Search

The aim of this study was to develop an in vitro drug permeability methodology which mimics the gastrointestinal environment more accurately than conventional 2D methodologies through a three-dimensional (3D) Caco-2 tubules using a microphysiological system. Such a system offers significant advantages, including accelerated cellular polarization and more accurate mimicry of the in vivo environment. This methodology was confirmed by measuring the permeability of propranolol as a model compound, and subsequently applied to those of solifenacin and bile acids for a comprehensive understanding of permeability for the drug product in the human gastrointestinal tract. To protect the Caco-2 tubules from bile acid toxicity, a mucus layer was applied on the surface of Caco-2 tubules and it enables to use simulated intestinal fluid. The assessment using propranolol reproduced results equivalent to those obtained from conventional methodology, while that using solifenacin indicated fluctuations in the permeability of solifenacin due to various factors, including interaction with bile acids. We therefore suggest that this model will serve as an alternative testing system for measuring drug absorption in an environment closely resembling that of the human gastrointestinal tract.

Acyl-CoAdiacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the fat absorption step in enterocytes. We previously reported that the pharmacological inhibition of DGAT1 increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity in corn oil-loaded rats without any sign of hepatotoxicity. In this study, we investigated this mechanism. We found that this elevation occurred only during the pharmacologically active period of a DGAT1 inhibitor and the magnitude did not depend on the volume of corn oil. In addition, this elevation was not accompanied by increases in ALT or AST mRNA levels in the small intestine and liver. To clarify a lipid component responsible for this elevation, rats were treated with free fatty acids instead of corn oil and no plasma ALT elevation was observed. Next, rats were pretreated with inhibitors of monoacylglycerol acyltransferase 2 and intestinal microsomal triglyceride transfer protein instead of the DGAT1 inhibitor, but no plasma ALT elevation was observed after corn oil loading. Since the results suggested a possible role of diacylglycerol (DAG), which activates protein kinase C (PKC), we measured PKC activity in the small intestine and found that the activity was increased by treatment with the DGAT1 inhibitor and corn oil. Moreover, rats pretreated with a PKC inhibitor in combination with the DGAT1 inhibitor showed suppression of plasma ALT elevation. Taken together, the present results suggest that DAG accumulation induced by pharmacological DGAT1 inhibition and resultant PKC activation in enterocytes are involved in the increase in plasma ALT and AST activity in rats.

Hepatotoxicity is one of the most common toxicities observed in non-clinical safety studies of drug candidates, and it is important to understand the hepatotoxicity mechanism to assess the risk of drug-induced liver injury in humans. In this study, we investigated the mechanism of hepatotoxicity caused by 2-[2-Methyl-1-(oxan-4-yl)-1H-benzimidazol-5-yl]-1,3-benzoxazole (DSP-0640), a drug candidate that showed hepatotoxicity characterized by centrilobular hypertrophy and vacuolation of hepatocytes in a 4-week oral repeated-dose toxicity study in male rats. In the liver of rats treated with DSP-0640, the expression of aryl hydrocarbon receptor (AHR) target genes, including Cyp1a1, was upregulated. In in vitro reporter assays, however, DSP-0640 showed only minimal AHR-activating potency. Therefore, we investigated the possibility that DSP-0640 indirectly activated AHR by inhibiting the CYP1 enzyme-dependent clearance of endogenous AHR agonists. In in vitro assays, DSP-0640 showed inhibitory effects on both rat and human CYP1A1 and enhanced rat and human AHR-mediated reporter gene expression induced by 6-formylindolo[3,2-b]carbazole, a well-known endogenous AHR agonist. The possible involvement of CYP1A1 inhibition in AHR activation was also demonstrated with other hepatotoxic compounds tacrine and albendazole. These results suggest that CYP1A1 inhibition-mediated AHR activation is involved in the hepatotoxicity caused by DSP-0640 and that DSP-0640 might induce hepatotoxicity in humans as well. We propose that CYP1A1 inhibition-mediated AHR activation is a novel mechanism for drug-induced hepatotoxicity.

The in vitro antitumor activity (e.g., IC50) of anticancer drugs is important for selecting candidate compounds for in vivo drug efficacy study in the early stage of drug discovery. In this study, ...

Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites.After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite.Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450.When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2. Nonclinical metabolite profiling of DS-1971a, a potent selective NaV1.7 inhibitor, was performed to predict human metabolites. After the oral administration of radiolabelled DS-1971a, the predominant metabolite in mouse plasma was M4, a monoxide at the pyrimidine ring, while the major metabolites with the first and second highest exposure in monkey plasma were M2, a monoxide at the cyclohexane ring, and M11, a demethylated pyrazole metabolite. Incubation studies with liver cytosolic and microsomal fractions in the absence or presence of NADPH indicated that the metabolising enzyme responsible for M4 formation was aldehyde oxidase (AO), while cytochrome P450s (P450s) were responsible for M2 and M11 formation. These results suggest that DS-1971a is a substrate for both AO and P450. When DS-1971a was incubated with liver S9 fractions and NADPH, the most abundant metabolites were M4 in mice, and M2 and M11 in monkeys, indicating that the results of in vitro incubation studies could provide information reflecting the in vivo plasma metabolite profiles in mice and monkeys. The results obtained from the incubation with the human liver S9 fraction and NADPH suggested that a major circulating metabolite in humans is M1, a regioisomer of M2.

Drug-induced liver injury (DILI) is one of the major causes for the discontinuation of drug development and withdrawal of drugs from the market. Since it is known that reactive metabolite formation and being substrates or inhibitors of cytochrome P450s (P450s) are associated with DILI, we systematically investigated the association between human P450 inhibition and DILI. The inhibitory activity of 266 DILI-positive drugs (DILI drugs) and 92 DILI-negative drugs (no-DILI drugs), which were selected from Liver Toxicity Knowledge Base (US Food and Drug Administration), against 8 human P450 forms was assessed using recombinant enzymes and luminescent substrates, and the threshold values showing the highest balanced accuracy for DILI discrimination were determined for each P450 enzyme using receiver operating characteristic analyses. The results showed that among the P450s tested, CYP1A1 and CYP1B1 were inhibited by DILI drugs more than no-DILI drugs with a statistical significance. We found that 91% of drugs that showed inhibitory activity greater than the threshold values against CYP1A1 or CYP1B1 were DILI drugs. The results of internal 5-fold cross-validation confirmed the usefulness of CYP1A1 and CYP1B1 inhibition data for the threshold-based discrimination of DILI drugs. Although the contribution of these P450s to drug metabolism in the liver is considered minimal, our present findings suggest that the assessment of CYP1A1 and CYP1B1 inhibition is useful for screening DILI risk of drug candidates at the early stage of drug development.

The liver plays critical roles to maintain homeostasis of living organisms and is also a major target organ of chemical toxicity. Meanwhile, nuclear receptors (NRs) are known to regulate major liver functions and also as a critical target for hepatotoxic compounds. In this study, we established mammalian one-hybrid assay systems for five rat-derived NRs, namely PXR, PPARα, LXRα, FXR and RXRα, and evaluated a total of 326 compounds for their NR-activating profiles. Then, we assessed the association between their NR-activating profile and hepatotoxic endpoints in repeated-dose toxicity data of male rats from Hazard Evaluation Support System. In the in vitro cell-based assays, 68, 38, 20, 17 and 17 compounds were identified as positives for PXR, PPARα, LXRα, FXR and RXRα, respectively. The association analyses demonstrated that the PXR-positive compounds showed high frequency of endpoints related to liver hypertrophy, such as centrilobular hepatocellular hypertrophy, suggesting that PXR activation is involved in chemical-induced liver hypertrophy in rats. It is intriguing to note that the PXR-positive compounds also showed statistically significant associations with both prolonged activated partial thromboplastin time and prolonged prothrombin time, suggesting a possible involvement of PXR in the regulation of blood clotting factors. Collectively, our approach may be useful for discovering new functions of NRs as well as understanding the complex mechanism for hepatotoxicity caused by chemical compounds.

