Language：English   Publishing type：Research paper (scientific journal)  

Pharmacotherapy shows striking individual differences in pharmacokinetics and pharmacodynamics, involving drug efficacy and adverse reactions. Recent genetic research has revealed that genetic polymorphisms are important intrinsic factors for these inter-individual differences. This pharmacogenomic information could help develop safer and more effective precision pharmacotherapies and thus, regulatory guidance/guidelines were developed in this area, especially in the EU and US. The Project for the Promotion of Progressive Medicine, Medical Devices, and Regenerative Medicine by the Ministry of Health, Labour and Welfare, performed by Tohoku University, reported scientific information on the evaluation of genetic polymorphisms, mainly on drug metabolizing enzymes and transporters, during non-clinical studies and phase I clinical trials in Japanese subjects/patients. We anticipate that this paper will be helpful in drug development for the regulatory usage of pharmacogenomic information, most notably pharmacokinetics.

DOI： 10.1016/j.dmpk.2018.01.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER HEIDELBERG  

Perfluorocarboxylic acids (PFCAs) including perfluorooctanoic acid (PFOA) are environmental pollutants showing high accumulation, thermochemical stability and hepatocarcinogenicity. Peroxisome proliferator-activated receptor alpha 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.

DOI： 10.1007/s00204-016-1888-3

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

The pregnane X receptor (PXR) is well-known as a key regulator of drug/xenobiotic clearance. Upon activation by ligand, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters. Recent studies have revealed that PXR also plays a role in regulating immune/inflammatory responses. Specific PXR activators, including synthetic ligands and phytochemicals, have been shown to ameliorate chemically induced colitis in mice. In this study, we investigated an antiinflammatory effect of pregnenolone 16 alpha-carbonitrile (PCN), a prototypical activator for rodent PXR, in concanavalin A (Con A)-induced liver injury, a model of immune-mediated liver injury, using wild-type and Pxr (/) mice. Unexpectedly, pretreatment with PCN significantly ameliorated Con A-induced liver injury in not only wild-type but Pxr (/) mice as well, accompanied with lowered plasma ALT levels and histological improvements. Pretreatment with PCN was found to significantly repress the induction of Cxcl2 and Ccl2 mRNA expression and neutrophil infiltration into the liver of both wild-type and Pxr (/) mice at the early time point of Con A- induced liver injury. Our results indicate that PCN has unexpected immunosuppressive activity independent of PXR activation to protect mice from immune- mediated liver injury induced by Con A. (C) 2017 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.toxlet.2017.02.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnaneXreceptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16 alpha-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression.

DOI： 10.1042/BJ20150734

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

Upon treatment with the pregnane X receptor (PXR) activator rifampicin (RIF), human hepatocellular carcinoma HepG2-derived ShP51 cells that stably express PXR showed epithelialmesenchymal transition (EMT)-like morphological changes and migration. Our recent DNA microarrays have identified hepatocyte nuclear factor (HNF) 4 alpha and insulin-like growth factorbinding protein (IGFBP) 1 mRNAs to be downregulated and upregulated, respectively, in RIF-treated ShP51 cells, and these regulations were confirmed by the subsequent real-time polymerase chain reaction and Western blot analyses. Using this cell system, we demonstrated here that the PXR-HNF4 alpha-IGFBP1 pathway is an essential signal for PXR-induced morphological changes and migration. First, we characterized the molecular mechanism underlying the PXR-mediated repression of the HNF4 alpha gene. Chromatin conformation capture and chromatin immunoprecipitation (ChIP) assays revealed that PXR activation by RIF disrupted enhancer-promoter communication and prompted deacetylation of histone H3 in the HNF4 alpha P1 promoter. Cell-based reporter and ChIP assays showed that PXR targeted the distal enhancer of the HNF4 alpha P1 promoter and stimulated dissociation of HNF3 beta from the distal enhancer. Subsequently, small interfering RNA knockdown of HNF4 alpha connected PXR-mediated gene regulation with the PXRinduced cellular responses, showing that the knockdown resulted in the upregulation of IGFBP1 and EMT-like morphological changes without RIF treatment. Moreover, recombinant IGFBP1 augmented migration, whereas an anti-IGFBP1 antibody attenuated both PXR-induced morphological changes and migration in ShP51 cells. PXR indirectly activated the IGFBP1 gene by repressing the HNF4 alpha gene, thus enabling upregulation of IGFBP1 to change the morphology of ShP51 cells and cause migration. These results provide new insights into PXR-mediated cellular responses toward xenobiotics including therapeutics.

