Editor's Highlight: Neonatal Activation of the Xenobiotic-Sensors PXR and CAR Results in Acute and Persistent Down-regulation of PPAR?-Signaling in Mouse Liver.
ABSTRACT: Safety concerns have emerged regarding the potential long-lasting effects due to developmental exposure to xenobiotics. The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are critical xenobiotic-sensing nuclear receptors that are highly expressed in liver. The goal of this study was to test our hypothesis that neonatal exposure to PXR- or CAR-activators not only acutely but also persistently regulates the expression of drug-processing genes (DPGs). A single dose of the PXR-ligand PCN (75?mg/kg), CAR-ligand TCPOBOP (3?mg/kg), or vehicle (corn oil) was administered intraperitoneally to 3-day-old neonatal wild-type mice. Livers were collected 24?h post-dose or from adult mice at 60 days of age, and global gene expression of these mice was determined using Affymetrix Mouse Transcriptome Assay 1.0. In neonatal liver, PCN up-regulated 464 and down-regulated 449 genes, whereas TCPOBOP up-regulated 308 and down-regulated 112 genes. In adult liver, there were 15 persistently up-regulated and 22 persistently down-regulated genes following neonatal exposure to PCN, as well as 130 persistently up-regulated and 18 persistently down-regulated genes following neonatal exposure to TCPOBOP. Neonatal exposure to both PCN and TCPOBOP persistently down-regulated multiple Cyp4a members, which are prototypical-target genes of the lipid-sensor PPAR?, and this correlated with decreased PPAR?-binding to the Cyp4a gene loci. RT-qPCR, western blotting, and enzyme activity assays in livers of wild-type, PXR-null, and CAR-null mice confirmed that the persistent down-regulation of Cyp4a was PXR and CAR dependent. In conclusion, neonatal exposure to PXR- and CAR-activators both acutely and persistently regulates critical genes involved in xenobiotic and lipid metabolism in liver.
Project description:The pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known xenobiotic-sensing nuclear receptors with overlapping functions. However, there lacks a quantitative characterization to distinguish between the PXR and CAR target genes and signaling pathways in the liver. The present study performed a transcriptomic comparison of the PXR- and CAR-targets using RNA-Seq in livers of adult wild-type mice that were treated with the prototypical PXR ligand PCN (200mg/kg, i.p. once daily for 4days in corn oil) or the prototypical CAR ligand TCPOBOP (3mg/kg, i.p., once daily for 4days in corn oil). At the given doses, TCPOBOP differentially regulated many more genes (2125) than PCN (212), and 147 of the same genes were differentially regulated by both chemicals. As expected, the top pathways differentially regulated by both PCN and TCPOBOP were involved in xenobiotic metabolism, and they also up-regulated genes involved in retinoid metabolism, but down-regulated genes involved in inflammation and iron homeostasis. Regarding unique pathways, PXR activation appeared to overlap with the aryl hydrocarbon receptor signaling, whereas CAR activation appeared to overlap with the farnesoid X receptor signaling, acute-phase response, and mitochondrial dysfunction. The mRNAs of differentially regulated drug-processing genes (DPGs) partitioned into three patterns, namely TCPOBOP-induced, PCN-induced, as well as TCPOBOP-suppressed gene clusters. The cumulative mRNAs of the differentially regulated DPGs, phase-I and -II enzymes, as well as efflux transporters were all up-regulated by both PCN and TCPOBOPOP, whereas the cumulative mRNAs of the uptake transporters were down-regulated only by TCPOBOP. The absolute mRNA abundance in control and receptor-activated conditions was examined in each DPG category to predict the contribution of specific DPG genes in the PXR/CAR-mediated pharmacokinetic responses. The preferable differential regulation by TCPOBOP in the entire hepatic transcriptome correlated with a marked change in the expression of many DNA and histone epigenetic modifiers. In conclusion, the present study has revealed known and novel, as well as common and unique targets of PXR and CAR in mouse liver following pharmacological activation using their prototypical ligands. Results from this study will further support the role of these receptors in regulating the homeostasis of xenobiotic and intermediary metabolism in the liver, and aid in distinguishing between PXR and CAR signaling at various physiological and pathophysiological conditions. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.
Project description:Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR), constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor ? (PPAR?) play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPAR? activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital, or PPAR? activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16?-carbonitrile (PCN) alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPAR? is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.