Long-term administration of some antiepileptic drugs often increases blood lipid levels. In this study, we investigated its molecular mechanism by focusing on the nuclear receptors constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα), which are key transcription factors for enzyme induction and lipid metabolism, respectively, in the liver. Treatment of mice with the CAR activator phenobarbital, an antiepileptic drug, increased plasma triglyceride levels and decreased the hepatic expression of PPARα target genes related to lipid metabolism. The increase in PPARα target gene expression induced by fenofibrate, a PPARα ligand, was inhibited by cotreatment with phenobarbital. CAR suppressed PPARα-dependent gene transcription in HepG2 cells but not in COS-1 cells. The mRNA level of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α), a coactivator for both CAR and PPARα, in COS-1 cells was much lower than in HepG2 cells. In reporter assays with COS-1 cells overexpressing PGC1α, CAR suppressed PPARα-dependent gene transcription, depending on the coactivator-binding motif. In mammalian two-hybrid assays, CAR attenuated the interaction between PGC1α and PPARα. Chemical inhibition of PGC1α prevented phenobarbital-dependent increases in plasma triglyceride levels and the inhibition of PPARα target gene expression. These results suggest that CAR inhibits the interaction between PPARα and PGC1α, attenuating PPARα-dependent lipid metabolism. This might explain the antiepileptic drug-induced elevation of blood triglyceride levels. Significance Statement CAR activated by antiepileptic drugs inhibits the PPARα-dependent transcription of genes related to lipid metabolism and upregulates blood TG levels. The molecular mechanism of this inhibition involves competition between these nuclear receptors for coactivator PGC1α binding.

Introduction: The nuclear receptor CAR plays an important role in the regulation of hepatic responses to xenobiotic exposure, including the induction of hepatocyte proliferation and chemical carcinogenesis. Phenobarbital, a well-known liver cancer promoter, has been found to promote hepatocyte proliferation via CAR activation. However, the molecular mechanisms by which CAR induces liver carcinogenesis remain unknown. In addition, it is believed that CAR-mediated liver carcinogenesis shows a species difference; phenobarbital treatment induces hepatocyte proliferation and liver cancer in rodents but not in humans. However, the mechanisms are also unknown.Areas covered: Several reports indicate that the key oncogenic signaling pathways Wnt/β-catenin and Hippo/YAP are involved in CAR-mediated liver carcinogenesis. We introduce current data about the possible molecular mechanisms involved in CAR-mediated liver carcinogenesis and species differences by focusing on these two signaling pathways.Expert opinion: CAR may activate both the Wnt/β-catenin and Hippo/YAP signaling pathways. The synergistic activation of both signaling pathways seems to be important for CAR-mediated liver cancer development. Low homology between the ligand binding domains of human CAR and rodent CAR might cause species differences in the interactions with proteins that control the Wnt/β-catenin and Hippo/YAP pathways as well as liver cancer induction.

A simulation system for ligand interaction of human CYP1A1 has been developed using "Template" composed of hexagonal grids, as a modification of CYP1A2 system established previously. Differing from CYP1A2 Template, Site of Oxidation of CYP1A1 was located one-grid (Ring) away horizontally from Trigger-Region (Ring B) on CYP1A1 Template. Simultaneous interaction at Site of Oxidation and Trigger-Region as uni- or bi-molecule binding was maintained with CYP1A1 as well as CYP1A2 for the functional contributions. Reciprocal comparison of simulation results with experimental data suggested the access of ligands to Site of Oxidation inside of CYP1A1, through three distinct routes, termed Sideway, Center-Area and Thick-Area. To facilitate the verification of feasible placement(s), typical modes of the regional interactions have been defined and developed for prognostic devices. Simulation experiments of human and rat CYP1A1 offered possible causative mechanisms of the species difference as their distinct interactions near Site of Oxidation. The present CYP1A1 Template system has been proven to afford regio- and stereo-chemically feasible placements, through the use of the prognostic devices, of total of 353 CYP1A1-mediated reactions of 223 of distinct ligands, including substrates, inhibitors and poor substrates of drugs, environmental chemicals and endobiotics.

Some patients encounter hepatotoxicity after repeated acetaminophen (APAP) dosing even at therapeutic doses. In the present study, we focused on the diabetic state as one of the suggested risk factors of drug-induced liver injury in humans and investigated the contribution of accelerated gluconeogenesis to the susceptibility to APAP-induced hepatotoxicity using an animal model of type 2 diabetes patients. Sprague-Dawley (SD) rats and spontaneously diabetic torii (SDT) rats were each given APAP at 0 mg/kg, 300 and 500 mg/kg for 35 days by oral gavage. Plasma and urinary glutathione-related metabolites, liver function parameters, and hepatic glutathione levels were compared between the non-APAP-treated SDT and SD rats and between the APAP-treated SDT and SD rats. Hepatic function parameters were not increased at either dose level in the APAP-treated SD rats, but were increased at both dose levels in the APAP-treated SDT rats. Increases in hepatic glutathione levels attributable to the treatment of APAP were noted only in the APAP-treated SD rats. There were differences in the profiles of plasma and urinary glutathione-related metabolites between the non-APAP-treated SD and SDT rats and the plasma/urinary endogenous metabolite profile after treatment with APAP in the SDT rats indicated that hepatic glutathione synthesis was decreased due to accelerated gluconeogenesis. In conclusion, SDT rats were more sensitive to APAP-induced chronic hepatotoxicity than SD rats and the high susceptibility of SDT rats was considered to be attributable to lowered hepatic glutathione levels induced by accelerated gluconeogenesis.

Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.

The liver plays critical roles to maintain homeostasis of living organisms and is also a major target organ of chemical toxicity. Meanwhile, nuclear receptors (NRs) are known to regulate major liver functions and also as a critical target for hepatotoxic compounds. In this study, we established mammalian one-hybrid assay systems for five rat-derived NRs, namely PXR, PPARα, LXRα, FXR and RXRα, and evaluated a total of 326 compounds for their NR-activating profiles. Then, we assessed the association between their NR-activating profile and hepatotoxic endpoints in repeated-dose toxicity data of male rats from Hazard Evaluation Support System. In the in vitro cell-based assays, 68, 38, 20, 17 and 17 compounds were identified as positives for PXR, PPARα, LXRα, FXR and RXRα, respectively. The association analyses demonstrated that the PXR-positive compounds showed high frequency of endpoints related to liver hypertrophy, such as centrilobular hepatocellular hypertrophy, suggesting that PXR activation is involved in chemical-induced liver hypertrophy in rats. It is intriguing to note that the PXR-positive compounds also showed statistically significant associations with both prolonged activated partial thromboplastin time and prolonged prothrombin time, suggesting a possible involvement of PXR in the regulation of blood clotting factors. Collectively, our approach may be useful for discovering new functions of NRs as well as understanding the complex mechanism for hepatotoxicity caused by chemical compounds.

Recently, selective androgen receptor modulators (SARMs), which bind to AR and act in a tissue/effect-specific manner, have been developed, but the selective mechanism is not well understood. In this study, we investigated the selective mechanism using the synthetic steroid YK11, which showed AR-mediated gene-selective transactivation. In the AR-positive human breast cancer MDA-MB-453 cells, different patterns of AR-mediated target gene expression and AR recruitment to their enhancer regions were observed between DHT and YK11. A docking study suggested the helices 11 and 12 was moved by the sterically hindered C17-group of YK11. Furthermore, the mutational studies of AR Gln902 and mammalian two-hybrid assays suggested different cofactor recruitment between DHT and YK11. The results of this study suggest that gene selective regulation by SARMs results from differential DNA-binding and/or cofactor recruitment by ligands. These results provide novel insights into the mechanism of action of SARMs.

Nonclinical metabolite profiling of DS-1971a, a potent selective Na

Pregnane X receptor (PXR), a xenobiotic-responsive nuclear receptor, plays key roles in drug disposition. PXR activation induces liver hypertrophy in rodents, but the molecular mechanism of this effect remains unclear although the PXR-mediated induction of cytochrome P450s is proposed to be involved. Since yes-associated protein (YAP), an effector protein of the Hippo pathway, functions as a transcriptional cofactor that controls organ size via TEA domain family members (TEADs) or other transcription factors, we investigated the functional interaction of PXR with YAP in liver hypertrophy and drug metabolism in this study. The treatment of mice with a PXR activator induced liver hypertrophy, promoted nuclear YAP accumulation and increased the expression of YAP/TEAD target genes in the liver, suggesting the coactivation of PXR and YAP. Through chronological analyses of this in vivo model, no clear association between PXR-dependent liver hypertrophy and cytochrome P450 induction was observed. In reporter assays, ligand-activated PXR enhanced YAP-mediated gene transcription, whereas YAP overexpression inhibited PXR-dependent gene transcription. No clear species differences in these transcriptional interactions between humans and mice were observed. Furthermore, in human hepatocarcinoma and primary hepatocyte-like cells, YAP suppressed the expression of liver-enriched transcription factors, including hepatocyte nuclear factor 4α (HNF4α), PXR and constitutive androstane receptor (CAR), and their target genes. These results suggest that YAP is involved in PXR-induced liver hypertrophy and that YAP activation interferes with gene expression associated with various liver functions. SIGNIFICANCE STATEMENT We have investigated the functional interaction between PXR and YAP, an effector protein of the Hippo pathway. PXR plays central roles in various liver functions including drug metabolism, and the Hippo pathway and YAP regulate organ size through interacting with several transcription factors, including TEADs. Our results suggest that YAP is involved in PXR-mediated liver hypertrophy and that YAP activation interferes with the expression of liver-enriched transcription factors and thus drug-metabolizing enzymes.