DOI： 10.1124/mol.115.099341

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

Severe adverse drug reactions are an important issue to be considered during proper drug usage in postmarketing period. Most severe adverse reactions are idiosyncratic and unrelated to their pharmacological actions via primary targets. Although these reactions were not predictable, recent developments in the field of genomics have revealed closely associated markers responsible for some severe adverse reactions, including Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). This review demonstrates genomic biomarkers for SJS/TEN and drug-induced liver injury (DILI) that were found mainly in Japanese patients and reveal ethnic differences. We and other groups have found the following associations of SJS/TEN with susceptible drugs: 1) HLA-B*58:01 for allopurinol-related cases; 2) HLA-B*15:11 and HLA-A*31:01 for carbamazepine-related cases; 3) HLA-B*51:01 for phenobarbital-related cases; 4) HLA-A*02:07 for zonisamide-related cases; 5) CYP2C9*3 for phenytoin-related cases; and 6) HLA-A*02:06 for cold medicine-related cases. The allele frequencies of these related HLA types vary among Asian populations. In addition, direct (non-covalent) binding of carbamazepine or an allopurinol metabolite, oxypurinol, to the associated HLA-type proteins was suggested. Associated genomic biomarkers are also summarized for DILI in Japanese and Caucasian populations. The application of these genomic biomarkers to prevent the onset of a reaction has been utilized in a few countries. However, in Japan, the package inserts only contain precautions that cite the research findings. To overcome this limitation, the following points should be addressed: 1) factors responsible for the development of SJS/TEN should be identified in addition to the above-mentioned HLA alleles; and 2) an inexpensive genotyping strategy and assay methods should be developed to provide a pharmacoeconomical viewpoint. Further research on severe adverse reactions is warranted.

DOI： 10.1248/yakushi.14-00249-3

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Language：Japanese   Publisher：(公社)日本薬学会  

国立医薬品食品衛生研究所では、収集した重症薬疹を対象に、被疑薬別にゲノムバイオマーカー探索を行っている。全ゲノムにわたる網羅的な遺伝子多型解析も行っているが、これまでに見出されたマーカーとしては、ヒト白血球抗原型が多い。特に、HLAクラスI蛋白質は、皮膚を含む全身の細胞に発現しており、表皮細胞壊死との関連が注目されている。重症薬疹の発症報告数と被疑薬・バイオマーカー研究・分子論的機構、薬剤性肝障害発症と関連するヒト白血球抗原型について述べた。

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Other Link： https://search-tp.jamas.or.jp/index.php?module=Default&action=Link&pub_year=2015&ichushi_jid=J01459&link_issn=&doc_id=20150407300006&doc_link_id=130005060821&url=https%3A%2F%2Fci.nii.ac.jp%2Fnaid%2F130005060821&type=CiNii&icon=https%3A%2F%2Fjk04.jamas.or.jp%2Ficon%2F00003_3.gif

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR), members of the nuclear receptor superfamily, are two major xeno-sensing transcription factors. They can be activated by a broad range of lipophilic xenobiotics including therapeutics drugs. In addition to xenobiotics, endogenous compounds such as steroid hormones and bile acids can also activate PXR and/or CAR. These nuclear receptors regulate genes that encode enzymes and transporters that metabolize and excrete both xenobiotics and endobiotics. Sulfotransferases (SULTs) are a group of these enzymes and sulfate xenobiotics for detoxification. In general, inactivation by sulfation constitutes the mechanism to maintain homeostasis of endobiotics. Thus, deciphering the molecular mechanism by which PXR and CAR regulate SULT genes is critical for understanding the roles of SULTs in the alterations of physiological and pathophysiological processes caused by drug treatment or environmental exposures.