Project description:The xenobiotic-sensing transcription factors (xeno-sensors) AhR, CAR, and PXR upregulate the expression of many drug-processing genes (DPGs) in liver. Previous studies have unveiled profound changes in the basal expression of DPGs during development; however, knowledge on the ontogeny of the inducibility of DPGs in response to pharmacological activation of xeno-sensors is still limited. The goal of this study was to investigate the age-specific regulation of DPGs by prototypical xeno-sensor ligands: 2,3,7,8-tetrachlorodibenzodioxin (TCDD) for AhR; 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) for CAR; and pregnane-16?-carbonitrile (PCN) for PXR during mouse liver development. The basal mRNAs of most DPGs were low during neonatal age, but gradually increased to adult levels, whereas some DPGs (Cyp1a2, Cyp2b10, Cyp3a11, Gstm2, Gstm3, Papss2, and Oatp1a4) exhibited an adolescent-predominant expression pattern. The inducibility of DPGs was age-specific: 1) during neonatal age, the highest fold increase in the mRNA expression was observed for Cyp1a2, Sult5a1, and Ugt1a9 by TCDD; Cyp3a11 and Mrp2 by TCPOBOP; as well as Gstm2 and Gstm3 by PCN; 2) during adolescent age, the highest fold increase in the mRNA expression was observed for Ugt1a6 and Mrp4 by TCDD, Cyp2b10, Ugt2b34, and Ugt2b35 by TCPOBOP, as well as Gsta1, Gsta4, Sult1e1, Ugt1a1, Mrp3, and Mrp4 by PCN; 3) in adults, the highest fold increase in the mRNA expression was observed for Aldh1a1, Aldh1a7, and Ugt2b36 by TCPOBOP, as well as Papss2 and Oatp1a4 by PCN. In conclusion, the inducibility of hepatic DPGs following the pharmacological activation of xeno-sensors is age specific.
Project description:Altered expression of long noncoding RNAs (lncRNAs) by environmental chemicals modulates the expression of xenobiotic biotransformation-related genes and may serve as therapeutic targets and novel biomarkers of exposure. The pregnane X receptor (PXR/NR1I2) is a critical xenobiotic-sensing nuclear receptor that regulates the expression of many drug-processing genes, and it has similar target-gene profiles and DNA-binding motifs with another xenobiotic-sensing nuclear receptor, namely, constitutive andronstrane receptor (CAR/Nr1i3). To test our hypothesis that lncRNAs are regulated by PXR in concert with protein-coding genes (PCGs) and to compare the PXR-targeted lncRNAs with CAR-targeted lncRNAs, RNA-Seq was performed from livers of adult male C57BL/6 mice treated with corn oil, the PXR agonist PCN, or the CAR agonist 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). Among 125,680 known lncRNAs, 3843 were expressed in liver, and 193 were differentially regulated by PXR (among which 40% were also regulated by CAR). Most PXR- or CAR-regulated lncRNAs were mapped to the introns and 3'-untranslated regions (UTRs) of PCGs, as well as intergenic regions. Combining the RNA-Seq data with a published PXR chromatin immunoprecipitation coupled with high-throughput sequencing; cytochrome P450 (P450; ChIP-Seq) data set, we identified 774 expressed lncRNAs with direct PXR-DNA binding sites, and 26.8% of differentially expressed lncRNAs had changes in PXR-DNA binding after PCN exposure. De novo motif analysis identified colocalization of PXR with liver receptor homolog (LRH-1), which regulates bile acid synthesis after PCN exposure. There was limited overlap of PXR binding with an epigenetic mark for transcriptional activation (histone-H3K4-di-methylation, H3K4me2) but no overlap with epigenetic marks for transcriptional silencing [H3 lysine 27 tri-methylation (H3K27me3) and DNA methylation]. Among differentially expressed lncRNAs, 264 were in proximity of PCGs, and the lncRNA-PCG pairs displayed a high coregulatory pattern by PXR and CAR activation. This study was among the first to demonstrate that lncRNAs are regulated by PXR and CAR activation and that they may be important regulators of PCGs involved in xenobiotic metabolism.
Project description:The gut microbiome regulates important host metabolic pathways including xenobiotic metabolism and intermediary metabolism, such as the conversion of primary bile acids (BAs) into secondary BAs. The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known regulators for xenobiotic biotransformation in liver. However, little is known regarding the potential effects of PXR and CAR on the composition and function of the gut microbiome. To test our hypothesis that activation of PXR and CAR regulates gut microbiota and secondary BA synthesis, 9-week-old male conventional and germ-free mice were orally gavaged with corn oil, PXR agonist PCN (75 mg/kg), or CAR agonist TCPOBOP (3 mg/kg) once daily for 4 days. PCN and TCPOBOP decreased two taxa in the Bifidobacterium genus, which corresponded with decreased gene abundance of the BA-deconjugating enzyme bile salt hydrolase. In liver and small intestinal content of germ-free mice, there was a TCPOBOP-mediated increase in total, primary, and conjugated BAs corresponding with increased Cyp7a1 mRNA. Bifidobacterium, Dorea, Peptociccaceae, Anaeroplasma, and Ruminococcus positively correlated with T-UDCA in LIC, but negatively correlated with T-CDCA in serum. In conclusion, PXR and CAR activation downregulates BA-metabolizing bacteria in the intestine and modulates BA homeostasis in a gut microbiota-dependent manner.