Differences in CYP1A2-mediated metabolisms in human, rat and mouse have been analyzed with Template of human CYP1A2 established in our previous studies (Drug Metab Pharmacokinet 31:363, 2016 and 32:229, 2017). Using more than 25 chemicals including phenanthrene, MeIQx, PhIP, caffeine and furafylline, Template for human CYP1A2 was found to be applicable for rat and mouse CYP1A2 reactions with the consideration of five distinct regional interactions: 1) Expanded use of Ring D region of pro-metabolized molecules and also of trigger molecules, 2) acceptance of secondary amino groups at Position 31 of Ring eC1, 3) overlapping of pro-metabolized and trigger molecules at Ring eC4, 4) restricted maneuvering of substrates into Bay 1 region, and 5) allowance of passage of slightly large ligands in Thin-Area. These distinction points were found to be mutual for both substrates and inhibitors. In the present study, the decision-tree for substrates entering from Thin-Area has been reevaluated in consideration of species differences in human and rodent CYP1A2 forms. As the results, five steps of verification procedures have been introduced to predict the occurrence order of the regioselective metabolisms.

Nuclear receptors pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR) are xenobiotic-responsible transcriptional factors that belong to the same subfamily and are expressed abundantly in the liver. They play crucial roles in various liver functions including xenobiotic disposition and energy metabolism. CAR is also involved in xenobiotic-induced hepatocyte proliferation and hepatocarcinogenesis in rodents. However, there are some open questions on the association between chemical carcinogenesis and these nuclear receptors. These include the molecular mechanism for CAR-mediated hepatocyte proliferation and hepatocarcinogenesis. Another important question is whether PXR is associated with hepatocyte proliferation. We have recently reported a novel and unique function of PXR associated with murine hepatocyte proliferation: PXR activation alone does not induce hepatocyte proliferation but accelerates hepatocyte proliferation induced by various types of stimuli including CAR- or peroxisome proliferator-activated receptor alpha activating compounds, liver injury, and growth factors. We have also reported a role of yes-associated protein (YAP), a transcriptional cofactor controlling organ size and cell growth under the Hippo pathway, in CAR-mediated hepatocyte proliferation in mice. In this review, I will introduce our recent results as well as related studies on the roles of PXR and CAR in xenobiotic-induced hepatocyte proliferation and their molecular mechanisms.

Pregnane X receptor (PXR) is a xenobiotic-responsive transcription factor that plays a pivotal role in drug metabolism and disposition in livers and intestines through regulating the expression of drug metabolizing enzymes and transporters. It is well known that PXR-mediated induction of CYP3A enzymes is downregulated under inflammatory conditions. Although some reports suggest that the downregulation is caused by an inhibition of transcriptional activity of PXR by nuclear factor-κB (NF-κB), a key inflammation-associated transcription factor, the detailed mechanism remains unclear. In reporter assays using the CYP3A4 promoter, the PXR-mediated transcription was suppressed by inflammatory stimuli and co-expression of NF-κB or activator protein-1 (AP-1), suggesting that not only NF-κB but also AP-1 play a key role in the suppression of CYP3A induction. We also revealed that PXR, NF-κB and AP-1 commonly use the coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) for their transcriptional activation and that inflammatory stimuli inhibited the GRIP1-mediated enhancement of the transcription by PXR. These results suggest that under inflammatory conditions activated NF-κB and/or AP-1 compete with PXR for GRIP1 usage to reduce the PXR/GRIP1-mediated transcription of CYP3A genes.

