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Ikoma K, Hosaka T, Ooka A, Shizu R, Yoshinari K. Machine Learning on Toxicogenomic Data Reveals a Strong Association Between the Induction of Drug-Metabolizing Enzymes and Centrilobular Hepatocyte Hypertrophy in Rats. Int J Mol Sci 2025; 26:4886. [PMID: 40430025 PMCID: PMC12112521 DOI: 10.3390/ijms26104886] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2025] [Revised: 05/18/2025] [Accepted: 05/18/2025] [Indexed: 05/29/2025] Open
Abstract
Centrilobular hepatocyte hypertrophy is frequently observed in animal studies for chemical safety assessment. Although its toxicological significance and precise mechanism remain unknown, it is considered an adaptive response resulting from the induction of drug-metabolizing enzymes (DMEs). This study aimed to elucidate the association between centrilobular hepatocyte hypertrophy and DME induction using machine learning on toxicogenomic data. Utilizing publicly available gene expression data and pathological findings from rat livers of 134 compounds, we developed six different types of machine learning models to predict the occurrence of centrilobular hepatocyte hypertrophy based on gene expression data as explanatory variables. Among these, a LightGBM-based model demonstrated the best performance with an accuracy of approximately 0.9. With this model, we assessed each gene's contribution to predicting centrilobular hepatocyte hypertrophy using mean absolute SHAP values. The results revealed that Cyp2b1 had an extremely significant contribution, while other DME genes also displayed positive contributions. Additionally, enrichment analysis of the top 100 genes based on mean absolute SHAP values identified "Metabolism of xenobiotics by cytochrome P450" as the most significantly enriched term. In conclusion, the current results suggest that the induction of multiple DMEs, including CYP2B1, is crucial for the development of centrilobular hepatocyte hypertrophy.
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Affiliation(s)
| | | | | | | | - Kouichi Yoshinari
- Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan (T.H.); (A.O.); (R.S.)
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Kurosawa K, Nakano M, Yokoseki I, Tomii M, Higuchi Y, Uehara S, Yoneda N, Suemizu H, Fukami T, Nakajima M. Switch/sucrose non-fermentable complex interacts with constitutive androstane receptor to regulate drug-metabolizing enzymes and transporters in the liver. Drug Metab Dispos 2025; 53:100057. [PMID: 40158296 DOI: 10.1016/j.dmd.2025.100057] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2024] [Revised: 02/18/2025] [Accepted: 02/26/2025] [Indexed: 04/02/2025] Open
Abstract
Constitutive androstane receptor (CAR) is a nuclear receptor that plays an important role in regulating drug metabolism and bile acid homeostasis in the liver. Recently, it was revealed that the switch/sucrose non-fermentable (SWI/SNF) complex, a chromatin remodeler, regulates transactivation by nuclear receptors, such as the pregnane X receptor and vitamin D receptor. However, studies on the involvement of the SWI/SNF complex in CAR-mediated transactivation are limited. Here, we demonstrated that the induction of cytochrome P450 CYP2B6 expression by CAR activators, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime and phenobarbital, was enhanced by the inhibition of AT-rich interactive domain-containing protein (ARID) 1A, a canonical brahma-related gene 1-associated factor (cBAF) component, one of the SWI/SNF complexes, and was attenuated by inhibition of bromodomain-containing protein (BRD) 9, a noncanonical BAF (ncBAF) component, in primary hepatocytes from humanized mice. Coimmunoprecipitation assays revealed that ARID1A and BRD9 interacted with CAR. Chromatin immunoprecipitation assay revealed that the 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime-induced binding of CAR to the 5'-flanking region of CYP2B6 gene increased with ARID1A inhibition and reduced with BRD9 inhibition. These results suggest that cBAF negatively regulates CAR-mediated transactivation by attenuating CAR binding to its response element, whereas ncBAF positively regulates it by facilitating CAR binding. Furthermore, ARID1A inhibition enhanced phenobarbital-induced increases in UDP-glucuronosyltransferase 1A1 expression and multidrug resistance-associated protein 2 mRNA level and activity. Collectively, our findings indicate that cBAF and ncBAF play essential roles in xenobiotic metabolism by regulating CAR-mediated transactivation and that ARID1A inhibitors may offer therapeutic benefits for hyperbilirubinemia and cholestasis by inducing UDP-glucuronosyltransferase 1A1 and multidrug resistance-associated protein 2 expression. SIGNIFICANCE STATEMENT: This study revealed that canonical brahma-related gene 1-associated factor and noncanonical brahma-related gene 1-associated factor, members of the switch/sucrose non-fermentable family, negatively and positively regulate constitutive androstane receptor (CAR) transactivation, respectively, through changes in the chromatin structure around the CAR response element in the 5'-flanking regions of CAR target genes. The inhibition of AT-rich interactive domain-containing protein 1A may be beneficial for cholestasis treatment by enhancing CAR-mediated transactivation.
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Affiliation(s)
- Kiamu Kurosawa
- Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan
| | - Masataka Nakano
- Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan; WPI Nano Life Science Institute (WPI-NanoLSI) Kanazawa University, Kakuma-machi, Kanazawa, Japan.
| | - Itsuki Yokoseki
- Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan
| | - Mei Tomii
- Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan
| | - Yuichiro Higuchi
- Liver Engineering Laboratory, Department of Applied Research for Laboratory Animals, Central Institute for Experimental Medicine and Life Science (CIEM), Kawasaki, Japan
| | - Shotaro Uehara
- Liver Engineering Laboratory, Department of Applied Research for Laboratory Animals, Central Institute for Experimental Medicine and Life Science (CIEM), Kawasaki, Japan
| | - Nao Yoneda
- Liver Engineering Laboratory, Department of Applied Research for Laboratory Animals, Central Institute for Experimental Medicine and Life Science (CIEM), Kawasaki, Japan
| | - Hiroshi Suemizu
- Liver Engineering Laboratory, Department of Applied Research for Laboratory Animals, Central Institute for Experimental Medicine and Life Science (CIEM), Kawasaki, Japan
| | - Tatsuki Fukami
- Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan; WPI Nano Life Science Institute (WPI-NanoLSI) Kanazawa University, Kakuma-machi, Kanazawa, Japan
| | - Miki Nakajima
- Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan; WPI Nano Life Science Institute (WPI-NanoLSI) Kanazawa University, Kakuma-machi, Kanazawa, Japan.
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Liu JJ, Mei HW, Jing YY, Li ZL, Wu SG, Yuan HX, Zhang XB. Yinchenhao decoction alleviates obstructive jaundice liver injury by modulating epidermal growth factor receptor and constitutive androstane receptor signaling. World J Hepatol 2025; 17:101724. [PMID: 40177192 PMCID: PMC11959654 DOI: 10.4254/wjh.v17.i3.101724] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Revised: 01/18/2025] [Accepted: 03/04/2025] [Indexed: 03/26/2025] Open
Abstract
BACKGROUND Yinchenhao decoction (YCHD) is a traditional Chinese medicine widely used to treat liver damage caused by obstructive jaundice (OJ). Although YCHD has demonstrated protective effects against liver damage, reduced apoptosis, and mitigated oxidative stress in OJ, the precise molecular mechanisms involved remain poorly understood. AIM To investigate the beneficial effects of YCHD on OJ and elucidate the underlying mechanisms. METHODS The active constituents of YCHD were identified using liquid chromatography-tandem mass spectrometry, and their potential targets for OJ treatment were predicted through network pharmacology. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed. An OJ rat model was established by common bile duct ligation. Rats were divided into three groups: Sham surgery (S Group), model (O Group), and YCHD (Y Group). YCHD was administered to Group Y for one week. Bilirubin levels, liver function parameters, and bile acid concentrations in blood and urine were measured by enzyme-linked immunosorbent assay. The bile acid renal clearance rate (Clr) was calculated. Histopathological evaluation of liver and kidney tissues was performed using hematoxylin-eosin staining. Western blotting was utilized to assess the expression of key bile acid metabolism and transport proteins in both liver and kidney tissues. The expression of the constitutive androstane receptor (CAR) and its nuclear localization were evaluated by immunohistochemistry. Molecular docking studies identified the epidermal growth factor receptor (EGFR) as a potential target of YCHD's active components. An OJ cell model was created using human liver (L02) and renal tubular epithelial (HK-2) cells, which were treated with YCHD-containing serum. Western blotting and immunofluorescence assays were employed to evaluate CAR expression and its nuclear localization in relation to EGFR activation. RESULTS Network analysis identified the EGFR signaling pathway as a key mechanism through which YCHD exerts its effects on OJ. In vivo experiments showed that YCHD improved liver function, reduced OJ-induced pathology in liver and kidney tissues, and decreased serum bile acid content by enhancing bile acid Clr and urine output. YCHD also increased CAR expression and nuclear heterotopy, upregulating proteins involved in bile acid metabolism and transport, including CYP3A4, UGT1A1, MRP3, and MRP4 in the liver, and MRP2 and MRP4 in the kidneys. In vitro, YCHD increased CAR expression and nuclear heterotopy in L02 and HK-2 cells, an effect that was reversed by EGFR agonists. CONCLUSION YCHD enhances bile acid metabolism in the liver and promotes bile acid excretion in the kidneys, ameliorating liver damage caused by OJ. These effects are likely mediated by the upregulation of CAR and its nuclear translocation.
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Affiliation(s)
- Jun-Jian Liu
- Department of Hepatobiliary and Pancreatic Surgery 2, Tianjin Nankai Hospital, Tianjin Medical University, Tianjin 300102, China
- Tianjin Key Laboratory, Acute Abdomen Disease Associated Organ Injury and ITCWM Repair, Tianjin 300102, China
| | - Han-Wei Mei
- Department of Gastrointestinal Surgery 3, Tianjin Nankai Hospital, Tianjin Medical University, Tianjin 301617, China
| | - Yan-Yan Jing
- Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
| | - Zhong-Lian Li
- Department of Hepatobiliary and Pancreatic Surgery 2, Tianjin Nankai Hospital, Tianjin Medical University, Tianjin 300102, China
| | - Su-Guo Wu
- Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
| | - Hong-Xia Yuan
- College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
| | - Xi-Bo Zhang
- Department of Hepatobiliary and Pancreatic Surgery 2, Tianjin Nankai Hospital, Tianjin Medical University, Tianjin 300102, China.
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Sondermann NC, Momin AA, Arold ST, Haarmann-Stemmann T. Polybrominated diphenyl ether flame retardants inhibit growth factor-induced activation of EGFR by binding to its extracellular domain. Arch Toxicol 2025; 99:745-753. [PMID: 39668266 DOI: 10.1007/s00204-024-03926-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2024] [Accepted: 11/28/2024] [Indexed: 12/14/2024]
Abstract
For many years, polybrominated diphenyl ethers (PBDEs) were used as flame retardants in a large number of consumer products. Even though international law meanwhile prohibits the production and usage of PBDEs, these persistent and bioaccumulative chemicals still leak into the environment, and are frequently detected in wildlife and humans. Population-based studies reveal positive correlations between human PBDE exposure and various adverse health effects, emphasizing that a better understanding of the mode of action of these polybrominated chemicals is urgently needed. Therefore, we investigated the effect of two widespread PBDEs, namely BDE-47 and BDE-99, on epidermal growth factor receptor (EGFR) activity in human cells. Recent studies showed that the EGFR is not only orchestrating cellular functions, but also serves as a cell-surface receptor for dioxins, phenolic benzotriazoles and related organic pollutants. Results from in silico docking analyses, AlphaLISA-based receptor binding studies and SDS-PAGE/Western blot analyses revealed that BDE-47 and BDE-99 inhibit the growth factor-triggered activation of EGFR by binding to its extracellular domain. In keratinocytes, PBDEs also inhibit amphiregulin-induced and EGFR-mediated DNA synthesis as well as the EGFR-triggered trans-repression of the aryl hydrocarbon receptor signaling pathway. Our data identify EGFR as a cell-surface receptor for PBDEs and shed light on a novel mode of action of these ubiquitous and persistent chemicals. This finding may contribute to an improved hazard assessment of PBDEs and structurally related flame retardants.
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Affiliation(s)
- Natalie C Sondermann
- IUF - Leibniz-Research Institute for Environmental Medicine, Auf'm Hennekamp 50, 40225, Düsseldorf, Germany
| | - Afaque A Momin
- Biological and Environmental Science and Engineering Division, Center of Excellence On Smart Health, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia
| | - Stefan T Arold
- Biological and Environmental Science and Engineering Division, Center of Excellence On Smart Health, King Abdullah University of Science and Technology (KAUST), Thuwal, 23955-6900, Kingdom of Saudi Arabia
| | - Thomas Haarmann-Stemmann
- IUF - Leibniz-Research Institute for Environmental Medicine, Auf'm Hennekamp 50, 40225, Düsseldorf, Germany.
