

During middle age, women are less susceptible to nonalcoholic steatohepatitis (NASH) than men. Thus, we investigated the underlying molecular mechanisms behind these sexual differences using an established rat model of NASH. Mature female and male stroke-prone spontaneously hypertensive 5/Dmcr rats were fed control or high-fat-cholesterol (HFC) diets for 2, 8, and 14 weeks. Although HFC-induced hepatic fibrosis was markedly less severe in females than in males, only minor gender differences were observed in expression levels of cytochrome P450 enzymes (CYP)7A1, CYP8B1 CYP27A1, and CYP7B1, and multidrug resistance-associated protein 3, and bile salt export pump, which are involved in fibrosis-related bile acid (BA) kinetics. However, the BA detoxification-related enzymes UDP-glucuronosyltransferase (UGT) and sulfotransferase (SULT) 2A1, and the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR), were strongly suppressed in HFC-fed males, and were only slightly changed in HFC-diet fed females. Expression levels of the farnesoid X receptor and its small heterodimer partner were similarly regulated in a gender-dependent fashion following HFC feeding. Hence, the pronounced female resistance to HFC-induced liver damage likely reflects sustained expression of the nuclear receptors CAR and PXR and the BA detoxification enzymes UGT and SULT.

Molecular interactions of dioxins and DLCs with the xenosensors (PXR and CAR): An in silico risk assessment approach.

Dioxins and dioxin-like compounds (DLCs) are known to cause endocrine disruption in humans and animals. Being lipophilic xenobiotic chemicals, they can be easily absorbed into the biological system from the surrounding environments, thereby causing various health dysfunctions. In the present study, a total of 100 dioxins and DLCs were taken, and their binding pattern was assessed with the xenosensors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in comparison with the corresponding known inhibitors and a well-studied endocrine disrupting xenobiotic, bisphenol A (BPA). The nuclear receptors CAR and PXR are known to play a significant role in handling potential toxins by coordinating cellular transport and metabolic functions of the same. Among different endocrine-disrupting chemicals used in the present study, DLCs (PCDFs and PCBs) elicited better interactions in comparison with the parent dioxin (polychlorinated dibenzodioxins) compounds. On comparing D scores of all the compounds against both the receptors, PCDF 8-hydroxy-3,4-dichlorodibenzofuran (8-OH-DCDF) and PCB tetrachlorobenzyltoluene (TCBT) exhibited significant molecular interactions against PXR (-7.633kcal mol-1 ) and CAR (-8.389kcal mol-1 ), respectively. Predominant interactions were found to be H-bonding, π-π stacking, hydrophobic, polar, and van der Waals. By contrast, BPA and some natural ligands tested in this study showed lower binding affinities with these receptors than certain DLCs reported herein, ie, certain DLCs might be more toxic than the proven toxic agent, BPA. Such studies play a pivotal role in the risk assessment of exposure to dioxins and DLCs on human health.

Thiazolidinedione Class of Anti-Diabetic Drugs Modulate Nuclear Receptor CAR Function

The human body has developed a defence system to prevent the accumulation of endogenous (bile acids, steroids, cholesterol metabolites, neurotransmitters, etc.) as well as exogenous (xenobiotics, clinical drugs, etc.) small molecules at toxic levels. This task is accomplished by ‘drug metabolism and disposition (DMD) machinery’ which entails phase I and phase II enzymes, and phase III transporter proteins. The components of this machinery act in a coordinated manner to biotransform and facilitate the elimination of small toxic molecules from the cellular milieu. Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, acts as one of the major transcriptional regulators of the DMD machinery. Prescription of combination therapy is a common regimen during the treatment of diverse metabolic disorders and infectious diseases. In such combination therapies one drug may modulate the expression of genes of DMD, influencing the metabolism of another co-administered drug. This leads to decreased bioavailability or increased toxicity of the latter. Evaluation of drug-drug interactions (DDIs) has now become a major safety concern during drug discovery and development processes. Pre-assessment of the small molecules for modulatory effects on CAR and induction of the components of DMD can resolve the safety concerns, treatment failures and drug withdrawals due to the harmful DDIs. In the present study, we have followed a ‘reverse approach’ to assess CAR activation by drugs previously withdrawn from clinical practices. We selected three redundant members of thiazolidinedione family of anti-diabetic drugs and examined their potential in regulation of CAR and its target gene CYP2B6. These drugs showed differential transcriptional activation of CAR. Two of the TZD i.e., rosiglitazone and pioglitazone enhanced CAR activity by behaving as receptor ligands while the other (troglitazone) did not influence the receptor function and was justly withdrawn since it inflicted cytotoxicity.