DOI： 10.3109/03602532.2013.835630

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：INFORMA HEALTHCARE  

Pregnane X receptor (PXR), an orphan member of the nuclear receptor superfamily, is a major xeno-sensing transcription factor. In response to xenobiotic exposure, PXR regulates genes involved in the metabolism and transport of xenobiotics to protect the body from their harmful effects. Recent progress has revealed that PXR responds not only to such external signals but also to internal signals to help the body adapt to changes in the internal environment, including dysregulation of the immune system. PXR responds to external and internal signals by up-or down-regulating certain metabolic pathways and cellular signals through gene regulation. PXR is a potential therapeutic target for inflammatory as well as metabolic diseases, although its activation may also have unfavorable effects on human health. This review will discuss the recent progress in the understanding of the physiological and pathophysiological roles of PXR and their implications in human diseases and drug therapy by elucidating the molecular mechanisms underlying PXR-mediated gene regulation.

DOI： 10.3109/03602532.2013.795585

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR), constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor alpha (PPAR alpha) play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPAR alpha 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 alpha 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 alpha-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 alpha 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.

DOI： 10.1371/journal.pone.0061802

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Language：Japanese   Publisher：(一社)レギュラトリーサイエンス学会  

発生頻度や患者背景の多様性の点から、市販後に明らかになる副作用は比較的多く、特に重篤な副作用は医薬品の適正使用の観点から問題となっている。バイオマーカーは副作用の予測および早期診断に有用と期待され、ゲノムを中心に解析が進展している。例えば、カルバマゼピン、アロプリノール等による重症薬疹に関しては、その発症と強く関連するヒト白血球抗原(HLA)型が、日本人を含む多くの人種で見いだされている。また薬物性肝障害に関しても、予測ゲノムバイオマーカーが明らかになってきている。一方で、環境的要因を反映しうるタンパク質および体内代謝物マーカーについても期待が寄せられており、少数であるが報告例もある。本項では、著者らが行っている研究を含め、これらの最新の知見を、民族差を含めて紹介する。バイオマーカー研究がさらに進み、医薬品のより安全な使用に応用されることを期待したい。一方で、これらを実臨床に応用するためには、前向き研究により診断の有用性を示すことが極めて重要であり、その進展が期待される。(著者抄録)

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Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated beta-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

DOI： 10.1080/03601234.2012.611018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Certain congeners of polychlorinated biphenyls (PCBs) and organochlorine insecticides are ligands of aryl hydrocarbon receptors (AhRs) in animals. A recombinant guinea pig (g) AhR, XgDV, was constructed by fusing the ligand-binding domain of gAhR, the DNA-binding domain of LexA, and the transactivating domain of VP16. Then, the expression unit of beta-glucuronidase (GUS) reporter gene regulated by XgDV was introduced into Arabidopsis and tobacco plants. When the transgenic Arabidopsis XgDV plants were cultured on Murashige-Skoog (MS) medium containing PCB congeners, the GUS activity in the plants increased toxic equivalent (TEQ)-dependently. The GUS activity in the transgenic Arabidopsis XgDV plants cultured on MS medium containing the organochlorine insecticide dieldrin was also induced. On the other hand, in the case of DDT, the GUS activity induced by 3-methylcholanthere in the plants decreased. The transgenic Arabidopsis XgDV plants detected 1000 ng g(-1) PCB126 in 1 g of soils. Thus the XgDV plants seemed to be useful for convenient assays of PCB congeners and organochlorine insecticides, without any extraction and purification steps.