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Sondermann NC, Vogel CFA, Haarmann-Stemmann T. Dioxins do not only bind to AHR but also team up with EGFR at the cell-surface: a novel mode of action of toxicological relevance? EXCLI JOURNAL 2025; 24:184-197. [PMID: 39996234 PMCID: PMC11847957 DOI: 10.17179/excli2024-8038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Figures] [Subscribe] [Scholar Register] [Received: 11/21/2024] [Accepted: 12/18/2024] [Indexed: 02/26/2025]
Abstract
Dioxins and dioxin-like compounds (DLCs) are highly toxic organic pollutants whose production and use are prohibited by international law. Despite this, these biopersistent and lipophilic chemicals are prevalent in the environment and accumulate in the food chain, posing significant health risks to consumers even at low exposure levels. Acute dioxin intoxication can cause chloracne, while chronic exposure has been associated with a wide range of adverse health effects, including carcinogenicity, reproductive and developmental disorders, immunotoxicity, and endocrine disruption. In the mid-1970s, scientists identified a transcription factor known as the aryl hydrocarbon receptor (AHR), which becomes activated upon binding of dioxins. AHR orchestrates numerous adaptive and maladaptive stress responses and is believed to mediate most, if not all, of the toxic effects triggered by dioxins and DLCs. Recent studies have provided mounting evidence that dioxins and dioxin-like polychlorinated biphenyls can inhibit growth factor-induced activation of the epidermal growth factor receptor (EGFR) by directly binding to its extracellular domain. This interaction prevents the activation of EGFR by polypeptide growth factors and downstream signal transduction. In this article, we explain this newly identified mechanism of action for dioxins and DLCs in detail and discuss its potential toxicological relevance by using two examples, i.e. breast cancer development and placental toxicity. Finally, we briefly refer to other environmental chemicals of global concern that, based on first published data, may act via the same mode of action. See also the graphical abstract(Fig. 1).
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Affiliation(s)
- Natalie C. Sondermann
- IUF - Leibniz Research Institute for Environmental Medicine, 40225 Düsseldorf, Germany
| | - Christoph F. A. Vogel
- Department of Environmental Toxicology and Center for Health and the Environment, University of California, Davis, CA 95616, USA
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Sondermann NC, Momin AA, Arold ST, Haarmann-Stemmann T. Benzotriazole UV stabilizers disrupt epidermal growth factor receptor signaling in human cells. ENVIRONMENT INTERNATIONAL 2024; 190:108886. [PMID: 39024829 DOI: 10.1016/j.envint.2024.108886] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/23/2024] [Revised: 07/03/2024] [Accepted: 07/12/2024] [Indexed: 07/20/2024]
Abstract
Phenolic benzotriazole UV stabilizers (BUV) are commonly used additives in synthetic polymeric products, which constantly leak into the environment. They are persistent and bioaccumulative, and have been detected not only in fish, birds, and sea mammals, but also in humans, including breast milk samples. Several authorities including the European Chemical Agency already consider some BUVs as Substances of Very High Concern in need of further information, e.g. mechanistical studies and biomonitoring. In this study, we are addressing this need by investigating the effect of several BUVs on the activity of the human epidermal growth factor receptor (EGFR), an important regulator of cellular processes that has recently been identified as a cell-surface receptor for environmental organic chemicals. By combining in silico docking, mutant analyses, receptor binding and internalization assays, we demonstrate that BUVs, particularly the chlorinated variants, bind to the extracellular domain of EGFR and thereby prevent the binding of growth factors. Accordingly, BUVs can inhibit EGFR downstream events, such as ERK1/2 phosphorylation and DNA synthesis, in human keratinocytes. Our data establish EGFR as a plasma membrane receptor for BUVs, offering novel mechanistic insights into the biological effects induced by these widespread and persistent chemicals. The findings of this study may not only improve hazard assessment for BUVs, but also contribute to the development of novel EGFR-targeting drugs.
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Affiliation(s)
- Natalie C Sondermann
- IUF - Leibniz-Research Institute for Environmental Medicine, 40225 Düsseldorf, Germany
| | - Afaque A Momin
- Biological and Environmental Science and Engineering Division, Center of Excellence on Smart Health, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia
| | - Stefan T Arold
- Biological and Environmental Science and Engineering Division, Center of Excellence on Smart Health, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia
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7
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Sonkar R, Ma H, Waxman DJ. Steatotic liver disease induced by TCPOBOP-activated hepatic constitutive androstane receptor: primary and secondary gene responses with links to disease progression. Toxicol Sci 2024; 200:324-345. [PMID: 38710495 PMCID: PMC11285164 DOI: 10.1093/toxsci/kfae057] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/08/2024] Open
Abstract
Constitutive androstane receptor (CAR, Nr1i3), a liver nuclear receptor and xenobiotic sensor, induces drug, steroid, and lipid metabolizing enzymes, stimulates liver hypertrophy and hyperplasia, and ultimately, hepatocellular carcinogenesis. The mechanisms linking early CAR responses to later disease development are poorly understood. Here we show that exposure of CD-1 mice to TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene), a halogenated xenochemical and selective CAR agonist ligand, induces pericentral steatosis marked by hepatic accumulation of cholesterol and neutral lipid, and elevated circulating alanine aminotransferase, indicating hepatocyte damage. TCPOBOP-induced steatosis was weaker in the pericentral region but stronger in the periportal region in females compared with males. Early (1 day) TCPOBOP transcriptional responses were enriched for CAR-bound primary response genes, and for lipogenesis and xenobiotic metabolism and oxidative stress protection pathways; late (2 weeks) TCPOBOP responses included many CAR binding-independent secondary response genes, with enrichment for macrophage activation, immune response, and cytokine and reactive oxygen species production. Late upstream regulators specific to TCPOBOP-exposed male liver were linked to proinflammatory responses and hepatocellular carcinoma progression. TCPOBOP administered weekly to male mice using a high corn oil vehicle induced carbohydrate-responsive transcription factor (MLXIPL)-regulated target genes, dysregulated mitochondrial respiratory and translation regulatory pathways, and induced more advanced liver pathology. Overall, TCPOBOP exposure recapitulates histological and gene expression changes characteristic of emerging steatotic liver disease, including secondary gene responses in liver nonparenchymal cells indicative of transition to a more advanced disease state. Upstream regulators of both the early and late TCPOBOP response genes include novel biomarkers for foreign chemical-induced metabolic dysfunction-associated steatotic liver disease.
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Affiliation(s)
- Ravi Sonkar
- Department of Biology and Bioinformatics Program, Boston University, Boston, Massachusetts 02215, USA
| | - Hong Ma
- Department of Biology and Bioinformatics Program, Boston University, Boston, Massachusetts 02215, USA
| | - David J Waxman
- Department of Biology and Bioinformatics Program, Boston University, Boston, Massachusetts 02215, USA
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Hafiz MZ, Pan J, Gao Z, Huo Y, Wang H, Liu W, Yang J. Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway. J Biomed Res 2024; 38:382-396. [PMID: 38817007 PMCID: PMC11300519 DOI: 10.7555/jbr.38.20240055] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2024] [Revised: 05/06/2024] [Accepted: 05/10/2024] [Indexed: 06/01/2024] Open
Abstract
The current study aimed to assess the effect of timosaponin AⅢ (T-AⅢ) on drug-metabolizing enzymes during anticancer therapy. The in vivo experiments were conducted on nude and ICR mice. Following a 24-day administration of T-AⅢ, the nude mice exhibited an induction of CYP2B10, MDR1, and CYP3A11 expression in the liver tissues. In the ICR mice, the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration. The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6, MDR1, and CYP3A4, along with constitutive androstane receptor (CAR) activation. Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression. Furthermore, other CAR target genes also showed a significant increase in the expression. The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice. Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation, with this effect being partially reversed by the ERK activator t-BHQ. Inhibition of the ERK1/2 signaling pathway was also observed in vivo. Additionally, T-AⅢinhibited the phosphorylation of EGFR at Tyr1173 and Tyr845, and suppressed EGF-induced phosphorylation of EGFR, ERK, and CAR. In the nude mice, T-AⅢ also inhibited EGFR phosphorylation. These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.
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Affiliation(s)
- Muhammad Zubair Hafiz
- Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211166, China
| | - Jie Pan
- Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211166, China
| | - Zhiwei Gao
- Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211166, China
| | - Ying Huo
- Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211166, China
| | - Haobin Wang
- Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211166, China
| | - Wei Liu
- Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211166, China
| | - Jian Yang
- Department of Pharmacology, Nanjing Medical University, Nanjing, Jiangsu 211166, China
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9
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Liu J, Malekoltojari A, Asokakumar A, Chow V, Li L, Li H, Grimaldi M, Dang N, Campbell J, Barrett H, Sun J, Navarre W, Wilson D, Wang H, Mani S, Balaguer P, Anakk S, Peng H, Krause HM. Diindoles produced from commensal microbiota metabolites function as endogenous CAR/Nr1i3 ligands. Nat Commun 2024; 15:2563. [PMID: 38519460 PMCID: PMC10960024 DOI: 10.1038/s41467-024-46559-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2023] [Accepted: 02/27/2024] [Indexed: 03/25/2024] Open
Abstract
Numerous studies have demonstrated the correlation between human gut bacteria and host physiology, mediated primarily via nuclear receptors (NRs). Despite this body of work, the systematic identification and characterization of microbe-derived ligands that regulate NRs remain a considerable challenge. In this study, we discover a series of diindole molecules produced from commensal bacteria metabolites that act as specific agonists for the orphan constitutive androstane receptor (CAR). Using various biophysical analyses we show that their nanomolar affinities are comparable to those of synthetic CAR agonists, and that they can activate both rodent and human CAR orthologues, which established synthetic agonists cannot. We also find that the diindoles, diindolylmethane (DIM) and diindolylethane (DIE) selectively up-regulate bona fide CAR target genes in primary human hepatocytes and mouse liver without causing significant side effects. These findings provide new insights into the complex interplay between the gut microbiome and host physiology, as well as new tools for disease treatment.
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Affiliation(s)
- Jiabao Liu
- Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 3E1, Canada
| | - Ainaz Malekoltojari
- Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 3E1, Canada
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Anjana Asokakumar
- Department of Molecular and Integrative Physiology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA
| | - Vimanda Chow
- Department of Chemistry, York University, Toronto, ON, M3J 1P3, Canada
| | - Linhao Li
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn Street, Baltimore, MD, 21201, USA
| | - Hao Li
- Department of Molecular Pharmacology; Department of Genetics; Department of Medicine; Albert Einstein College of Medicine, Bronx, NY, 10461, USA
| | - Marina Grimaldi
- Institut de Recherche en Cancérologie de Montpellier (IRCM), Université Montpellier, Institut régional du Cancer de Montpellier (ICM), Montpellier, Inserm, U1194, France
| | - Nathanlown Dang
- Department of Molecular and Integrative Physiology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA
| | - Jhenielle Campbell
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Holly Barrett
- Department of Chemistry, University of Toronto, Toronto, ON, M5S 3H6, Canada
| | - Jianxian Sun
- Department of Chemistry, University of Toronto, Toronto, ON, M5S 3H6, Canada
- School of the Environment, University of Toronto, Toronto, ON, M5S 3H6, Canada
| | - William Navarre
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada
| | - Derek Wilson
- Department of Chemistry, York University, Toronto, ON, M3J 1P3, Canada
| | - Hongbing Wang
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, 20 Penn Street, Baltimore, MD, 21201, USA
| | - Sridhar Mani
- Department of Molecular Pharmacology; Department of Genetics; Department of Medicine; Albert Einstein College of Medicine, Bronx, NY, 10461, USA
| | - Patrick Balaguer
- Institut de Recherche en Cancérologie de Montpellier (IRCM), Université Montpellier, Institut régional du Cancer de Montpellier (ICM), Montpellier, Inserm, U1194, France
| | - Sayeepriyadarshini Anakk
- Department of Molecular and Integrative Physiology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA
| | - Hui Peng
- Department of Chemistry, University of Toronto, Toronto, ON, M5S 3H6, Canada.
- School of the Environment, University of Toronto, Toronto, ON, M5S 3H6, Canada.
| | - Henry M Krause
- Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, M5S 3E1, Canada.
- Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
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Ho YS, Torres-Vergara P, Penny J. Regulation of the ATP-binding cassette transporters ABCB1, ABCG2 and ABCC5 by nuclear receptors in porcine blood-brain barrier endothelial cells. Br J Pharmacol 2023; 180:3092-3109. [PMID: 37476954 DOI: 10.1111/bph.16196] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2022] [Revised: 05/26/2023] [Accepted: 06/22/2023] [Indexed: 07/22/2023] Open
Abstract
BACKGROUND AND PURPOSE Blood-brain barrier (BBB) ABCB1, ABCG2 and ABCC5 transporters influence central therapeutic drug distribution. Transporter expression is regulated by the NR3C1, NR1I3 and NR1I2 nuclear receptors, but their precise roles in brain are poorly understood. We investigated the effects of selective ligand-based activation of NR3C1, NR1I3, NR1I2 and NR2B1 in porcine brain endothelial cells (PBECs). EXPERIMENTAL APPROACH Primary cultures of PBECs were exposed to NR3C1, NR1I3 and NR1I2 ligands and ABCB1, ABCG2 and ABCC5 transporter activities determined by measuring intracellular accumulation of fluorescent probes. Western blotting was used to determine the effects of receptor ligands on expression of ABCB1, ABCG2, ABCC5, NR1I2, NR1I3, NR3C1 and NR2B1. Fluorescent immunocytochemistry was employed to assess the effects of receptor ligands on the cellular localisation of NR1I2 and NR1I3. KEY RESULTS The NR1I2 agonist rifampicin significantly up-regulated ABCG2 activity, which is counteracted by co-treatment with NR1I2 antagonist l-sulforaphane. The NR1I3 agonist 6-(4-chlorophenyl)-imidazo[2,1-b]thiazole-5-carbaldehyde and inverse agonist meclizine significantly down-regulated ABCB1, ABCG2 and ABCC5 activity. NR3C1 agonist dexamethasone significantly increased ABCB1, ABCG2 and ABCC5 activity and ABCG2 and ABCC5 protein expression, which was counteracted by co-treatment with the NR3C1 antagonist mifepristone. This first study demonstrates that NR1I3 and NR3C1 regulate ABCC5 activity and protein expression in BBB endothelial cells. CONCLUSIONS AND IMPLICATIONS In PBECs, expression of key ATP-binding cassette (ABC) transporters and nuclear receptors is differentially regulated by NR1I3, NR1I2, NR3C1 and NR2B1. This will help to better understand the response of the BBB to physiological and pharmacological activation of nuclear receptors.
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Affiliation(s)
- Yu Siong Ho
- Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK
| | - Pablo Torres-Vergara
- Departamento de Farmacia, Facultad de Farmacia, Universidad de Concepción, Concepción, Chile
| | - Jeffrey Penny
- Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, UK
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11
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Oliva-Vilarnau N, Vorrink SU, Büttner FA, Heinrich T, Sensbach J, Koscielski I, Wienke D, Petersson C, Perrin D, Lauschke VM. Comparative analysis of YAP/TEAD inhibitors in 2D and 3D cultures of primary human hepatocytes reveals a novel non-canonical mechanism of CYP induction. Biochem Pharmacol 2023; 215:115755. [PMID: 37607620 DOI: 10.1016/j.bcp.2023.115755] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2023] [Revised: 08/16/2023] [Accepted: 08/16/2023] [Indexed: 08/24/2023]
Abstract
Induction of cytochrome P450 (CYP) genes constitutes an important cause of drug-drug interactions and preclinical evaluation of induction liability is mandatory for novel drug candidates. YAP/TEAD signaling has emerged as an attractive target for various oncological indications and multiple chemically distinct YAP/TEAD inhibitors are rapidly progressing towards clinical stages. Here, we tested the liability for CYP induction of a diverse set of YAP/TEAD inhibitors with different modes of action and TEAD isoform selectivity profiles in monolayers and 3D spheroids of primary human hepatocytes (PHH). We found that YAP/TEAD inhibition resulted in broad induction of CYPs in 2D monolayers, whereas, if at all, only marginal induction was seen in spheroid culture. Comprehensive RNA-Seq indicated that YAP/TEAD signaling was increased in 2D culture compared to spheroids, which was paralleled by elevated activities of the interacting transcription factors LXR and ESRRA, likely at least in part due to altered mechanosensing. Inhibition of this YAP/TEAD hyperactivation resulted in an overall reduction of hepatocyte dedifferentiation marked by increased hepatic functionality, including CYPs. These results thus demonstrate that the observed induction is due to on-target effects of the compounds rather than direct activation of xenobiotic sensing nuclear receptors. Combined, the presented data link hepatocyte dedifferentiation to YAP/TEAD dysregulation, reveal a novel non-canonical pathway of CYP induction and highlight the advantage of organotypic 3D cultures to predict clinically relevant pharmacokinetic properties, particularly for atypical induction mechanisms.
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Affiliation(s)
- Nuria Oliva-Vilarnau
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
| | | | - Florian A Büttner
- Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany; University of Tuebingen, Tuebingen, Germany
| | - Timo Heinrich
- Department of Medicinal Chemistry and Drug Design, The Healthcare Business of Merck KGaA, Darmstadt, Germany
| | - Janike Sensbach
- Department of Chemical and Pre-Clinical Safety, The Healthcare Business of Merck KGaA, Darmstadt, Germany
| | - Isabel Koscielski
- Department of Chemical and Pre-Clinical Safety, The Healthcare Business of Merck KGaA, Darmstadt, Germany
| | - Dirk Wienke
- Department of Drug Metabolism and Pharmacokinetics (DMPK), The Healthcare Business of Merck KGaA, Darmstadt, Germany
| | - Carl Petersson
- Department of Drug Metabolism and Pharmacokinetics (DMPK), The Healthcare Business of Merck KGaA, Darmstadt, Germany
| | - Dominique Perrin
- Department of Drug Metabolism and Pharmacokinetics (DMPK), The Healthcare Business of Merck KGaA, Darmstadt, Germany
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; HepaPredict AB, Stockholm, Sweden; Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany; University of Tuebingen, Tuebingen, Germany.
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12
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Shindo S, Kakizaki S, Sakaki T, Kawasaki Y, Sakuma T, Negishi M, Shizu R. Phosphorylation of nuclear receptors: Novelty and therapeutic implications. Pharmacol Ther 2023; 248:108477. [PMID: 37330113 DOI: 10.1016/j.pharmthera.2023.108477] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Revised: 05/20/2023] [Accepted: 06/06/2023] [Indexed: 06/19/2023]
Abstract
Nuclear receptors (NR) collectively regulate several biological functions in various organs. While NRs can be characterized by activation of the transcription of their signature genes, they also have other diverse roles. Although most NRs are directly activated by ligand binding, which induces cascades of events leading to gene transcription, some NRs are also phosphorylated. Despite extensive investigations, primarily focusing on unique phosphorylation of amino acid residues in different NRs, the role of phosphorylation in the biological activity of NRs in vivo has not been firmly established. Recent studies on the phosphorylation of conserved phosphorylation motifs within the DNA- and ligand-binding domains confirmed has indicated the physiologically relevance of NR phosphorylation. This review focuses on estrogen and androgen receptors, and highlights the concept of phosphorylation as a drug target.
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Affiliation(s)
- Sawako Shindo
- Department of Environmental Toxicology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan
| | - Satoru Kakizaki
- Department of Clinical Research, National Hospital Organization Takasaki General Medical Center, 36 Takamatsu-cho, Takasaki, Gunma 370-0829, Japan
| | - Toshiyuki Sakaki
- Department of Pharmaceutical Engineering, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
| | - Yuki Kawasaki
- Laboratory of Public Health, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaourui-machi, Takasaki, Gunma 370-0033, Japan
| | - Tsutomu Sakuma
- School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611, Japan
| | - Masahiko Negishi
- Reproductive and Developmental Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
| | - Ryota Shizu
- Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
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13
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Murase W, Kubota A, Ikeda-Araki A, Terasaki M, Nakagawa K, Shizu R, Yoshinari K, Kojima H. Effects of perfluorooctanoic acid (PFOA) on gene expression profiles via nuclear receptors in HepaRG cells: Comparative study with in vitro transactivation assays. Toxicology 2023:153577. [PMID: 37302725 DOI: 10.1016/j.tox.2023.153577] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Revised: 05/31/2023] [Accepted: 06/08/2023] [Indexed: 06/13/2023]
Abstract
Perfluorooctanoic acid (PFOA), a synthetic perfluorinated eight-carbon organic chemical, has been reported to induce hepatotoxicity, including increased liver weight, hepatocellular hypertrophy, necrosis, and increased peroxisome proliferation in rodents. Epidemiological studies have demonstrated associations between serum PFOA levels and various adverse effects. In this study, we investigated the gene expression profiles of human HepaRG cells exposed to 10 and 100 μM PFOA for 24h. Treatment with 10 and 100 μM PFOA significantly modulated the expression of 190 genes and 996 genes, respectively. In particular, genes upregulated or downregulated by 100µM PFOA included peroxisome proliferator-activated receptor (PPAR) signaling genes related to lipid metabolism, adipocyte differentiation, and gluconeogenesis. In addition, we identified the "Nuclear receptors-meta pathways" following the activation of other nuclear receptors: constitutive androstane receptor (CAR), pregnane X receptor (PXR) and farnesoid X receptor (FXR), and the transcription factor, nuclear factor E2-related factor 2 (Nrf2). The expression levels of some target genes (CYP4A11, CYP2B6, CYP3A4, CYP7A1, and GPX2) of these nuclear receptors and Nrf2 were confirmed using quantitative reverse transcription polymerase chain reaction. Next, we performed transactivation assays using COS-7 or HEK293 cells to investigate whether these signaling-pathways were activated by the direct effects of PFOA on human PPARα, CAR, PXR, FXR and Nrf2. PFOA activated PPARα in a concentration-dependent manner, but did not activate CAR, PXR, FXR, or Nrf2. Taken together, these results suggest that PFOA affects the hepatic transcriptomic responses of HepaRG cells through direct activation of PPARα and indirect activation of CAR, PXR FXR and Nrf2. Our finding indicates that PPARα activation found in the "Nuclear receptors-meta pathways" functions as a molecular initiating event for PFOA, and indirect activation of alternative nuclear receptors and Nrf2 also provide important molecular mechanisms in PFOA-induced human hepatotoxicity.
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Affiliation(s)
- Wataru Murase
- School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
| | - Atsuhito Kubota
- School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
| | - Atsuko Ikeda-Araki
- Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060-0812, Japan; Center for Environmental and Health Sciences, Hokkaido University, Kita-12, Nishi-7, Kita-ku, Sapporo 060-0812, Japan
| | - Masaru Terasaki
- School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan; Advanced Research Promotion Center, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
| | - Koji Nakagawa
- School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan; Advanced Research Promotion Center, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan
| | - Ryota Shizu
- School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
| | - Kouichi Yoshinari
- School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
| | - Hiroyuki Kojima
- School of Pharmaceutical Sciences, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan; Advanced Research Promotion Center, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan.
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14
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Miners JO, Polasek TM, Hulin JA, Rowland A, Meech R. Drug-drug interactions that alter the exposure of glucuronidated drugs: Scope, UDP-glucuronosyltransferase (UGT) enzyme selectivity, mechanisms (inhibition and induction), and clinical significance. Pharmacol Ther 2023:108459. [PMID: 37263383 DOI: 10.1016/j.pharmthera.2023.108459] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Revised: 05/18/2023] [Accepted: 05/22/2023] [Indexed: 06/03/2023]
Abstract
Drug-drug interactions (DDIs) arising from the perturbation of drug metabolising enzyme activities represent both a clinical problem and a potential economic loss for the pharmaceutical industry. DDIs involving glucuronidated drugs have historically attracted little attention and there is a perception that interactions are of minor clinical relevance. This review critically examines the scope and aetiology of DDIs that result in altered exposure of glucuronidated drugs. Interaction mechanisms, namely inhibition and induction of UDP-glucuronosyltransferase (UGT) enzymes and the potential interplay with drug transporters, are reviewed in detail, as is the clinical significance of known DDIs. Altered victim drug exposure arising from modulation of UGT enzyme activities is relatively common and, notably, the incidence and importance of UGT induction as a DDI mechanism is greater than generally believed. Numerous DDIs are clinically relevant, resulting in either loss of efficacy or an increased risk of adverse effects, necessitating dose individualisation. Several generalisations relating to the likelihood of DDIs can be drawn from the known substrate and inhibitor selectivities of UGT enzymes, highlighting the importance of comprehensive reaction phenotyping studies at an early stage of drug development. Further, rigorous assessment of the DDI liability of new chemical entities that undergo glucuronidation to a significant extent has been recommended recently by regulatory guidance. Although evidence-based approaches exist for the in vitro characterisation of UGT enzyme inhibition and induction, the availability of drugs considered appropriate for use as 'probe' substrates in clinical DDI studies is limited and this should be research priority.