Project description:TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene) and PCN (pregnenolone 16α-carbonitrile) are inducers of drug metabolism through activation of nuclear receptors CAR (constitutive androstane receptor) and PXR (pregnane X receptor), respectively. Mouse experiment was designed to study the effect of CAR and PXR activation on cholesterol homeostasis genes and other genes, which are present on the Steroltalk v2 microarray. Treatments were combined with standard and high-cholesterol diet to observe the interference of high liver cholesterol on nuclear receptor transcription regulation. All experiments were done within the European sixth Framework program “Steroltalk” (www.steroltalk.net). Results form these experiments give new knowledge about involvement of ‘xenosensors’ CAR and PXR in regulation of endogenous liver metabolism. Animals were injected i.p. 3mg/kg TCPOBOP, 40 mg/kg PCN or vehicle (corn oil) in combination of one week of standard or 1% cholesterol diet prior tretament. After 24h they were sacrificed and total RNA was isolated from the livers. Each sample was hybridized with a reference sample to Steroltalk v2 microarrays.
Project description:UDP-glucuronosyltransferases (UGTs) catalyze the addition of UDP-glucuronic acid to endo- and xenobiotics, enhancing their water solubility and elimination. Many exogenous compounds, such as microsomal enzyme inducers (MEIs), alter gene expression through xenobiotic-responsive transcription factors, namely, the aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear factor erythroid 2-related factor 2 (Nrf2). These transcription factors regulate xenobiotic-inducible expression of hepatic and intestinal biotransformation enzymes and transporters. The purpose of this study was to determine hepatic and intestinal inducibility of mouse Ugt mRNA by MEIs. Male C57BL/6 mice were treated for four consecutive days with activators of AhR [2,3,7,8-tetrachlorodibenzodioxin (TCDD), polychlorinated biphenyl 126, and beta-naphthoflavone], CAR [1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), phenobarbital, and diallyl sulfide], PXR [pregnenolone-16alpha-carbonitrile (PCN), spironolactone, and dexamethasone], PPARalpha (clofibrate, ciprofibrate, and diethylhexylphthalate), and Nrf2 (oltipraz, ethoxyquin, and butylated hydroxyanisole), respectively. Ugt1a1 mRNA expression in liver was induced by activators of all five transcription factor pathways, Ugt1a5 by Nrf2 activators, Ugt1a6 by all the pathways except CAR, and Ugt1a9 by all the pathways except Nrf2. Ugt2b35 mRNA in liver was induced by AhR activators and Ugt2b36 by CAR and PPARalpha activators. Throughout the small and large intestine, the AhR ligand TCDD increased Ugt1a6 and Ugt1a7 mRNA. In small intestine, the PXR activator PCN increased Ugt1a1, Ugt1a6, Ugt1a7, Ugt2b34, and Ugt2b35 mRNA in the duodenum. In conclusion, chemical activation of AhR, CAR, PXR, PPARalpha, and Nrf2 in mouse results in induction of distinct Ugt gene sets in liver and intestine, predominantly the Ugt1a isoforms.
Project description:Aberrant epigenetic alterations during development may result in long-term epigenetic memory and have a permanent effect on the health of subjects. Constitutive androstane receptor (CAR) is a central regulator of drug/xenobiotic metabolism. Here, we report that transient neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism. CAR activation by neonatal exposure to the CAR-specific ligand 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) led to persistently induced expression of the CAR target genes Cyp2B10 and Cyp2C37 throughout the life of exposed mice. These mice showed a permanent reduction in sensitivity to zoxazolamine treatment as adults. Compared with control groups, the induction of Cyp2B10 and Cyp2C37 in hepatocytes isolated from these mice was more sensitive to low concentrations of the CAR agonist TCPOBOP. Accordingly, neonatal activation of CAR led to a permanent increase of histone 3 lysine 4 mono-, di-, and trimethylation and decrease of H3K9 trimethylation within the Cyp2B10 locus. Transcriptional coactivator activating signal cointegrator-2 and histone demethylase JMJD2d participated in this CAR-dependent epigenetic switch.Neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism.
Project description:This study aimed to quantify and compare the mRNA abundance of major xenobiotic processing genes in liver following activation of PXR and CAR using RNA-Seq Overall design: mRNA profiles of liver in adult male C57BL/6 mice treated with corn oil, PCN (mouse PXR-ligand), or TCPOBOP (mouse CAR ligand) were determined by RNA-Seq, n=3 per group, using an Illumina HiSeq 2000 sequencer.
Project description:Changes in gene expression were assayed in mouse liver nuclear RNA following a single injection of the CAR agonist TCPOBOP (1,4-Bis-[2-(3,5-dichloropyridyloxy)]benzene) or the PXR agonist PCN (pregnenolone 16α-carbonitrile) in 7-week old mice. This study is part of a larger study entitled Sex-Differential Responses of Tumor Promotion-Associated Genes and Dysregulation of Novel Long Noncoding RNAs in Constitutive Androstane Receptor-Activated Mouse Liver (PMID: 28903501). Overall design: Nuclear RNA was isolated from male and female CD1 mouse liver following either 3 hour exposure or 27 hour exposure to TCPOBOP; or from male liver following a 3 hour exposure to PCN. N=2-3 biological pools were sequenced per control or treatment group, with each pool comprised of nuclear RNA pooled from n=3-4 individual mouse livers.