DOI： 10.1080/03601234.2012.668453

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Pregnane X receptor (PXR), acting as a xenobiotic-activated transcription factor, regulates the hepatic metabolism of therapeutics as well as endobiotics such as steroid hormones. Given our finding that PXR activation by rifampicin (RIF) represses the estrogen sulfotransferase (SULT1E1) gene in human primary hepatocytes and hepatocellular carcinoma Huh7 cells, here we have investigated the molecular mechanism of this repression. First the PXR-responsive enhancer was delineated to a 100 bp sequence (-1000/-901), which contains three half sites that constitute the overlapping direct repeat 1 (DR1) and direct repeat 2 (DR2) motifs and two forkhead factor binding sites. siRNA knockdown, chromatin immunoprecipitation and chromatin conformation capture assays were employed to demonstrate that hepatocyte nuclear factor 4 alpha (HNF4 alpha) bound to the PXR-responsive enhancer, and activated the enhancer by looping its position close to the proximal promoter. Upon activation by RIF, PXR indirectly interacted with the enhancer, decreasing the interaction with HNF4 alpha and dissolving the looped SULT1E1 promoter with deacetylation of histone 3. Removal of the DR sites from the enhancer hampers the ability of HNF4 alpha to loop the promoter and that of PXR to repress the promoter activity. Thus, PXR represses human SULT1E1, possibly attenuating the inactivation of estrogen.

DOI： 10.1093/nar/gkr458

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Pregnane X receptor (PXR) was originally characterized as a transcription factor that induces hepatic drug metabolism by activating cytochrome P450 genes. Here we have now demonstrated a novel function of PXR, that of eliciting p38 mitogen-activated protein kinase (MAPK) phosphorylation for cell migration. Upon xenobiotic activation of ectopic human PXR, human hepatocellular carcinoma HepG2 cells were found to exhibit increased phosphorylation of p38 MAPK and to subsequently change morphology and migrate. p38 MAPK was responsible for the regulation of these morphological changes and cell migration because the p38 MAPK inhibitor SB239063 repressed both. Prior to this phosphorylation, PXR directly activated the early response GADD45 beta gene by binding to a distal direct repeat 4 site of the GADD45 beta promoter. Ectopic expression of GADD45 beta increased p38 MAPK phosphorylation, whereas siRNA knockdown of GADD45 beta decreased the PXR-induced p38 MAPK phosphorylation, confirming that GADD45 beta can regulate PXR-induced p38 MAPK phosphorylation in HepG2 cells. These results indicate that PXR activates the GADD45 beta gene, increasing p38 MAPK phosphorylation, and leading HepG2 cells to change morphology and migrate. The GADD45 beta gene is a direct target for PXR, eliciting cell signals to regulate various cellular functions.

DOI： 10.1074/jbc.M110.179812

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS  

We have studied the molecular mechanism by which the nuclear xenobiotic receptors pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR) regulate transcription of the vitamin D-3 24-hydroxylase (CYP24A1) gene. In the absence of vitamin D-3, PXR activates the CYP24A1 gene by directly binding to and transactivating vitamin D-response elements (VDREs) within its promoter. Vitamin D-3 activates the CYP24A1 promoter by dissociating the corepressor silencing mediator for retinoid and thyroid hormone receptors ( SMRT) from the vitamin D receptor (VDR) on those VDREs. PXR strongly represses vitamin D-3 activation of the CYP24A1 gene, in which PXR indirectly binds to and prevents vitamin D-3-dependent dissociation of SMRT from the CYP24A1 promoter. The degree of the PXR-mediated locking of SMRT depends on the relative concentration of vitamin D 3 to the human PXR activator rifampicin; SMRT increased its dissociation as this ratio increased. CAR is also found to prevent dissociation of SMRT from the CYP24A1 promoter. Thus, our present study defines the novel molecular mechanism by which PXR and CAR mediate drug interactions with vitamin D 3 to regulate the CYP24A1 gene. Pxr(+/+) and Pxr(-/-) mice were continuously treated with mouse PXR activator PCN to evaluate the hypothesis that induction of the Cyp24a1 gene is responsible for the loss of bone mineral density often observed in patients treated continuously with PXR-activating drugs. PCN-dependent loss of mineral density is observed in the metaphyseal bones of only the Pxr(+/+) mice. This loss, however, does not correlate with the expression levels of the Cyp24a1 gene in these mice.