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Affiliation(s)
- John O Miners
- Discipline of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University College of Medicine and Public Health, Flinders University, Adelaide, Australia.
| | - Thomas M Polasek
- Certara, Princeton, NJ, USA; Centre for Medicines Use and Safety, Monash University, Melbourne, Australia
| | - Julie-Ann Hulin
- Discipline of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University College of Medicine and Public Health, Flinders University, Adelaide, Australia
| | - Andrew Rowland
- Discipline of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University College of Medicine and Public Health, Flinders University, Adelaide, Australia
| | - Robyn Meech
- Discipline of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University College of Medicine and Public Health, Flinders University, Adelaide, Australia
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15
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Men S, Wang H. Phenobarbital in Nuclear Receptor Activation: An Update. Drug Metab Dispos 2023; 51:210-218. [PMID: 36351837 PMCID: PMC9900862 DOI: 10.1124/dmd.122.000859] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2022] [Revised: 09/26/2022] [Accepted: 09/27/2022] [Indexed: 11/11/2022] Open
Abstract
Phenobarbital (PB) is a commonly prescribed anti-epileptic drug that can also benefit newborns from hyperbilirubinemia. Being the first drug demonstrating hepatic induction of cytochrome P450 (CYP), PB has since been broadly used as a model compound to study xenobiotic-induced drug metabolism and clearance. Mechanistically, PB-mediated CYP induction is linked to a number of nuclear receptors, such as the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and estrogen receptor α, with CAR being the predominant regulator. Unlike prototypical agonistic ligands, PB-mediated activation of CAR does not involve direct binding with the receptor. Instead, dephosphorylation of threonine 38 in the DNA-binding domain of CAR was delineated as a key signaling event underlying PB-mediated indirect activation of CAR. Further studies revealed that such phosphorylation sites appear to be highly conserved among most human nuclear receptors. Interestingly, while PB is a pan-CAR activator in both animals and humans, PB activates human but not mouse PXR. The species-specific role of PB in gene regulation is a key determinant of its implication in xenobiotic metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In this review, we summarize the recent progress in our understanding of PB-provoked transactivation of nuclear receptors with a focus on CAR and PXR. SIGNIFICANCE STATEMENT: Extensive studies using PB as a research tool have significantly advanced our understanding of the molecular basis underlying nuclear receptor-mediated drug metabolism, drug-drug interactions, energy homeostasis, and cell proliferation. In particular, CAR has been established as a cell signaling-regulated nuclear receptor in addition to ligand-dependent functionality. This mini-review highlights the mechanisms by which PB transactivates CAR and PXR.
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Affiliation(s)
- Shuaiqian Men
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland (S.M., H.W.)
| | - Hongbing Wang
- Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland (S.M., H.W.)
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16
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Abstract
Dehydroepiandrosterone (3β-hydroxy-5-androsten-17-one, DHEA) and its sulfated metabolite DHEA-S are the most abundant circulating steroids and are precursors for active sex steroid hormones, estradiol and testosterone. DHEA has a broad range of reported effects in the central nervous system (CNS), cardiovascular system, adipose tissue, kidney, liver, and in the reproductive system. The mechanisms by which DHEA and DHEA-S initiate their biological effects are diverse. DHEA and DHEA-S may directly bind to plasma membrane (PM) receptors, including a DHEA-specific, G-protein coupled receptor (GPCR) in endothelial cells; various neuroreceptors, e.g., aminobutyric-acid-type A (GABA(A)), N-methyl-d-aspartate (NMDA) and sigma-1 (S1R) receptors (NMDAR and SIG-1R). DHEA and DHEA-S directly bind the nuclear androgen and estrogen receptors (AR, ERα, or ERβ) although with significantly lower binding affinities compared to the steroid hormones, e.g., testosterone, dihydrotestosterone, and estradiol, which are the cognate ligands for AR and ERs. Thus, extra-gonadal metabolism of DHEA to the sex hormones must be considered for many of the biological benefits of DHEA. DHEA also actives GPER1 (G protein coupled estrogen receptor 1). DHEA activates constitutive androstane receptor CAR (CAR) and proliferator activated receptor (PPARα) by indirect dephosphorylation. DHEA affects voltage-gated sodium and calcium ion channels and DHEA-2 activates TRPM3 (Transient Receptor Potential Cation Channel Subfamily M Member 3). This chapter updates our previous 2018 review pertaining to the physiological, biochemical, and molecular mechanisms of DHEA and DHEA-S activity.
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Affiliation(s)
- Barbara J Clark
- Department of Biochemistry & Molecular Genetics, Center for Integrative Environmental Health Sciences (CIEHS), University of Louisville School of Medicine, Louisville, KY, United States
| | - Carolyn M Klinge
- Department of Biochemistry & Molecular Genetics, Center for Integrative Environmental Health Sciences (CIEHS), University of Louisville School of Medicine, Louisville, KY, United States.
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17
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Heintz MM, Eccles JA, Olack EM, Maner-Smith KM, Ortlund EA, Baldwin WS. Human CYP2B6 produces oxylipins from polyunsaturated fatty acids and reduces diet-induced obesity. PLoS One 2022; 17:e0277053. [PMID: 36520866 PMCID: PMC9754190 DOI: 10.1371/journal.pone.0277053] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2022] [Accepted: 10/18/2022] [Indexed: 12/23/2022] Open
Abstract
Multiple factors in addition to over consumption lead to obesity and non-alcoholic fatty liver disease (NAFLD) in the United States and worldwide. CYP2B6 is the only human detoxification CYP whose loss is associated with obesity, and Cyp2b-null mice show greater diet-induced obesity with increased steatosis than wildtype mice. However, a putative mechanism has not been determined. LC-MS/MS revealed that CYP2B6 metabolizes PUFAs, with a preference for metabolism of ALA to 9-HOTrE and to a lesser extent 13-HOTrE with a preference for metabolism of PUFAs at the 9- and 13-positions. To further study the role of CYP2B6 in vivo, humanized-CYP2B6-transgenic (hCYP2B6-Tg) and Cyp2b-null mice were fed a 60% high-fat diet for 16 weeks. Compared to Cyp2b-null mice, hCYP2B6-Tg mice showed reduced weight gain and metabolic disease as measured by glucose tolerance tests, however hCYP2B6-Tg male mice showed increased liver triglycerides. Serum and liver oxylipin metabolite concentrations increased in male hCYP2B6-Tg mice, while only serum oxylipins increased in female hCYP2B6-Tg mice with the greatest increases in LA oxylipins metabolized at the 9 and 13-positions. Several of these oxylipins, specifically 9-HODE, 9-HOTrE, and 13-oxoODE, are PPAR agonists. RNA-seq data also demonstrated sexually dimorphic changes in gene expression related to nuclear receptor signaling, especially CAR > PPAR with qPCR suggesting PPARγ signaling is more likely than PPARα signaling in male mice. Overall, our data indicates that CYP2B6 is an anti-obesity enzyme, but probably to a lesser extent than murine Cyp2b's. Therefore, the inhibition of CYP2B6 by xenobiotics or dietary fats can exacerbate obesity and metabolic disease potentially through disrupted PUFA metabolism and the production of key lipid metabolites.
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Affiliation(s)
- Melissa M. Heintz
- Biological Sciences, Clemson University, Clemson, South Carolina, United States of America
| | - Jazmine A. Eccles
- Biological Sciences, Clemson University, Clemson, South Carolina, United States of America
| | - Emily M. Olack
- Biological Sciences, Clemson University, Clemson, South Carolina, United States of America
| | - Kristal M. Maner-Smith
- Emory Integrated Metabolomics and Lipodomics Core, Emory University, Atlanta, Georgia, United States of America
| | - Eric A. Ortlund
- Department of Biochemistry, Emory University School of Medicine, Emory University, Atlanta, Georgia, United States of America
| | - William S. Baldwin
- Biological Sciences, Clemson University, Clemson, South Carolina, United States of America
- * E-mail:
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18
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Sato T, Shizu R, Miura Y, Hosaka T, Kanno Y, Sasaki T, Yoshinari K. Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats. Food Chem Toxicol 2022; 170:113510. [DOI: 10.1016/j.fct.2022.113510] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2022] [Revised: 10/25/2022] [Accepted: 11/03/2022] [Indexed: 11/09/2022]
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Banerjee B, Olajide OJ, Bortolussi G, Muro AF. Activation of Alternative Bilirubin Clearance Pathways Partially Reduces Hyperbilirubinemia in a Mouse Model Lacking Functional Ugt1a1 Activity. Int J Mol Sci 2022; 23:ijms231810703. [PMID: 36142606 PMCID: PMC9505366 DOI: 10.3390/ijms231810703] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 09/08/2022] [Accepted: 09/11/2022] [Indexed: 11/21/2022] Open
Abstract
Bilirubin is a heme catabolite and Ugt1a1 is the only enzyme involved in the biological elimination of bilirubin. Partially functional or non-functional Ugt1a1 may result in neuronal damage and death due to the accumulation of unconjugated bilirubin in the brain. The understanding of the role of alternative bilirubin detoxification mechanisms that can reduce bilirubin toxicity risk is crucial for developing novel therapeutic strategies. To provide a proof-of-principle showing whether activation of alternative detoxification pathways could lead to life-compatible bilirubin levels in the absence of Ugt1a1 activity, we used Ugt1−/− hyperbilirubinemic mice devoid of bilirubin glucuronidation activity. We treated adult Ugt1−/− mice with TCPOBOP, a strong agonist of the constitutive androstane receptor (CAR). TCPOBOP treatment decreased plasma and liver tissue bilirubin levels by about 38%, and resulted in the transcriptional activation of a vast array of genes involved in bilirubin transport and metabolism. However, brain bilirubin level was unaltered. We observed ~40% degradation of bilirubin in the liver microsomes from TCPOBOP treated Ugt1−/− mice. Our findings suggest that, in the absence of Ugt1a1, the activation of alternative bilirubin clearance pathways can partially improve hyperbilirubinemic conditions. This therapeutic approach may only be considered in a combinatorial manner along with other treatments.
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Proteomic analysis of hepatic effects of phenobarbital in mice with humanized liver. Arch Toxicol 2022; 96:2739-2754. [PMID: 35881160 PMCID: PMC9352639 DOI: 10.1007/s00204-022-03338-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2022] [Accepted: 07/13/2022] [Indexed: 11/25/2022]
Abstract
Activation of the constitutive androstane receptor (CAR) may induce adaptive but also adverse effects in rodent liver, including the induction of drug-metabolizing enzymes, transient hepatocellular proliferation, and promotion of liver tumor growth. Human relevance of CAR-related adverse hepatic effects is controversially debated. Here, we used the chimeric FRG-KO mouse model with livers largely repopulated by human hepatocytes, in order to study human hepatocytes and their response to treatment with the model CAR activator phenobarbital (PB) in vivo. Mice received an intraperitoneal injection with 50 mg/kg body weight PB or saline, and were sacrificed after 72–144 h. Non-repopulated FRG-KO mice were used as additional control. Comprehensive proteomics datasets were generated by merging data obtained by targeted as well as non-targeted proteomics approaches. For the first time, a novel proteomics workflow was established to comparatively analyze the effects of PB on human and murine proteins within one sample. Analysis of merged proteome data sets and bioinformatics data mining revealed comparable responses in murine and human hepatocytes with respect to nuclear receptor activation and induction of xenobiotic metabolism. By contrast, activation of MYC, a key regulator of proliferation, was predicted only for mouse but not human hepatocytes. Analyses of 5-bromo-2′-deoxyuridine incorporation confirmed this finding. In summary, this study for the first time presents a comprehensive proteomic analysis of CAR-dependent effects in human and mouse hepatocytes from humanized FRG-KO mice. The data support the hypothesis that PB does induce adaptive metabolic responses, but not hepatocellular proliferation in human hepatocytes in vivo.