Drug-induced liver injury (DILI) is one of the major causes for the discontinuation of drug development and withdrawal of drugs from the market. Since it is known that reactive metabolite formation and being substrates or inhibitors of cytochrome P450s (P450s) are associated with DILI, we systematically investigated the association between human P450 inhibition and DILI. The inhibitory activity of 266 DILI-positive drugs (DILI drugs) and 92 DILI-negative drugs (no-DILI drugs), which were selected from Liver Toxicity Knowledge Base (US Food and Drug Administration), against 8 human P450 forms was assessed using recombinant enzymes and luminescent substrates, and the threshold values showing the highest balanced accuracy for DILI discrimination were determined for each P450 enzyme using receiver operating characteristic analyses. The results showed that among the P450s tested, CYP1A1 and CYP1B1 were inhibited by DILI drugs more than no-DILI drugs with a statistical significance. We found that 91% of drugs that showed inhibitory activity greater than the threshold values against CYP1A1 or CYP1B1 were DILI drugs. The results of internal 5-fold cross-validation confirmed the usefulness of CYP1A1 and CYP1B1 inhibition data for the threshold-based discrimination of DILI drugs. Although the contribution of these P450s to drug metabolism in the liver is considered minimal, our present findings suggest that the assessment of CYP1A1 and CYP1B1 inhibition is useful for screening DILI risk of drug candidates at the early stage of drug development.

We had previously reported that treatment with the aryl hydrocarbon receptor (AHR) agonist β-naphthoflavone (βNF) suppressed mammosphere formation derived from cancer stem cells in human breast cancer MCF-7 cells (Cancer Lett., 317, 2012, Zhao et al.). Here, using several AHR agonists, we have investigated the association of this suppression with the classical ability to induce AHR-mediated gene transcription in the xenobiotic response element (XRE). The mammosphere formation assays were performed using wild-type and AHR-knockout MCF-7 cells in the presence of AHR agonists including 3-methylcholanthrene (3MC), benzo[a]pyrene (BaP), 7,12-dimethylbenz[a]anthracene (DMBA), 6-formylindolo[3,2-b]carbazole (FICZ), indirubin, indole-3-carbinol (I3C), indole-3-acetic acid (IAA), and kynurenine (KYN), followed by the XRE-reporter gene assays of the agonists. We showed that treatments with 3MC, BaP, and DMBA strongly suppressed mammosphere formation of the stem cells in an AHR-dependent manner, while other agonists showed weaker suppression. In reporter gene assays, the strength or duration of AHR/XRE-mediated gene transcription was found to be dependent on the agonist. Although strong transcriptional activation was observed with 3MC, FICZ, indirubin, I3C, IAA, or KYN after 6 h of treatment, only weak activation was seen with BaP or DMBA. While transcriptional activation was sustained or increased at 24 h with 3MC, BaP, or DMBA, appreciable reduction was observed with the other agonists. In conclusions, the results demonstrated that the suppressive effects of AHR agonists on mammosphere formation do not necessarily correlate with their abilities to induce AHR-mediated gene transcription. Hence, different AHR functions may be differentially induced in an agonist-dependent manner.

Pregnane X receptor (PXR) and constitutively active receptor/constitutive androstane receptor (CAR) are xenobiotic-responsible transcription factors belonging to the same nuclear receptor gene subfamily and highly expressed in the liver. These receptors are activated by a variety of chemicals and play pivotal roles in many liver functions, including xenobiotic metabolism and disposition. Phenobarbital, an enzyme inducer and liver tumor promoter, activates both rodent and human CAR but causes liver tumors only in rodents. Although the precise mechanism for phenobarbital/CAR-mediated liver tumor formation remains to be established, intracellular pathways, including the Hippo pathway/Yes-associated protein-TEA-domain family members system and

A Template system for a prediction of human CYP2E1-mediated reactions (Drug Metab Rev 2011) has been refined with the introduction of ideas of Trigger-residue and the residue-initiated movement of ligands in the active site. The refined system also includes ideas of bi-molecule binding and angled-placement, which allow to sit diverse types of ligands on Template. With the use of these ideas in common with other Template systems for human CYP1A1, CYP1A2 and CYP3A4 (Drug Metab Pharmacokinet 2016, 2017, 2019, and 2020), 349 reactions of 192 distinct chemicals published as CYP2E1 ligands were examined in the refined system. Verifications of good and poor substrates, regioselectivity and also inhibitory interaction were available faithfully for these ligands from their placements on the refined Template and rules for interaction modes, accompanied with their deciphering information to lead to the judgements. The refined CYP2E1 Template system will thus offer more reliable estimations of human CYP2E1 catalysis toward ligands of diverse structures.

Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are nuclear receptors that are highly expressed in the liver and activated by numerous chemicals. While CAR activation by its activators, such as phenobarbital (PB), induces hepatocyte proliferation and liver carcinogenesis in rodents, it remains unclear whether PXR activation drives liver cancer. To investigate the influence of PXR activation on liver carcinogenesis, we treated mice with the PXR activator pregnenolone 16α-carbonitrile (PCN) with or without PB following tumor initiation with diethylnitrosamine (DEN). After 20 weeks of treatment, preneoplastic lesions detected by immunostaining with an anti-KRT8/18 antibody were observed in PB-treated but not PCN-treated mice, and PCN cotreatment augmented the formation of preneoplastic lesions by PB. After 35 weeks of treatment, macroscopic observations indicated that PB-treated and PB/PCN-cotreated mice had increased numbers of liver tumors compared to control and PCN-treated mice. In the pathological analyses of liver sections, all the mice in the PB and PB/PCN groups developed carcinoma and/or eosinophilic adenoma, but in the PB/PCN group, the multiplicity of carcinoma and eosinophilic adenoma was significantly reduced and the size of carcinoma showed a tendency to decrease. No mouse in the control or PCN-treated group developed such tumors. Differentially expressed gene (DEG) and gene set enrichment analyses in combination with RNA sequencing suggested the increased expression of genes related to epithelial–mesenchymal transition (EMT) in mice cotreated with PCN and PB compared to those treated with PB alone. Changes in the hepatic mRNA levels of epithelial marker genes supported the results of the transcriptome analyses. In conclusion, the present results suggest that PXR activation does not promote hepatocarcinogenesis in contrast to CAR and rather attenuates CAR-mediated liver cancer development by suppressing the EMT of liver cancer cells in rodents.

Long-term administration of some antiepileptic drugs often increases blood lipid levels. In this study, we investigated its molecular mechanism by focusing on the nuclear receptors constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor

The activation of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2)/death receptor 5 (DR5) induces apoptosis in various tumor cells but not in normal human cells. Because some therapeutic antibodies targeting TRAIL-R2 have demonstrated severe hepatotoxicity in clinical applications, novel in vivo models reflecting clinical hepatotoxicity are now required. In this study, we investigated the hepatotoxicity caused by KMTR2, an anti-human TRAIL-R2 monoclonal antibody, in chimeric mice with humanized livers (PXB-mice). PXB-mice were exposed to KMTR2 by single or repeated (weekly for 4 weeks) intravenous administrations, and the analyses of blood chemistry, liver histopathology, hepatic gene expression, and toxicokinetics were performed. Treatment with 1 or 10 mg/kg of KMTR2 increased alanine transaminase (ALT) activity and human ALT1 levels in blood. Histopathological analysis revealed that cell death and degeneration with the infiltration of inflammatory cells in human but not mouse hepatocytes were increased in a time-dependent manner after KMTR2 administration. Furthermore, increases in TdT-mediated dUTP nick end labeling (TUNEL)-positive human hepatocytes and serum concentration of cleaved cytokeratin 18, a human-specific apoptosis marker, were observed. RNA sequence analysis showed that the gene expression profile changed in different manners between human and mouse hepatocytes and the up-regulation of TRAIL-R2-related genes was observed only in human hepatocytes. Taken together, these results indicate that KMTR2-mediated TRAIL-R2 activation induces apoptosis of human hepatocytes and hepatotoxicity in PXB-mice and suggest that chimeric mice with humanized liver can be novel tools for the evaluation of in vivo human-specific hepatotoxicity induced by therapeutic antibodies in pre-clinical studies.

CYP2C enzymes play key roles in drug metabolism, and clinical drug-drug interactions caused by CYP2C induction have been reported. The aim of this study was to establish a method to predict the potency of CYP2C inductions considering the mechanism. We first investigated the relations of CYP2C induction with CYP3A4 or CYP2B6 induction in human hepatocytes after 48-h exposure with 19 inducers. The fold-induction values of CYP2C8 and CYP2C9 were well correlated with those of CYP3A4, whereas the inducers were separated into 2 groups showing different correlations with CYP2B6 induction for CYP2C8 and CYP2C9 induction. In the regression models established, the fold-induction values of CYP2C8 and CYP2C9 were well expressed as the functions of those of CYP3A4 and CYP2B6, while no such obvious correlation was observed for CYP2C19 induction. These results suggest that CYP2Cs are not simply coinduced with CYP3A4 and that CYP2C8 and CYP2C9 inductions are regulated by both pregnane X receptor and constitutive androstane receptor with different contributions. Finally, simple correlations were proposed using the experimental Emax values obtained and plasma concentrations of CYP2C9 substrates from the literature, and positive correlations were observed. These data provide methods to estimate the clinical impact of CYP2C9 induction from in vitro data.