DOI： 10.1124/mol.108.051904

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

Dioxin residues widely contaminate soil and agricultural products at low concentrations and may accumulate in organisms at the top of food chains owing to their physicochemical properties. In this study, we have developed novel, dioxin-inducible, reporter gene expression systems regulated by recombinant aryl hydrocarbon receptors (AhRs). The recombinant AhRs, referred to as XDVs, consist of the DNA-binding domain of the bacterial repressor protein LexA, a 90-kDa heat shock protein- and ligand-binding regulatory domain from mouse AhR, and the transactivation domain of herpes simplex virus regulatory protein VP16. Transgenic tobacco plants carrying XDVs absorb various AhR ligands, including 3-methylcholanthrene, beta-naphthoflavone and indigo from solid medium and vermiculite, and show dose- and time-dependent expression of the beta-glucuronidase reporter gene. The results clearly suggest that XDVs are functional transcription factors that respond to AhR ligands, and that the XDV-mediated reporter gene expression system is applicable to bioassays for dioxin residues in the environment.

DOI： 10.1111/j.1467-7652.2008.00378.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAP SOC STUDY XENOBIOTICS  

Nuclear receptors constitutive active/androstane receptor (CAR) and pregnane X receptor (PXR) were originally characterized as transcription factors regulating the hepatic genes that encode drug metabolizing enzymes. Recent works have now revealed that these nuclear receptors also play the critical roles in modulating hepatic energy metabolism. While CAR and PXR directly bind to their response sequences phenobarbital-responsive enhancer module (PBREM) and xenobiotic responsive enhancer module (XREM) in the promoter of target genes to increase drug metabolism, the receptors also cross talk with various hormone responsive transcription factors such as forkhead box O1 (FoxO1), forkhead box A2 (FoxA2), cAMP-response element binding protein, and peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC 1 alpha) to decrease energy metabolism through down-regulating gluconeogenesis, fatty acid oxidation and ketogenesis and up-regulating lipogenesis. In addition, CAR modulates thyroid hormone activity by regulating type 1 deiodinase in the regenerating liver. Thus, CAR and PXR are now placed at the crossroad where both xenobiotics and endogenous stimuli co-regulate liver function.

DOI： 10.2133/dmpk.23.8

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

In mammals, the aryl hydrocarbon receptor (AhR) mediates expression of certain genes, including CYP1A1, in response to exposure to dioxins and related compounds. We have constructed a mouse AhR-mediated gene expression systems for a beta-glucuronidase (GUS) reporter gene consisting of an AhR, an AhR nuclear translocator (Arnt), and a xenobiotic response element (XRE)-driven promoter in transgenic tobacco plants. On treatment with the AhR ligands 3-methyl-cholanthrene (MC), beta-naphthoflavone (beta NF), and indigo, the transgenic tobacco plants exhibited enhanced GUS activity, presumably by inducible expression of the reporter gene. The recombinant AhR (AhRV), with the activation domain replaced by that of the Herpes simplex virus protein VP16, induced GUS activity much more than the wild-type AhR in the transgenic tobacco plants. Plants carrying AhRV expressed the GUS reporter gene in a dose- and time-dependent manner when treated with MC; GUS activity was detected at 5 nM MC on solid medium and at 12 h after soaking in 25 mu M MC. Histochemical GUS staining showed that this system was active mainly in leaf and stem. These results suggest that the AhR-mediated reporter gene expression system has potential for the bioassay of dioxins in the environment and as a novel gene expression system in plants.

DOI： 10.1007/s00425-007-0592-1

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

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 by 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 16 alpha-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 wildtype, 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.

DOI： 10.1042/BJ20070481

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

In the early 1960s, phenobarbital (PB) was shown to induce hepatic drug metabolism and the induction was implicated in the molecular mechanism of drug tolerance development. Since then, it has become evident that PB not only induces drug metabolism, but also triggers pleiotropic effects on liver function., such as cell growth and communication, proliferation of the endoplasmic reticulum, tumor promotion, glucose metabolism, steroid/thyroid hormone metabolism, and bile acid synthesis. Upon activation by PB and numerous PB-type inducers, the nuclear receptor CAR mediates those pleiotropic actions by regulating various hepatic genes, utilizing multiple regulatory mechanisms.