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21
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Guengerich FP. Roles of cytochrome P450 enzymes in pharmacology and toxicology: Past, present, and future. ADVANCES IN PHARMACOLOGY (SAN DIEGO, CALIF.) 2022; 95:1-47. [PMID: 35953152 PMCID: PMC9869358 DOI: 10.1016/bs.apha.2021.12.001] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The development of the cytochrome P450 (P450) field has been remarkable in the areas of pharmacology and toxicology, particularly in drug development. Today it is possible to use the knowledge base and relatively straightforward assays to make intelligent predictions about drug disposition prior to human dosing. Much is known about the structures, regulation, chemistry of catalysis, and the substrate and inhibitor specificity of human P450s. Many aspects of drug-drug interactions and side effects can be understood in terms of P450s. This knowledge has also been useful in pharmacy practice, as well as in the pharmaceutical industry and medical practice. However, there are still basic and practical questions to address regarding P450s and their roles in pharmacology and toxicology. Another aspect is the discovery of drugs that inhibit P450 to treat diseases.
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Affiliation(s)
- F Peter Guengerich
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, United States.
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22
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Zhong XB, Lai Y. Special Section on Drug Metabolism and Regulation-Editorial. Drug Metab Dispos 2022; 50:998-999. [PMID: 35817440 DOI: 10.1124/dmd.122.000925] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2022] [Accepted: 05/17/2022] [Indexed: 11/22/2022] Open
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23
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Honkakoski P. Searching for Constitutive Androstane Receptor Modulators. Drug Metab Dispos 2022; 50:1002-1009. [PMID: 35184042 DOI: 10.1124/dmd.121.000482] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Accepted: 02/01/2022] [Indexed: 02/13/2025] Open
Abstract
The constitutive androstane receptor (CAR; NR1I3) has been established as one of the main drug- and xenobiotic-responsive transcriptional regulators, collectively called xenosensors. CAR activates the expression of several oxidative, hydrolytic, and conjugative drug-metabolizing enzymes and drug transporters, and therefore, it contributes to drug and xenobiotic elimination, drug interactions, and toxicological processes. This minireview introduces mechanisms that modulate CAR activity and focuses on the recent approaches used to search and characterize CAR agonists, inverse agonists, and indirect activators. This minireview is dedicated to Dr. Masahiko Negishi to celebrate his scientific achievements during his long service at the National Institutes of Health. SIGNIFICANCE STATEMENT: Discovery and characterization of human constitutive androstane receptor (CAR) modulators is important for drug development, toxicity studies, and in generation of chemical tools to dissect biological functions of CAR. This minireview focuses on the main methods used to search for these compounds and discusses their essential features.
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Affiliation(s)
- Paavo Honkakoski
- School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio, Finland
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24
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Yoshinari K, Shizu R. Distinct Roles of the Sister Nuclear Receptors PXR and CAR in Liver Cancer Development. Drug Metab Dispos 2022; 50:1019-1026. [PMID: 35184041 DOI: 10.1124/dmd.121.000481] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2021] [Accepted: 02/08/2022] [Indexed: 02/13/2025] Open
Abstract
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 β-catenin signaling, seem to be involved. In contrast to CAR, previous findings by our group suggest that PXR activation does not promote hepatocyte proliferation but it enhances the proliferation induced by various stimuli. Moreover, and surprisingly, PXR may have antitumor effects in both rodents and humans by targeting inflammatory cytokine signals, angiogenesis and epithelial-mesenchymal transition. In this review, we summarize the current knowledge on the associations of PXR and CAR with hepatocyte proliferation and liver tumorigenesis and their molecular mechanisms and species differences. SIGNIFICANCE STATEMENT: Pregnane X receptor and constitutively active receptor/constitutive androstane receptor have very similar functions in the gene regulation associated with xenobiotic disposition, as suggested by their identification as xenosensors for enzyme induction. In contrast, recent reports clearly suggest that these receptors play distinct roles in the control of hepatocyte proliferation and liver cancer development. Understanding these differences at the molecular level may help us evaluate the human safety of chemical compounds and develop novel drugs targeting liver cancers.
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Affiliation(s)
- Kouichi Yoshinari
- Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
| | - Ryota Shizu
- Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
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25
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Chen L, Guo P, Li W, Jiang X, Zhao Q, Li D, Wang Q, Xiao Y, Xing X, Pang Y, Aschner M, Zhang L, Chen W. Protein phosphatase 2A regulates cytotoxicity and drug resistance by dephosphorylating xenobiotic metabolism enzymes AHR and MDR1. J Biol Chem 2022; 298:101918. [PMID: 35405096 PMCID: PMC9118923 DOI: 10.1016/j.jbc.2022.101918] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Revised: 03/26/2022] [Accepted: 03/28/2022] [Indexed: 11/20/2022] Open
Abstract
Protein phosphatase 2A (PP2A) is a serine/threonine dephosphorylating enzyme complex that plays numerous roles in biological processes, including cell growth and metabolism. However, its specific actions in many of these critical pathways are unclear. To explore mechanisms underlying metabolic enzyme regulation in the liver, we investigated the key pathways involved in regulation of xenobiotic-metabolizing enzymes in a mouse model with hepatocyte-specific deletion of Ppp2r1a, encoding the Aα subunit of PP2A. We performed transcriptome and phosphoproteome analysis in mouse livers at the age of 3 months and identified 2695 differentially expressed genes and 549 upregulated phosphoproteins in homozygous knockout mouse livers compared with WT littermates. In particular, the expression of metabolic enzymes Cyp2e1, Cyp1a1, Cyp1a2, Mdr1a, and Abcg2 was dramatically altered in homozygous knockout mouse livers. We also demonstrated that activation of PP2A reversed the decline of metabolic enzyme expression in primary mouse hepatocytes. We found that specific PP2A holoenzymes were involved in metabolic enzyme induction through dephosphorylation of transcription factors, nuclear receptors, or the target enzymes themselves, leading to dysregulation of xenobiotic metabolism or drug-induced hepatotoxicity. Notably, we confirmed that a regulatory axis, PP2A B56α–aryl hydrocarbon receptor–Cyp1a1, was involved in benzo(a)pyrene-induced cytotoxicity through dephosphorylation of the metabolic nuclear receptor, aryl hydrocarbon receptor, at serine 36. In addition, we showed that PP2A B56δ complexes directly dephosphorylated the multidrug efflux pump MDR1 (encoded by multi-drug resistance gene 1), contributing to drug resistance against the chemotherapeutic 5-fluorouracil. Taken together, these novel findings demonstrate the involvement of PP2A in the regulation of liver metabolism.
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Affiliation(s)
- Liping Chen
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Ping Guo
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Wenxue Li
- Department of Toxicology, Guangzhou Center for Disease Control and Prevention, Guangzhou 510440, China
| | - Xinhang Jiang
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Qun Zhao
- Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, Dalian 116023, China
| | - Daochuan Li
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Qing Wang
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Yongmei Xiao
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Xiumei Xing
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China
| | - Yaqin Pang
- Faculty of Toxicology, School of Public Health, Youjiang Medical College for Nationalities, Guangxi 533000, China
| | - Michael Aschner
- Department of Molecular Pharmacology, Albert Einstein College of Medicine, Forchheimer 209, 1300 Morris Park Avenue, Bronx, NY 10461, USA
| | - Lihua Zhang
- Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic Research and Analysis Center, Dalian 116023, China.
| | - Wen Chen
- Guangdong Provincial Key Laboratory of Food, Nutrition and Health, Department of Toxicology, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China.
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26
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Andersen ME, Guerrero T. Assessing Modes of Action, Measures of Tissue Dose and Human Relevance of Rodent Toxicity Endpoints with Octamethylcyclotetrasiloxane (D4). Toxicol Lett 2022; 357:57-72. [PMID: 34995712 DOI: 10.1016/j.toxlet.2021.12.020] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2021] [Revised: 12/07/2021] [Accepted: 12/30/2021] [Indexed: 10/19/2022]
Abstract
Octamethylcyclotetrasiloxane (D4), a highly lipophilic, volatile compound with low water solubility, is metabolized to lower molecular weight, linear silanols. Toxicity has been documented in several tissues in animals following mixed vapor/aerosol exposures by inhalation at near saturating vapor concentrations or with gavage dosing in vegetable oil vehicles. These results, together with more mechanism-based studies and detailed pharmacokinetic information, were used to assess likely modes of action (MOAs) and the tissue dose measures of D4 and metabolites that would serve as key events leading to these biological responses. This MOA analysis indicates that pulmonary effects arise from direct epithelial contact with mixed vapor/aerosol atmospheres of D4; liver hypertrophy and hepatocyte proliferation arise from adaptive, rodent-specific actions of D4 with nuclear receptor signaling pathways; and, nephropathy results from silanol metabolites binding with alpha-2μ globulin (a rat specific protein). At this time, the MOAs of other liver effects - pigment accumulation and bile duct hyperplasia (BDH) preferentially observed in Sprague-Dawley (SD) rats- are not known. Hypothalamic actions of D4 delaying the rat mid-cycle gonadotrophin releasing hormone (GnRH) surge that result in reproductive effects and subsequent vaginal/uterine/ovarian tissue responses, including small increases in incidence of benign endometrial adenomas, are associated with prolongation of endogenous estrogen exposures due to delays in ovulation. Human reproduction is not controlled by a mid-cycle GnRH surge. Since the rodent-specific reproductive and the vaginal/uterine/ovarian tissue responses are not relevant for risk assessments in human populations, D4 should neither be classified as a CMR (i.e., carcinogenic, mutagenic, or toxic for reproduction) substance nor be regarded as an endocrine disruptor. Bile duct hyperplasia (BDH) and pigment accumulation in liver seen in SD rats are endpoints that could serve to define a Benchmark Dose or No-Observed-Effect-Level (NOEL) for D4.
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Affiliation(s)
- Melvin E Andersen
- Andersen ToxConsulting LLC, 424 Granite Lake Ct., Denver, NC 28037, United States.
| | - Tracy Guerrero
- American Chemistry Council Director, Silicones, Environmental, Health, and Safety Center, 700 2nd Street, NE, Washington, DC, 20002, United States.
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27
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Shindo S, Moore R, Yi M, Negishi M. Detection and Functional Analysis of Estrogen Receptor α Phosphorylated at Serine 216 in Mouse Neutrophils. Methods Mol Biol 2022; 2418:63-75. [PMID: 35119660 DOI: 10.1007/978-1-0716-1920-9_5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Serine 216 constitutes a protein kinase C phosphorylation motif located within the DNA binding domain of estrogen receptor α (ERα). In this chapter, we present experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in peripheral blood neutrophils and in neutrophils that infiltrate the uterus, as well as the role of phosphoserine 216 in neutrophil migration. A phospho-peptide antibody (αP-S216) was utilized in Western blot, immunohistochemistry, and double immunofluorescence staining to detect this phosphorylation of an endogenous ERα. Both immunohistochemistry (with αP-S216 or neutrophil marker Ly6G antibody) and double immunofluorescence staining of mouse uterine sections prepared from C3H/HeNCrIBR females revealed that phosphorylated ERα was expressed in all infiltrating neutrophils during hormonal cycles but not in any other of the other uterine cells. Neutrophils infiltrate the uterus from the bloodstream. White blood cells (WBC) were prepared from peripheral blood of C3H/HeNCrIBR females or males and double immunostained. Blood neutrophils also expressed phosphorylated ERα but in only about 20% of cells in both sexes. Only the neutrophils expressing phosphorylated ERα spontaneously migrated in in vitro Transwell migration assays and infiltrated the uterus in mice.