Activation of nuclear receptor CAR by an environmental pollutant perfluorooctanoic acid.

Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor α is suggested to mediate their toxicities, but the precise mechanism remains unclear. Previous reports also imply a possible role of constitutive androstane receptor (CAR), a key transcription factor for the xenobiotic-induced expression of various genes involved in drug metabolism and disposition as well as hepatocarcinogenesis. Therefore, we have investigated whether PFCAs activate CAR. In wild-type but not Car-null mice, mRNA levels of Cyp2b10, a CAR target gene, were increased by PFOA treatment. PFCA treatment induced the nuclear translocation of CAR in mouse livers. Since CAR activators are divided into two types, ligand-type activators and phenobarbital-like indirect activators, we investigated whether PFCAs are CAR ligands or not using the cell-based reporter gene assay that can detect CAR ligands but not indirect activators. As results, neither PFCAs nor phenobarbital increased reporter activities. Interestingly, in mouse hepatocytes, pretreatment with the protein phosphatase inhibitor okadaic acid prevented an increase in Cyp2b10 mRNA levels induced by phenobarbital as reported, but not that by PFOA. Finally, in human hepatocyte-like HepaRG cells, PFOA treatment increased mRNA levels of CYP2B6, a CAR target gene, as did phenobarbital. Taken together, our present results suggest that PFCAs including PFOA are indirect activators of mouse and human CAR and that the mechanism might be different from that for phenobarbital. The results imply a role of CAR in the hepatotoxicity of PFCAs.

Serum serotonin reduced the expression of hepatic transporter Mrp2 and P-gp via regulating nuclear receptor CAR in PI-IBS rats

Hepatic transporters and drug metabolizing enzymes (DMEs) play important roles in the pharmacological effects and (or) side-effects of many drugs, and are regulated by several mediators, including neurotransmitters. This work aimed to investigate whether serum levels of 5-hydroxytryptamine (5-HT) affected the expression of hepatic transporters or DMEs. The expression of hepatic transporters was assessed using the Western-blot technique in a 2,4,6-trinitrobenzenesulfonic-acid-induced rat model of post-infectious irritable bowel syndrome (PI-IBS), in which serum levels of 5-HT were significantly elevated. To further clarify the underlying mechanism, the 5-HT precursor 5-hydroxytryptophan (5-HTP) and the 5-HT depleting agent parachlorophenylalanine (pCPA) were applied to adjust serum levels of 5-HT. Serum levels of 5-HT were measured using LC-MS/MS; the expression of hepatic transporters, DMEs, and nuclear receptors were examined by Western-blot technique. Our results showed that in PI-IBS rats the expression of multidrug resistance protein 2 (Mrp2) was significantly decreased, while colonic enterochromaffin cell density and serum levels of 5-HT were all significantly increased. Moreover, 5-HTP treatment significantly increased serum levels of 5-HT and decreased the expression of Mrp2 and glycoprotein P (P-gp), whereas treatment with pCPA markedly decreased serum levels of 5-HT and increased the expression of Mrp2 and P-gp. Our results indicated that serum 5-HT regulates the expression of Mrp2 and P-gp, and the underlying mechanism may be related to the altered expression of the nuclear receptor constitutive androstane receptor (CAR).