DOI： 10.1080/03602530600569851

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Transgenic plants carrying mammalian hormone receptor genes were examined for monitoring of environmental chemicals including dioxins and endocrine disruptors. The transgenic tobacco plants expressing mouse arylhydrocarbon receptor gene detected 1ppb of the dioxin-like compound 20-methylcholanthrene in the cultured plants. In addition, the transgenic Arabidopsis plants expressing human estrogen receptor gene detected 1ppt of 17 beta-estradiol and ppb levels of the other endocrine disruptors such as bisphenol A, 4-t-octylphenol, and others in potted plants. These transgenic plants appear to be useful for on site monitoring of dioxins and estrogenic contaminants in the environment. The transgenic plants expressing AhR and ER detected 1ppb of MC under cultured conditions and 1ppt of E-2 under potted conditions, respectively. Basic technology was established for monitoring dioxins and EDs in the transgenic plants with the receptor genes. However, increase of the sensitivity towards other EDs with low affinity towards receptors is necessary. In order to overcome this problem, (1)sensitive receptors may be useful instead of mouse AhR and human ER. It is known that fish ERs were more sensitive to alkylphenols such as nonylphenol and octylphenol than mammalian ones. (2)Increase of uptake efficiency of dioxins and EDs is another way to increase sensitivity. Zucchini plants are known to have efficient translocation of polychlorinated dibenzo-p-dioxins and dibenzofurans(7). Furthermore, (3)genes synthesizing flower colors as reporter genes may be useful for visible monitoring of contaminations on site.

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The nuclear receptors CAR and PXR activate hepatic genes in response to therapeutic drugs and xenobiotics, leading to the induction of drug-metabolizing enzymes, such as cytochrome P450. Insulin inhibits the ability of FOXO1 to express genes encoding gluconeogenic enzymes. Induction by drugs is known to be decreased by insulin, whereas gluconeogenic activity is often repressed by treatment with certain drugs, such as phenobarbital (PB). Performing cell-based transfection assays with drug-responsive and insulin-responsive enhancers, glutathione S-transferase pull down, RNA interference (RNAi), and mouse primary hepatocytes, we examined the molecular mechanism by which nuclear receptors and FOXO1 could coordinately regulate both enzyme pathways. FOXO1 was found to be a coactivator to CAR- and PXR-mediated transcription. In contrast, CAR and PXR, acting as corepressors, downregulated FOXO1-mediated transcription in the presence of their activators, such as 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and pregnenolone 16alpha-carbonitrile, respectively. A constitutively active mutant of the insulin-responsive protein kinase Akt, but not the kinase-negative mutant, effectively blocked FOXO1 activity in cell-based assays. Thus, insulin could repress the receptors by activating the Akt-FOXO1 signal, whereas drugs could interfere with FOXO1-mediated transcription by activating CAR and/or PXR. Treatment with TCPOBOP or PB decreased the levels of phosphoenolpyruvate carboxykinase 1 mRNA in mice but not in Car(-/-) mice. We conclude that FOXO1 and the nuclear receptors reciprocally coregulate their target genes, modulating both drug metabolism and gluconeogenesis.

DOI： 10.1128/MCB.24.18.7931.7940.2004

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1, The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv, Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did, The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.

DOI： 10.1271/bbb.64.2025

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Language：Japanese  

DOI： 10.14982/rsmp.9.95

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：JAPANESE SOC STUDY XENOBIOTICS  

DOI： 10.1016/j.dmpk.2016.10.119

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Language：Japanese   Publisher：(一社)日本毒性学会  

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Language：Japanese   Publisher：(一社)日本毒性学会  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：Japanese   Publisher：サイエンティスト社  

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Language：Japanese   Publisher：(公社)日本薬学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：INFORMA HEALTHCARE  