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Affiliation(s)
- Sawako Shindo
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA
- Department of Environmental Health, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Sendai, Japan
| | - Rick Moore
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA
| | - MyeongJin Yi
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA.
| | - Masahiko Negishi
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Durham, NC, USA
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28
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Lai B, Xie X, Li F, Cui Q, Dang E, Luo W, Wang N, Zheng Y, Wang G, Xiao L, Wang N. Xenobiotic Receptor CAR Is Highly Induced in Psoriasis and Promotes Keratinocyte Proliferation. J Invest Dermatol 2021; 141:2895-2907.e7. [PMID: 34097921 DOI: 10.1016/j.jid.2021.05.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 05/04/2021] [Accepted: 05/06/2021] [Indexed: 11/29/2022]
Abstract
Psoriasis is a chronic inflammatory skin disease with abnormal epidermal proliferation. Xenobiotics contribute to the pathogenesis of psoriasis. The mechanism linking xenobiotic stimuli with epidermal proliferation remains largely unknown. In this study, we investigated the role of CAR, a nuclear receptor (NR1I3) responsible for xenobiotics detoxification. We showed that CAR and its target genes were induced in the lesions from patients with psoriasis and imiquimod-treated mice. Proinflammatory cytokines (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α) synergistically increased the expressions of CAR and its target genes in both human and mouse keratinocytes. Overexpression of CAR promoted the G1/S transition by regulating cyclin E and c-Myc expressions, whereas the silencing of CAR attenuated it. Importantly, a selective CAR agonist 6-(4-chlorophenyl)imidazo(2,1-b)(1,3)thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime or the proinflammatory cytokines induced cyclin E and c-Myc, which were largely blocked by clotrimazole, a selective CAR antagonist, or CAR small interfering RNA. In addition, we showed that topical application of 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective agonist for mouse CAR, exacerbated the IMQ-induced psoriasis lesions with increased expressions of proliferative and inflammatory markers. In contrast, Car-knockout mice developed significantly milder lesions. In conclusion, these results showed that CAR plays a pathogenic role and, potentially, may be a target for the treatment of psoriasis.
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Affiliation(s)
- Baochang Lai
- Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, China
| | - Xinya Xie
- Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, China
| | - Fan Li
- Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, China
| | - Qi Cui
- Advanced Institute for Medical Sciences, Dalian Medical University, Dalian, China
| | - Erle Dang
- Department of Dermatology, Xijing Hospital, Air Force Medical University, Xi'an, China
| | - Wenhuan Luo
- Advanced Institute for Medical Sciences, Dalian Medical University, Dalian, China
| | - Ning Wang
- Department of Dermatology, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China
| | - Yan Zheng
- Department of Dermatology, The Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, China
| | - Gang Wang
- Department of Dermatology, Xijing Hospital, Air Force Medical University, Xi'an, China
| | - Lei Xiao
- Cardiovascular Research Center, School of Basic Medical Sciences, Xi'an Jiaotong University, Xi'an, China; Key Laboratory of Environment and Genes Related to Diseases, Xi'an Jiaotong University, Ministry of Education of China, Xi'an, China.
| | - Nanping Wang
- Advanced Institute for Medical Sciences, Dalian Medical University, Dalian, China
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29
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Cytochrome P450 Enzymes and Drug Metabolism in Humans. Int J Mol Sci 2021; 22:ijms222312808. [PMID: 34884615 PMCID: PMC8657965 DOI: 10.3390/ijms222312808] [Citation(s) in RCA: 400] [Impact Index Per Article: 100.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Revised: 11/24/2021] [Accepted: 11/24/2021] [Indexed: 01/07/2023] Open
Abstract
Human cytochrome P450 (CYP) enzymes, as membrane-bound hemoproteins, play important roles in the detoxification of drugs, cellular metabolism, and homeostasis. In humans, almost 80% of oxidative metabolism and approximately 50% of the overall elimination of common clinical drugs can be attributed to one or more of the various CYPs, from the CYP families 1–3. In addition to the basic metabolic effects for elimination, CYPs are also capable of affecting drug responses by influencing drug action, safety, bioavailability, and drug resistance through metabolism, in both metabolic organs and local sites of action. Structures of CYPs have recently provided new insights into both understanding the mechanisms of drug metabolism and exploiting CYPs as drug targets. Genetic polymorphisms and epigenetic changes in CYP genes and environmental factors may be responsible for interethnic and interindividual variations in the therapeutic efficacy of drugs. In this review, we summarize and highlight the structural knowledge about CYPs and the major CYPs in drug metabolism. Additionally, genetic and epigenetic factors, as well as several intrinsic and extrinsic factors that contribute to interindividual variation in drug response are also reviewed, to reveal the multifarious and important roles of CYP-mediated metabolism and elimination in drug therapy.
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30
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Kawase A, Mukai H, Tateishi S, Kuroda S, Kazaoka A, Satoh R, Shimada H, Sugiura R, Iwaki M. Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice. J Pharmacol Exp Ther 2021; 379:53-63. [PMID: 34312179 DOI: 10.1124/jpet.121.000790] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2021] [Accepted: 07/22/2021] [Indexed: 01/04/2023] Open
Abstract
In receptor-type transcription factors-mediated cytochrome P450 (P450) induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels and metabolic activity of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A; PKN3 -/-] were determined after treatment with activators for receptor-type transcription factors. mRNA and protein levels and metabolic activity of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB) but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene compared with WT mice. We examined the CAR-dependent pathway regulated by PKN after PB treatment because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with Src kinase inhibitor 1 (SKI-1) or U0126 (a mitogen-activated protein kinase inhibitor) were evaluated. Treatment of hepatocytes from D mice with the combination of PB with U0126 but not SKI-1 significantly increased the mRNA levels of Cyp2b10 compared with those from the corresponding WT mice. These findings suggest that PKN may have inhibitory effects on the Src-receptor for activated C kinase 1 (RACK1) pathway in the CAR-mediated induction of Cyp2b10 in mice livers. SIGNIFICANCE STATEMENT: This is the first report of involvement of PKN in the transcriptional regulation of P450s. The elucidation of mechanisms responsible for induction of P450s could help optimize the pharmacotherapy and improve drug development. We examined whether the mRNA and protein levels and activities of P450s were altered in double-mutant mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations. PKN1/3 negatively regulates CAR-mediated induction of Cyp2b10 through phosphorylation of a signaling molecule in the Src-RACK1 pathway.
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Affiliation(s)
- Atsushi Kawase
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Hideyuki Mukai
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Shunsuke Tateishi
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Shintaro Kuroda
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Akira Kazaoka
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Ryosuke Satoh
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Hiroaki Shimada
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Reiko Sugiura
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
| | - Masahiro Iwaki
- Department of Pharmacy, Faculty of Pharmacy, Kindai University, Osaka, Japan (A.Kaw., S.T., S.K., A.Kaz., H.S., M.I.); Biosignal Research Center, Kobe University, Hyogo, Japan (H.M.); Department of Clinical Laboratory, Kitano Hospital, Osaka, Japan (H.M.); Department of Pharmaceutical Sciences, Faculty of Pharmacy, Kindai University, Osaka, Japan (R.Sa., R.Su.); Pharmaceutical Research and Technology Institute, Kindai University, Osaka, Japan (R.Su., M.I.); and Antiaging Center, Kindai University, Osaka, Japan (R.Su., M.I.)
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31
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Preiss LC, Liu R, Hewitt P, Thompson D, Georgi K, Badolo L, Lauschke VM, Petersson C. Deconvolution of Cytochrome P450 Induction Mechanisms in HepaRG Nuclear Hormone Receptor Knockout Cells. Drug Metab Dispos 2021; 49:668-678. [PMID: 34035124 DOI: 10.1124/dmd.120.000333] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Accepted: 05/10/2021] [Indexed: 12/11/2022] Open
Abstract
Pregnane X receptor (PXR), constitutive androstane receptor (CAR), and PXR/CAR knockout (KO) HepaRG cells, as well as a PXR reporter gene assay, were used to investigate the mechanism of CYP3A4 and CYP2B6 induction by prototypical substrates and a group of compounds from the Merck KGaA oncology drug discovery pipeline. The basal and inducible gene expression of CYP3A4 and CYP2B6 of nuclear hormone receptor (NHR) KO HepaRG relative to control HepaRG was characterized. The basal expression of CYP3A4 was markedly higher in the PXR (10-fold) and CAR (11-fold) KO cell lines compared with control HepaRG, whereas inducibility was substantially lower. Inversely, basal expression of CYP3A4 in PXR/CAR double KO (dKO) was low (10-fold reduction). Basal CYP2B6 expression was high in PXR KO (9-fold) cells which showed low inducibility, whereas the basal expression remained unchanged in CAR and dKO cell lines compared with control cells. Most of the test compounds induced CYP3A4 and CYP2B6 via PXR and, to a lesser extent, via CAR. Furthermore, other non-NHR-driven induction mechanisms were implicated, either alone or in addition to NHRs. Notably, 5 of the 16 compounds (31%) that were PXR inducers in HepaRG did not activate PXR in the reporter gene assay, illustrating the limitations of this system. This study indicates that HepaRG is a highly sensitive system fit for early screening of cytochrome P450 (P450) induction in drug discovery. Furthermore, it shows the applicability of HepaRG NHR KO cells as tools to deconvolute mechanisms of P450 induction using novel compounds representative for oncology drug discovery. SIGNIFICANCE STATEMENT: This work describes the identification of induction mechanisms of CYP3A4 and CYP2B6 for an assembly of oncology drug candidates using HepaRG nuclear hormone receptor knockout and displays its advantages compared to a pregnane X receptor reporter gene assay. With this study, risk assessment of drug candidates in early drug development can be improved.
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Affiliation(s)
- Lena C Preiss
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
| | - Ruoqi Liu
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
| | - Philip Hewitt
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
| | - David Thompson
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
| | - Katrin Georgi
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
| | - Lassina Badolo
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
| | - Volker M Lauschke
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
| | - Carl Petersson
- Departments of Drug Metabolism and Pharmacokinetics (L.C.P., R.L., K.G., L.B., C.P.) and Early Chemical and Preclinical Safety (P.H.), Merck KGaA, Darmstadt, Germany; Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (L.C.P., V.M.L.); and Research & Development, In Vitro Safety Systems, MilliporeSigma, St. Louis, Missouri (D.T.)
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Song Y, Li C, Liu G, Liu R, Chen Y, Li W, Cao Z, Zhao B, Lu C, Liu Y. Drug-Metabolizing Cytochrome P450 Enzymes Have Multifarious Influences on Treatment Outcomes. Clin Pharmacokinet 2021; 60:585-601. [PMID: 33723723 DOI: 10.1007/s40262-021-01001-5] [Citation(s) in RCA: 53] [Impact Index Per Article: 13.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/09/2021] [Indexed: 02/06/2023]
Abstract
Drug metabolism is a critical process for the removal of unwanted substances from the body. In humans, approximately 80% of oxidative metabolism and almost 50% of the overall elimination of commonly used drugs can be attributed to one or more of various cytochrome P450 (CYP) enzymes from CYP families 1-3. In addition to the basic metabolic effects for elimination, CYP enzymes in vivo are capable of affecting the treatment outcomes in many cases. Drug-metabolizing CYP enzymes are mainly expressed in the liver and intestine, the two principal drug oxidation and elimination organs, where they can significantly influence the drug action, safety, and bioavailability by mediating phase I metabolism and first-pass metabolism. Furthermore, CYP-mediated local drug metabolism in the sites of action may also have the potential to impact drug response, according to the literature in recent years. This article underlines the ability of CYP enzymes to influence treatment outcomes by discussing CYP-mediated diversified drug metabolism in primary metabolic sites (liver and intestine) and typical action sites (brain and tumors) according to their expression levels and metabolic activity. Moreover, intrinsic and extrinsic factors of personal differential CYP phenotypes that contribute to interindividual variation of treatment outcomes are also reviewed to introduce the multifarious pivotal role of CYP-mediated metabolism and clearance in drug therapy.
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Affiliation(s)
- Yurong Song
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China
| | - Chenxi Li
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China
| | - Guangzhi Liu
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China
| | - Rui Liu
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China
| | - Youwen Chen
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China
| | - Wen Li
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China
| | - Zhiwen Cao
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China
| | - Baosheng Zhao
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China.
| | - Cheng Lu
- Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
| | - Yuanyan Liu
- School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 100029, China.