Expression of CYP3A4 and CYP3A7 in Human Foetal Tissues and its Correlation with Nuclear Receptors.

Previous reports have suggested that the nuclear receptors vitamin D receptor (VDR), peroxisome proliferator-activated receptor α (PPARα), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are involved in the regulation of the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 expression in adults. The aim of this study was to investigate the gene expression of CYP3A4 and the foetal CYP3A7 in human foetal tissues and their relation to gene expression and genetic variations in the nuclear receptors VDR, PPARα, PXR and CAR. We determined the relative expression of CYP3A4 and CYP3A7 and these nuclear receptors in foetal livers, intestines and adrenals, using quantitative PCR. In addition, the expression of these enzymes was also analysed in adult liver. There was a high interindividual variability in CYP3A4 and CYP3A7, 49 times and 326 times, respectively. Both CYP3A4 and CYP3A7 had the highest expression in the liver. There were significant correlations (p<0.001) between the nuclear receptors studied and the expression of CYP3A4 and CYP3A7 in foetal liver, as well as the expression of CYP3A4 in foetal intestine. Polymorphisms in the VDR gene, rs1544410 and rs1523130 (TaqI), in the PXR gene, rs1523130, and in the PPARα gene, rs4253728, were not correlated with CYP3A4 or CYP3A7 expression. However, C-homozygous individuals of the TaqI VDR polymorphism had 60% lower VDR gene expression (p<0.05), than individuals carrying one or two T alleles. In conclusion, differences in the expression of nuclear receptors might determine the variability in CYP3A4 and CYP3A7 expression observed in foetal liver.

Effect of a high-fat diet on the hepatic expression of nuclear receptors and their target genes: relevance to drug disposition.

More than 1·4 billion individuals are overweight or obese worldwide. While complications often require therapeutic intervention, data regarding the impact of obesity on drug disposition are scarce. As the influence of diet-induced obesity on drug transport and metabolic pathways is currently unclear, the objective of the present study was to investigate the effect of high fat feeding for 13 weeks in female Sprague-Dawley rats on the hepatic expression of the nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), liver X receptor (LXR) and farnesoid X receptor (FXR) and several of their target genes. We hypothesised that high fat feeding would alter the gene expression of major hepatic transporters through a dysregulation of the expression of the nuclear receptors. The results demonstrated that, along with a significant increase in body fat and weight, a high-fat diet (HFD) induced a significant 2-fold increase in the expression of PXR as well as a 2-, 5- and 2·5-fold increase in the hepatic expression of the PXR target genes Abcc2, Abcb1a and Cyp3a2, respectively (P<0·05). The expression levels of FXR were significantly increased in rats fed a HFD in addition to the increase in the expression levels of FXR target genes Abcb11 and Abcb4. The expression levels of both LXRα and LXRβ were slightly but significantly increased in rats fed a HFD, and the expression levels of their target genes Abca1 and Abcg5, but not Abcg8, were significantly increased. The expression of the nuclear receptor CAR was not significantly altered between the groups. This suggests that a HFD may induce changes in the hepatobiliary transport and metabolism of endogenous and exogenous compounds.

Phenobarbital-mediated tumor promotion in transgenic mice with humanized CAR and PXR.

The nuclear receptors CAR (constitutive androstane receptor) and possibly PXR (pregnane X receptor) mediate the hepatic effects of phenobarbital (PB) and similar-acting compounds. Although PB is a potent nongenotoxic tumor promoter in rodent liver, epidemiological data from epilepsy patients treated with phenobarbital do not show a specific role of PB in human liver cancer risk. That points to species differences in the susceptibility to tumor promotion by PB, which might be attributed to divergent functions of the PB receptors CAR and PXR in mice and humans. In the present study, male transgenic mice expressing human CAR and PXR were used to detect possible differences between wild-type (WT) and humanized mice in their response to CAR activation in a tumor initiation/promotion experiment with a single injection of the tumor initiator N-nitrosodiethylamine preceding chronic PB treatment for 10 months. Analysis of liver tumor burden revealed that PB strongly promoted the outgrowth of hepatocellular adenoma driven by activated β-catenin in WT mice, whereas the tumor-promoting effect of PB was much less pronounced in the humanized group. In conclusion, the present findings demonstrate that human CAR and PXR support tumor promotion by PB in mouse liver, but to a significantly lesser extent than the WT murine receptors.