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

核内受容体pregnane X receptor (PXR)は，肝臓や腸管に高いレベルで発現し，それら臓器の主要な生理機能の調節を担う低分子応答性の転写因子である。近年，PXRの機能喪失や異常と免疫系の活性亢進及び炎症性疾患との関連，またPXRの活性化による炎症軽減作用が報告され，PXRの新規生理機能として免疫系調節への関与が注目されている。しかし，その詳細な作用機序は依然として不明である。そこでコンカナバリンA(Con A)誘発性マウス肝障害モデルを用いて，PXRによる免疫系調節の作用機序の解明を目的に本研究を実施した。Con Aの単回静脈投与は，肝特異的に障害を誘発する。野生型マウス及びPXR欠損(PXR-KO)マウスにCon Aを同量投与した結果，野生型と比較して，PXR-KOマウスでは肝細胞壊死の増悪化や肝組織内への好中球の浸潤亢進が認められ，これらは血中ALT値の著しい上昇と正に相関していた。次いで，野生型マウスにげっ歯類特異的PXRリガンドPCNを前投与したところ，Con A投与9時間後において有意な肝細胞壊死の軽減，血中ALT値の改善，及び炎症関連遺伝子群のmRNAレベルの低下が認められた。更にCon A投与後，肝細胞壊死や血中ALT値の上昇が認められない早期の段階（3時間後）においては，ケモカインCCL2 やCXCL2のmRNAレベルの有意な上昇や肝組織内への好中球浸潤が認められた一方で，PCNの前投与によりこれらが著しく抑制されることを新たに見出した。以上本研究により，PXRの遺伝子欠損により，マウスのCon A誘発性肝障害に対する感受性が高まること，更にPXR活性化薬の投与は，Con A投与により惹起される一連の免疫反応の過程において，CCL2，CXCL2の発現，肝組織内への好中球の浸潤を抑制し，肝障害を軽減する可能性があることを新たに見出した。

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

核内受容体pregnene X receptor (PXR)およびconstitutive androstane receptor (CAR)は，肝に高発現し，生体異物に応答して異物除去など様々な生理機能を制御する転写因子である。CARの活性化はまた，肝細胞増殖や肝発がんと関連しており，このことが化学物質の安全性評価においてしばしば問題となる。最近我々は，PXRの活性化は単独では肝細胞増殖作用を示さないものの，CAR活性化薬や核内受容体PPARα活性化薬による肝細胞増殖を増強することを見出した（第39回日本毒性学会学術年会）。本研究では，この分子機序を明らかにするために，マウス肝細胞の細胞周期調節におけるPXRの機能を解析した。C57BL/6系雄性マウスにPXR活性化薬物であるpregnenolone 16α-carbonitrile (PCN; 100 mg/kg）を腹腔内投与し，その肝臓を試料として用いた。投与48時間後の肝細胞をコラゲナーゼ灌流法により分離し，propidium iodideによるDNA染色またはPyronin Yおよび7-aminoactinomycin DによるDNA/RNA二重染色を利用したフローサイトサイトメトリー法により細胞周期を解析した結果，PCN投与に伴う肝細胞のG0期からG1期への移行が確認された。また，G0/G1トランジションに関連する遺伝子のmRNAレベルを定量的逆転写PCR法により測定したところ，PCN処置24時間後において，Rbl2 (p130)とその転写調節因子をコードするFoxo1，Foxo3およびFoxo4遺伝子の発現低下が認められた。一方，PXR欠損マウスではそのような変化は認められなかった。さらにレポーターアッセイにおいてPXRの活性化はFOXO3依存的なRbl2の転写を抑制した。またゲルシフトアッセイにおいてPXRはRbl2プロモーター上のFOXO結合領域へのFOXO3の結合を抑制した。以上の結果から，PXRはマウス肝細胞において，FOXO3との相互作用によりRbl2の発現を抑制し，細胞周期をG0期からG1期に移行させることで様々な細胞増殖刺激に対する感受性を増加させる機能を有することが示唆された。