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Hardesty JE, Wahlang B, Prough RA, Head KZ, Wilkey D, Merchant M, Shi H, Jin J, Cave MC. Effect of Epidermal Growth Factor Treatment and Polychlorinated Biphenyl Exposure in a Dietary-Exposure Mouse Model of Steatohepatitis. ENVIRONMENTAL HEALTH PERSPECTIVES 2021; 129:37010. [PMID: 33788613 PMCID: PMC8011667 DOI: 10.1289/ehp8222] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/05/2020] [Revised: 02/26/2021] [Accepted: 03/03/2021] [Indexed: 05/12/2023]
Abstract
BACKGROUND Polychlorinated biphenyls (PCBs) are signaling disrupting chemicals that exacerbate nonalcoholic steatohepatitis (NASH) in mice. They are epidermal growth factor receptor (EGFR) inhibitors that enhance hepatic inflammation and fibrosis in mice. OBJECTIVES This study tested the hypothesis that epidermal growth factor (EGF) administration can attenuate PCB-related NASH by increasing hepatic EGFR signaling in a mouse model. METHODS C57BL/6 male mice were fed a 42% milk fat diet and exposed to Aroclor 1260 (20 mg / kg ) or vehicle for 12 wk. EGF (0.2 μ g / g ) or vehicle were administered daily for 10 d starting at study week 10. Liver and metabolic phenotyping were performed. The EGF dose was selected based on results of an acute dose-finding study (30 min treatment of EGF at 0.2, 0.02, 0.002 μ g / g of via intraperitoneal injection). Hepatic phosphoproteomic analysis was performed using liver tissue from this acute study to understand EGFR's role in liver physiology. RESULTS Markers of EGFR signaling were higher in EGF-treated mice. EGF + PCB -exposed mice had lower hepatic free fatty acids, inflammation, and fibrosis relative to PCB-only exposed mice. EGF-treated mice had higher plasma lipids, with no improvement in hepatic steatosis, and an association with higher LXR target gene expression and de novo lipogenesis. EGF-treated mice showed more severe hyperglycemia associated with lower adiponectin levels and insulin sensitivity. EGF-treated mice had higher hepatic HNF 4 α , NRF2, and AhR target gene expression but lower constitutive androstane receptor and farnesoid X receptor target gene expression. The hepatic EGF-sensitive phosphoproteome demonstrated a role for EGFR signaling in liver homeostasis. DISCUSSION These results validated EGFR inhibition as a causal mode of action for PCB-related hepatic inflammation and fibrosis in a mouse model of NASH. However, observed adverse effects may limit the clinical translation of EGF therapy. More data are required to better understand EGFR's underinvestigated roles in liver and environmental health. https://doi.org/10.1289/EHP8222.
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Affiliation(s)
- Josiah E. Hardesty
- Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, School of Medicine, University of Louisville, Louisville, Kentucky, USA
| | - Banrida Wahlang
- Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- University of Louisville Superfund Research Center, University of Louisville, Louisville, Kentucky, USA
| | - Russell A. Prough
- Department of Biochemistry and Molecular Genetics, School of Medicine, University of Louisville, Louisville, Kentucky, USA
| | - Kim Z. Head
- Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- The Animal Model and Biorepository Core of the Hepatobiology and Toxicology Center, University of Louisville, Louisville, Kentucky, USA
| | - Daniel Wilkey
- University of Louisville Superfund Research Center, University of Louisville, Louisville, Kentucky, USA
- Division of Nephrology and Hypertension, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- The ’Omics Core of the Hepatobiology and Toxicology Center, University of Louisville, Louisville, Kentucky, USA
| | - Michael Merchant
- University of Louisville Superfund Research Center, University of Louisville, Louisville, Kentucky, USA
- Division of Nephrology and Hypertension, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- The ’Omics Core of the Hepatobiology and Toxicology Center, University of Louisville, Louisville, Kentucky, USA
| | - Hongxue Shi
- Department of Pharmacology & Toxicology, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- Department of Medicine, Columbia University Irving Medical Center, New York, New York, USA
| | - Jian Jin
- Department of Pharmacology & Toxicology, School of Medicine, University of Louisville, Louisville, Kentucky, USA
| | - Matthew C. Cave
- Division of Gastroenterology, Hepatology, and Nutrition, Department of Medicine, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- University of Louisville Superfund Research Center, University of Louisville, Louisville, Kentucky, USA
- Department of Biochemistry and Molecular Genetics, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- The Animal Model and Biorepository Core of the Hepatobiology and Toxicology Center, University of Louisville, Louisville, Kentucky, USA
- Department of Pharmacology & Toxicology, School of Medicine, University of Louisville, Louisville, Kentucky, USA
- The Robley Rex Veterans Affairs Medical Center, U.S. Department of Veterans Affairs, Louisville, Kentucky, USA
- The Liver Transplant Program, Jewish Hospital Trager Transplant Center, UofL Health, Louisville, Kentucky, USA
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Juang SH, Hsieh MT, Hsu PL, Chen JL, Liu HK, Liang FP, Kuo SC, Chiu CY, Liu SH, Chou CH, Wu TS, Hung HY. Studies of Coumarin Derivatives for Constitutive Androstane Receptor (CAR) Activation. Molecules 2020; 26:164. [PMID: 33396516 PMCID: PMC7796031 DOI: 10.3390/molecules26010164] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2020] [Revised: 12/25/2020] [Accepted: 12/28/2020] [Indexed: 11/22/2022] Open
Abstract
Constitutive androstane receptor (CAR) activation has found to ameliorate diabetes in animal models. However, no CAR agonists are available clinically. Therefore, a safe and effective CAR activator would be an alternative option. In this study, sixty courmarin derivatives either synthesized or purified from Artemisia capillaris were screened for CAR activation activity. Chemical modifications were on position 5,6,7,8 with mono-, di-, tri-, or tetra-substitutions. Among all the compounds subjected for in vitro CAR activation screening, 6,7-diprenoxycoumarin was the most effective and was selected for further preclinical studies. Chemical modification on the 6 position and unsaturated chains were generally beneficial. Electron-withdrawn groups as well as long unsaturated chains were hazardous to the activity. Mechanism of action studies showed that CAR activation of 6,7-diprenoxycoumarin might be through the inhibition of EGFR signaling and upregulating PP2Ac methylation. To sum up, modification mimicking natural occurring coumarins shed light on CAR studies and the established screening system provides a rapid method for the discovery and development of CAR activators. In addition, one CAR activator, scoparone, did showed anti-diabetes effect in db/db mice without elevation of insulin levels.
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Affiliation(s)
- Shin-Hun Juang
- School of Pharmacy, China Medical University, Taichung 404, Taiwan; (S.-H.J.); (M.-T.H.); (P.-L.H.); (J.-L.C.); (F.-P.L.); (S.-C.K.)
| | - Min-Tsang Hsieh
- School of Pharmacy, China Medical University, Taichung 404, Taiwan; (S.-H.J.); (M.-T.H.); (P.-L.H.); (J.-L.C.); (F.-P.L.); (S.-C.K.)
- Chinese Medicine Research and Development Center, China Medical University Hospital, 2 Yude Road, Taichung 404, Taiwan
| | - Pei-Ling Hsu
- School of Pharmacy, China Medical University, Taichung 404, Taiwan; (S.-H.J.); (M.-T.H.); (P.-L.H.); (J.-L.C.); (F.-P.L.); (S.-C.K.)
| | - Ju-Ling Chen
- School of Pharmacy, China Medical University, Taichung 404, Taiwan; (S.-H.J.); (M.-T.H.); (P.-L.H.); (J.-L.C.); (F.-P.L.); (S.-C.K.)
- School of Pharmacy, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; (C.-H.C.); (T.-S.W.)
| | - Hui-Kang Liu
- National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112, Taiwan;
| | - Fong-Pin Liang
- School of Pharmacy, China Medical University, Taichung 404, Taiwan; (S.-H.J.); (M.-T.H.); (P.-L.H.); (J.-L.C.); (F.-P.L.); (S.-C.K.)
| | - Sheng-Chu Kuo
- School of Pharmacy, China Medical University, Taichung 404, Taiwan; (S.-H.J.); (M.-T.H.); (P.-L.H.); (J.-L.C.); (F.-P.L.); (S.-C.K.)
| | - Chen-Yuan Chiu
- Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; (C.-Y.C.); (S.-H.L.)
| | - Shing-Hwa Liu
- Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei 100, Taiwan; (C.-Y.C.); (S.-H.L.)
| | - Chen-Hsi Chou
- School of Pharmacy, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; (C.-H.C.); (T.-S.W.)
| | - Tian-Shung Wu
- School of Pharmacy, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; (C.-H.C.); (T.-S.W.)
| | - Hsin-Yi Hung
- School of Pharmacy, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan; (C.-H.C.); (T.-S.W.)
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Pande P, Zhong XB, Ku WW. Histone Methyltransferase G9a Regulates Expression of Nuclear Receptors and Cytochrome P450 Enzymes in HepaRG Cells at Basal Level and in Fatty Acid Induced Steatosis. Drug Metab Dispos 2020; 48:1321-1329. [PMID: 33077425 DOI: 10.1124/dmd.120.000195] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Accepted: 09/11/2020] [Indexed: 02/13/2025] Open
Abstract
Obesity and nonalcoholic fatty liver disease (NAFLD) affect expression and function of cytochrome P450 genes (P450s). The increased expression of inflammatory cytokines is a major driver of the downregulation of P450 expression in NAFLD. Decrease in P450 expression could potentially lead to drug-drug interaction, inefficient pharmacological effect of a drug, or hepatotoxicity. An epigenetic modifier, histone 3 lysine 9 methyl transferase enzyme (G9a), known to increase histone 3 lysine 9 methylation, is downregulated in diet-induced obesity animal models. In a liver-specific G9a knockout animal model, expression of P450s was downregulated. Currently, the role of G9a in regulation of P450s in steatosis is unknown. Our hypothesis is that in steatosis G9a plays a role in downregulation of P450 expression. In this study, we used HepaRG cells to induce steatosis using a combination of free fatty acids oleic acid and palmitic acid. The G9a was knocked down and overexpressed using small interfering RNA and adenovirus mediated approaches, respectively. Knockdown and overexpression of G9a in the absence of steatosis decreased and increased expression of nuclear receptors constitutive androstane receptor (CAR), pregnane X receptor, small heterodimer partner, and CYP2B6, 2E1, 2C8, 2C9, and 3A4, respectively. In steatotic conditions, overexpression of G9a prevented fatty acid mediated decreased expression of CAR, CYP2C19, 2C8, 7A1, and 3A4. Our current study suggests that G9a might serve as a key regulator of P450 expression at both the basal level and in early steatotic conditions. Single nucleotide polymorphism of G9a leading to loss/gain of function could lead to the poor metabolizer or ultrarapid metabolizer phenotypes. SIGNIFICANCE STATEMENT: The current study demonstrates that histone modification enzyme G9a is involved in the regulation of expression of nuclear receptors constitutive androstane receptor, pregnane X receptor, and small heterodimer partner as well as drug-metabolizing cytochrome P450s (P450s) at basal conditions and in fatty acid induced cellular model of steatosis. Histone 3 lysine 9 methylation should be considered together with histone 3 lysine 4 and histone 3 lysine 27 methylation as the epigenetic mechanisms controlling gene expression of P450s.
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Affiliation(s)
- Parimal Pande
- Non-Clinical Drug Safety, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut (P.P., W.W.K.) and Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut (P.P., X.-b.Z.)
| | - Xiao-Bo Zhong
- Non-Clinical Drug Safety, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut (P.P., W.W.K.) and Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut (P.P., X.-b.Z.)
| | - Warren W Ku
- Non-Clinical Drug Safety, Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut (P.P., W.W.K.) and Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut (P.P., X.-b.Z.)
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Skoda J, Dusek J, Drastik M, Stefela A, Dohnalova K, Chalupsky K, Smutny T, Micuda S, Gerbal-Chaloin S, Pavek P. Diazepam Promotes Translocation of Human Constitutive Androstane Receptor (CAR) via Direct Interaction with the Ligand-Binding Domain. Cells 2020; 9:cells9122532. [PMID: 33255185 PMCID: PMC7761063 DOI: 10.3390/cells9122532] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Revised: 10/07/2020] [Accepted: 11/20/2020] [Indexed: 11/17/2022] Open
Abstract
The constitutive androstane receptor (CAR) is the essential regulator of genes involved both in xenobiotic and endobiotic metabolism. Diazepam has been shown as a potent stimulator of CAR nuclear translocation and is assumed as an indirect CAR activator not interacting with the CAR cavity. In this study, we sought to determine if diazepam is a ligand directly interacting with the CAR ligand binding domain (LBD) and if it regulates its target genes in a therapeutically relevant concentration. We used different CAR constructs in translocation and luciferase reporter assays, recombinant CAR-LBD in a TR-FRET assay, and target genes induction studied in primary human hepatocytes (PHHs), HepaRG cells, and in CAR humanized mice. We also used in silico docking and CAR-LBD mutants to characterize the interaction of diazepam and its metabolites with the CAR cavity. Diazepam and its metabolites such as nordazepam, temazepam, and oxazepam are activators of CAR+Ala in translocation and two-hybrid assays and fit the CAR cavity in docking experiments. In gene reporter assays with CAR3 and in the TR-FRET assay, only diazepam significantly interacts with CAR-LBD. Diazepam also promotes up-regulation of CYP2B6 in PHHs and in HepaRG cells. However, in humanized CAR mice, diazepam significantly induces neither CYP2B6 nor Cyp2b10 genes nor does it regulate critical genes involved in glucose and lipids metabolism and liver proliferation. Thus, we demonstrate that diazepam interacts with human CAR-LBD as a weak ligand, but it does not significantly affect expression of tested CAR target genes in CAR humanized mice.