A sensitive analytical method was developed for the simultaneous detection of 11 chiral pharmaceuticals and their hepatic metabolites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an ovomucoid chiral column. After optimization of the LC conditions, all pharmaceuticals examined were enantio-separated with Rs of >0.82 in LC-MS/MS analysis. The limit of detections of all pharmaceuticals by MS/MS detection ranged from 1.2 to 92.3 nM, which is approximately 1000 - 25000 times lower than those obtained by UV detection. From hepatic metabolite analyses in P450-expressing cells, metabolites of three pharmaceuticals were detected and enantio-separated. By using the proposed method, changes in the optical isomer ratio of the hepatic metabolites chlorpheniramine and verapamil caused by differential cytochrome P450 enzyme expression for each isomer, could be successfully traced.

The demand for alternatives to animal experiment-based assessment is increasing. Alternatives for assessing repeated-dose toxicity, however, have yet to be developed. Our aim was to develop discriminative models for predicting an increase in serum ALT levels in rats, using molecular descriptors. In vivo data for rats in the training data sets were obtained using the Hazard Evaluation Support System Integrated Platform (HESS), and molecular descriptors were calculated using DRAGON 6. We developed the discriminative models based on logistic regression models; however, there were two statistical difficulties to be overcome: (i) the number of molecular descriptors was much greater than the number of compounds; (ii) the training data sets were imbalanced. In order to overcome these difficulties, the k-medoids method was employed in the case of the first difficulty, and the Synthetic Minority Over-sampling Technique (SMOTE) algorithm in the case of the second. One of the resulting models showed predictive capability, with sensitivity of 0.783, specificity of 0.745, and concordance of 0.750. Our results show that a statistical learning approach can create a discriminative model with high predictive capability using only information on the molecular descriptors of chemicals.

The nuclear receptor pregnane X receptor (PXR) plays a major role in the xenobiotic-induced expression of drug-metabolizing enzymes. PXR activation is also associated with several adverse events in the liver. Especially, the receptor enhances hepatocyte proliferation mediated by chemical liver tumor promoters, suggesting that exposure to PXR activators increases the risk of liver cancer. In this study, we have investigated the influences of food additives on PXR to understand their potential adverse effects when they are taken in combination with other chemical compounds. We first screened 25 food additives and related compounds for their PXR-activating ability using reporter assays in HepG2 cells expressing mouse PXR, and found that imazalil dose-dependently activated mouse PXR. Next, to investigate whether imazalil could activate mouse PXR in vivo, mice were treated with imazalil and we found that imazalil treatment increased hepatic mRNA levels of Cyp3a11, a PXR target gene. Finally, to investigate the influence of imazalil exposure on the hepatocyte proliferation induced by nuclear receptor constitutive active/androstane receptor (CAR), mice were treated with imazalil with or without mouse CAR activator TCPOBOP. Although imazalil alone did not induce hepatocyte proliferation, co-treatment with imazalil facilitated the TCPOBOP-dependent proliferation, indicated by the increases in cell proliferation marker levels, Ki-67-positive nuclei and Mcm2 mRNA levels. These results suggest that in mice imazalil activates PXR to enhance hepatocyte proliferation mediated by CAR-activating liver tumor promoters.

A Template system for a prediction of human CYP2E1-mediated reactions (Drug Metab Rev 2011) has been refined with the introduction of ideas of Trigger-residue and the residue-initiated movement of ligands in the active site. The refined system also includes ideas of bi-molecule binding and angled-placement, which allow to sit diverse types of ligands on Template. With the use of these ideas in common with other Template systems for human CYP1A1, CYP1A2 and CYP3A4 (Drug Metab Pharmacokinet 2016, 2017, 2019, and 2020), 349 reactions of 192 distinct chemicals published as CYP2E1 ligands were examined in the refined system. Verifications of good and poor substrates, regioselectivity and also inhibitory interaction were available faithfully for these ligands from their placements on the refined Template and rules for interaction modes, accompanied with their deciphering information to lead to the judgements. The refined CYP2E1 Template system will thus offer more reliable estimations of human CYP2E1 catalysis toward ligands of diverse structures.