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

【目的】Constitutive androstane receptor (CAR)およびpregnene X receptor (PXR)は、肝に高発現し生体異物により活性化される核内受容体である。両受容体は薬物代謝酵素など異物除去と関連する種々の遺伝子の転写制御に中心的に働いている。また、両受容体は類似したプロモーター配列に結合するため、多くの標的遺伝子を共有し、上記の遺伝子発現において協調的に機能していると考えられている。CARは、薬物代謝酵素の誘導に加えて、齧歯動物における肝細胞増殖にも働くことが知られており、CAR活性化作用が化学物質のヒトでの安全性評価において問題となっている。一方、PXRと肝細胞増殖に関する報告はほとんどなく、肝細胞増殖作用における両核内受容体の協調性は明確ではない。本研究では、CARとPXRが、薬物代謝酵素の遺伝子発現と同様に、肝細胞増殖作用においても協調的に機能しているか否かを検討した。【方法】8週齢の雄性C57BL/6系マウスにCAR活性化薬物のTCPOBOP (3 mg/kg)とPXR活性化薬物のPCN (100 mg/kg）を単独または同時に腹腔内投与し、4、24、48時間後の肝臓を試料とした。細胞増殖は抗Ki-67抗体および抗PCNA抗体を用いた免疫染色により評価した。細胞周期関連遺伝子のmRNA発現レベルは定量的逆転写PCR法により測定した。【結果・考察】TCPOBOP処置24および48時間後においてKi-67またはPCNA陽性細胞数の増加、細胞周期関連遺伝子発現レベルの増加が認められ、CARの肝細胞増殖作用が確認された。一方、PCN処置ではいずれの時間においてもそのような変化は認められなかった。しかし、PCNをTCPOBOPと併用処置した場合には、24および48時間後における細胞増殖陽性細胞数や細胞周期関連遺伝子の発現レベルは、TCPOBOP単独処置時に比べて増加した。以上の結果から、マウス肝において、PXRの活性化は、単独では細胞増殖を引き起こさないがCARによる細胞増殖作用を増強させる可能性が示された。

DOI： 10.14869/toxpt.39.1.0.P-180.0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：INFORMA HEALTHCARE  

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Language：Japanese   Publisher：The Japanese Society of Toxicology  

【背景・目的】Constitutive androstane receptor (CAR)とpregnane X receptor (PXR)は、同じ核内受容体遺伝子ファミリーに属し、類似した機能を有している。両者は共に肝に高発現し、薬物などの生体外異物により活性化される。CARとPXRが結合する塩基配列は類似しており、異物代謝関連遺伝子など多くの標的遺伝子は両者により活性化される。CARまたはPXRを活性化する薬物はいずれも齧歯動物において肝肥大を起こすことも知られている。しかし、詳細な分子機構は不明であるが、CARの活性化は、齧歯動物において肝細胞増殖作用を示すのに対し、PXRの活性化は肝細胞増殖および肝発癌プロモーション作用を示さないと考えられている。このため、CAR選択的な発現制御を受ける遺伝子が肝細胞増殖作用に関わるのではないかと考えた。そこで本研究では、CARまたはPXRの活性化に伴う、細胞周期関連遺伝子の発現プロファイルの変化を比較解析した。【方法】8週齢の雄性C57BL/6NマウスにマウスCAR活性化物質のTCPOBOP (3 mg/kg)、マウスPXR活性化物質のPCN (100 mg/kg)または溶媒を経時的に投与し、肝から調製したRNAを用いて、PCRアレイ法 (細胞周期関連の84遺伝子)および定量的逆転写PCR法により細胞周期関連遺伝子の発現変動を解析した。【結果・考察】TCPOBOPとPCN投与に伴う細胞周期関連遺伝子の発現変動プロファイルは大きく異なることが示され、CAR選択的に発現が制御されている可能性がある遺伝子を7遺伝子同定した。その中にはNek2やCdc20などのG2期からM期への移行に関与する遺伝子が含まれており、これらの遺伝子の発現変動がCARを介した肝細胞増殖に関与している可能性が考えられた。

DOI： 10.14869/toxpt.39.1.0.P-181.0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：TAYLOR & FRANCIS INC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：TAYLOR & FRANCIS INC  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

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Language：Japanese   Publisher：Pesticide Science Society of Japan  

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