The Constitutive Androstane Receptor (CAR): A Nuclear Receptor in Health and Disease

Constitutive Androstane Receptor (CAR, NR1I3), a member of the nuclear receptor superfamily of transcription factors, has emerged as one of the key regulators of the drug and xenobiotic metabolism. The unique feature that separates CAR from other members of the superfamily is that it remains active in the absence of ligand and is further regulated by activators. From its first isolation in 1994, a number of studies related to its distribution, characteristics, functions, and relation to other members of the superfamily have been conducted that place it centrally, governing many key events of the body. Human CAR is expressed relatively higher in liver and epithelial cells of the small intestine villi and less in heart, muscle, kidney, brain and lung. Though there are some controversies regarding its subcellular localization in different cell lines, in general, the subcellular localization of CAR is reported to be predominantly cytoplasmic, in complex with co-chaperone partners HSP90 and CCRP (cytoplasmic CAR retention protein). To execute transcription functions, nuclear translocation is a prerequisite event for a NR, including CAR. In this context, existence of two pathways is suggested, i) direct mechanism of action; and ii) indirect mechanism of action that is governed via nuclear translocation of CAR. Additionally, existence of species-specific differences in its modulation with ligands acting either as an agonist, antagonist or inverse agonist is also apparent. Like the other xenobiotic receptor PXR, CAR also functions as an alternative 'xenosensor' to defend the body against persistent chemical insults. It responds to diverse array of chemically distinct compounds, including endobiotics and xenobiotics, to regulate the clearance of noxious chemicals and toxic metabolites in liver and intestine via induction of genes involved in their metabolism. The usefulness of targeting CAR in metabolic diseases including bilirubinemia, obesity, type 2 diabetes mellitus, atherosclerosis, preeclampsia, hypertension, cholestasis and also in liver cancer is being extensively studied in animal models. However, to determine the human relevance it requires further investigation. Though a large number of natural and synthetic compounds act as modulators of CAR, designing new derivatives with defined therapeutic benefit need to be investigated. The purpose of this review is to highlight the general aspects of nuclear receptor CAR, its mechanism of action and importance in human health and disease.

The influence of standardized Valeriana officinalis extract on the CYP3A1 gene expression by nuclear receptors in in vivo model.

Valeriana officinalis is one of the most popular medicinal plants commonly used as a sedative and sleep aid. It is suggested that its pharmacologically active compounds derived from the root may modulate the CYP3A4 gene expression by activation of pregnane X receptor (PXR) or constitutive androstane receptor (CAR) and lead to pharmacokinetic herb-drug interactions. The aim of the study was to determine the influence of valerian on the expression level of CYP3A1 (homologue to human CYP3A4) as well as nuclear receptors PXR, CAR, RXR, GR, and HNF-4α. Male Wistar rats were given standardized valerian extract (300 mg/kg/day, p.o.) for 3 and 10 days. The expression in liver tissue was analyzed by using real-time PCR. Our result showed a decrease of CYP3A1 expression level by 35% (P = 0.248) and 37% (P < 0.001), respectively. Moreover, Valeriana exhibited statistically significant reduction in RXR (approximately 28%) only after 3-day treatment. We also demonstrated a decrease in the amount HNF-4α by 22% (P = 0.005) and 32% (P = 0.012), respectively. In case of CAR, the increase of expression level by 46% (P = 0.023) was noted. These findings suggest that Valeriana officinalis extract can decrease the CYP3A4 expression and therefore may lead to interactions with synthetic drugs metabolized by this enzyme.