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Affiliation(s)
- Josef Skoda
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic; (J.S.); (J.D.); (A.S.); (T.S.)
| | - Jan Dusek
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic; (J.S.); (J.D.); (A.S.); (T.S.)
| | - Martin Drastik
- Department of Physical Chemistry and Biophysics, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic;
| | - Alzbeta Stefela
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic; (J.S.); (J.D.); (A.S.); (T.S.)
| | - Klara Dohnalova
- 1 Medical Faculty, Charles University, Katerinská 32, 121 08 Prague, Czech Republic;
- Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic;
| | - Karel Chalupsky
- Czech Centre for Phenogenomics, Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic;
| | - Tomas Smutny
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic; (J.S.); (J.D.); (A.S.); (T.S.)
| | - Stanislav Micuda
- Department of Pharmacology, Medical Faculty in Hradec Kralove, Charles University, Simkova 870, 500 03 Hradec Kralove, Czech Republic;
| | | | - Petr Pavek
- Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic; (J.S.); (J.D.); (A.S.); (T.S.)
- Correspondence: ; Tel.: +420-495-067-334
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Braeuning A, Pavek P. β-catenin signaling, the constitutive androstane receptor and their mutual interactions. Arch Toxicol 2020; 94:3983-3991. [PMID: 33097968 PMCID: PMC7655584 DOI: 10.1007/s00204-020-02935-8] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2020] [Accepted: 10/08/2020] [Indexed: 12/24/2022]
Abstract
Aberrant signaling through β-catenin is an important determinant of tumorigenesis in rodents as well as in humans. In mice, xenobiotic activators of the constitutive androstane receptor (CAR), a chemo-sensing nuclear receptor, promote liver tumor growth by means of a non-genotoxic mechanism and, under certain conditions, select for hepatocellular tumors which contain activated β-catenin. In normal hepatocytes, interactions of β-catenin and CAR have been demonstrated with respect to the induction of proliferation and drug metabolism-related gene expression. The molecular details of these interactions are still not well understood. Recently it has been hypothesized that CAR might activate β-catenin signaling, thus providing a possible explanation for some of the observed phenomena. Nonetheless, many aspects of the molecular interplay of the two regulators have still not been elucidated. This review briefly summarizes our current knowledge about the interplay of CAR and β-catenin. By taking into account data and observations obtained with different mouse models and employing different experimental approaches, it is shown that published data also contain substantial evidence that xenobiotic activators of CAR do not activate, or do even inhibit signaling through the β-catenin pathway. The review highlights new aspects of possible ways of interaction between the two signaling cascades and will help to stimulate scientific discussion about the crosstalk of β-catenin signaling and the nuclear receptor CAR.
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Affiliation(s)
- Albert Braeuning
- Department Food Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Str. 8-10, 10589, Berlin, Germany.
| | - Petr Pavek
- Department of Pharmacology and Toxicology, Charles University, Faculty of Pharmacy, Heyrovskeho 1203, Hradec Kralove, 500 05, Prague, Czech Republic
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Küblbeck J, Niskanen J, Honkakoski P. Metabolism-Disrupting Chemicals and the Constitutive Androstane Receptor CAR. Cells 2020; 9:E2306. [PMID: 33076503 PMCID: PMC7602645 DOI: 10.3390/cells9102306] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2020] [Revised: 10/13/2020] [Accepted: 10/13/2020] [Indexed: 02/07/2023] Open
Abstract
During the last two decades, the constitutive androstane receptor (CAR; NR1I3) has emerged as a master activator of drug- and xenobiotic-metabolizing enzymes and transporters that govern the clearance of both exogenous and endogenous small molecules. Recent studies indicate that CAR participates, together with other nuclear receptors (NRs) and transcription factors, in regulation of hepatic glucose and lipid metabolism, hepatocyte communication, proliferation and toxicity, and liver tumor development in rodents. Endocrine-disrupting chemicals (EDCs) constitute a wide range of persistent organic compounds that have been associated with aberrations of hormone-dependent physiological processes. Their adverse health effects include metabolic alterations such as diabetes, obesity, and fatty liver disease in animal models and humans exposed to EDCs. As numerous xenobiotics can activate CAR, its role in EDC-elicited adverse metabolic effects has gained much interest. Here, we review the key features and mechanisms of CAR as a xenobiotic-sensing receptor, species differences and selectivity of CAR ligands, contribution of CAR to regulation hepatic metabolism, and evidence for CAR-dependent EDC action therein.
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Affiliation(s)
- Jenni Küblbeck
- A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, P.O. Box 1627, FI-70210 Kuopio, Finland;
- School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70210 Kuopio, Finland;
| | - Jonna Niskanen
- School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70210 Kuopio, Finland;
| | - Paavo Honkakoski
- School of Pharmacy, University of Eastern Finland, P.O. Box 1627, FI-70210 Kuopio, Finland;
- Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Campus Box 7569, Chapel Hill, NC 27599-7569, USA
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Noncanonical Constitutive Androstane Receptor Signaling in Gene Regulation. Int J Mol Sci 2020; 21:ijms21186735. [PMID: 32937916 PMCID: PMC7555422 DOI: 10.3390/ijms21186735] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2020] [Revised: 09/04/2020] [Accepted: 09/10/2020] [Indexed: 11/17/2022] Open
Abstract
The constitutive androstane receptor (CAR, NR1I3) is extremely important for the regulation of many physiological processes, especially xenobiotic (drug) metabolism and transporters. CAR differs from steroid hormone receptors in that it can be activated using structurally unrelated chemicals, both through direct ligand-binding and ligand-independent (indirect) mechanisms. By binding to specific responsive elements on DNA, CAR increases the expression of its target genes encoding drug-metabolizing enzymes and transporters. Therefore, CAR is mainly characterized as a ligand-dependent or ligand-independent transcription factor, and the induction of gene expression is considered the canonical mode of CAR action. Consistent with its central role in xenobiotic metabolism, CAR signaling includes a collection of mechanisms that are employed alongside the core transcriptional machinery of the receptor. These so-called noncanonical CAR pathways allow the receptor to coordinate the regulation of many aspects of cell biology. In this mini-review, we review noncanonical CAR signaling, paying special attention to the role of CAR in energy homeostasis and cell proliferation.
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How Dysregulated Ion Channels and Transporters Take a Hand in Esophageal, Liver, and Colorectal Cancer. Rev Physiol Biochem Pharmacol 2020; 181:129-222. [PMID: 32875386 DOI: 10.1007/112_2020_41] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Over the last two decades, the understanding of how dysregulated ion channels and transporters are involved in carcinogenesis and tumor growth and progression, including invasiveness and metastasis, has been increasing exponentially. The present review specifies virtually all ion channels and transporters whose faulty expression or regulation contributes to esophageal, hepatocellular, and colorectal cancer. The variety reaches from Ca2+, K+, Na+, and Cl- channels over divalent metal transporters, Na+ or Cl- coupled Ca2+, HCO3- and H+ exchangers to monocarboxylate carriers and organic anion and cation transporters. In several cases, the underlying mechanisms by which these ion channels/transporters are interwoven with malignancies have been fully or at least partially unveiled. Ca2+, Akt/NF-κB, and Ca2+- or pH-dependent Wnt/β-catenin signaling emerge as cross points through which ion channels/transporters interfere with gene expression, modulate cell proliferation, trigger epithelial-to-mesenchymal transition, and promote cell motility and metastasis. Also miRs, lncRNAs, and DNA methylation represent potential links between the misexpression of genes encoding for ion channels/transporters, their malfunctioning, and cancer. The knowledge of all these molecular interactions has provided the basis for therapeutic strategies and approaches, some of which will be broached in this review.
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Negishi M, Kobayashi K, Sakuma T, Sueyoshi T. Nuclear receptor phosphorylation in xenobiotic signal transduction. J Biol Chem 2020; 295:15210-15225. [PMID: 32788213 DOI: 10.1074/jbc.rev120.007933] [Citation(s) in RCA: 37] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2020] [Revised: 08/05/2020] [Indexed: 12/11/2022] Open
Abstract
Nuclear pregnane X receptor (PXR, NR1I2) and constitutive active/androstane receptor (CAR, NR1I3) are nuclear receptors characterized in 1998 by their capability to respond to xenobiotics and activate cytochrome P450 (CYP) genes. An anti-epileptic drug, phenobarbital (PB), activates CAR and its target CYP2B genes, whereas PXR is activated by drugs such as rifampicin and statins for the CYP3A genes. Inevitably, both nuclear receptors have been investigated as ligand-activated nuclear receptors by identifying and characterizing xenobiotics and therapeutics that directly bind CAR and/or PXR to activate them. However, PB, which does not bind CAR directly, presented an alternative research avenue for an indirect ligand-mediated nuclear receptor activation mechanism: phosphorylation-mediated signal regulation. This review summarizes phosphorylation-based mechanisms utilized by xenobiotics to elicit cell signaling. First, the review presents how PB activates CAR (and other nuclear receptors) through a conserved phosphorylation motif located between two zinc fingers within its DNA-binding domain. PB-regulated phosphorylation at this motif enables nuclear receptors to form communication networks, integrating their functions. Next, the review discusses xenobiotic-induced PXR activation in the absence of the conserved DNA-binding domain phosphorylation motif. In this case, phosphorylation occurs at a motif located within the ligand-binding domain to transduce cell signaling that regulates hepatic energy metabolism. Finally, the review delves into the implications of xenobiotic-induced signaling through phosphorylation in disease development and progression.
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Affiliation(s)
- Masahiko Negishi
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA.
| | - Kaoru Kobayashi
- Department of Biopharmaceutics, Meiji Pharmaceutical University, Kiyose, Tokyo, Japan
| | - Tsutomu Sakuma
- School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima, Japan
| | - Tatsuya Sueyoshi
- Pharmacogenetics Section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina, USA
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42
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Shindo S, Chen SH, Gotoh S, Yokobori K, Hu H, Ray M, Moore R, Nagata K, Martinez J, Hong JS, Negishi M. Estrogen receptor α phosphorylated at Ser216 confers inflammatory function to mouse microglia. Cell Commun Signal 2020; 18:117. [PMID: 32727504 PMCID: PMC7390202 DOI: 10.1186/s12964-020-00578-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2019] [Accepted: 04/13/2020] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Estrogen receptor α (ERα) has been suggested to regulate anti-inflammatory signaling in brain microglia, the only resident immune cells in the brain. ERα conserves the phosphorylation motif at Ser216 within the DNA binding domain. Previously, Ser216 was found to be phosphorylated in neutrophils infiltrating into the mouse uterus and to enable ERα to regulate migration. Given the implication of this phosphorylation in immune regulation, ERα was examined in mouse microglia to determine if Ser216 is phosphorylated and regulates microglia's inflammation. It was found that Ser216 was constitutively phosphorylated in microglia and demonstrated that in the absence of phosphorylated ERα in ERα KI brains microglia inflamed, confirming that phosphorylation confers ERα with anti-inflammatory capability. ERα KI mice were obese and weakened motor ability. METHODS Mixed glia cells were prepared from brains of 2-days-old neonates and cultured to mature and isolate microglia. An antibody against an anti-phospho-S216 peptide of ERα (αP-S216) was used to detect phosphorylated ERα in double immunofluorescence staining with ERα antibodies and a microglia maker Iba-1 antibody. A knock-in (KI) mouse line bearing the phosphorylation-blocked ERα S216A mutation (ERα KI) was generated to examine inflammation-regulating functions of phosphorylated ERα in microglia. RT-PCR, antibody array, ELISA and FACS assays were employed to measure expressions of pro- or anti-inflammatory cytokines at their mRNA and protein levels. Rotarod tests were performed to examine motor connection ability. RESULTS Double immune staining of mixed glia cells showed that ERα is phosphorylated at Ser216 in microglia, but not astrocytes. Immunohistochemistry with an anti-Iba-1 antibody showed that microglia cells were swollen and shortened branches in the substantial nigra (SN) of ERα KI brains, indicating the spontaneous activation of microglia as observed with those of lipopolysaccharide (LPS)-treated ERα WT brains. Pro-inflammatory cytokines were up-regulated in the brain of ERα KI brains as well as cultured microglia, whereas anti-inflammatory cytokines were down-regulated. FACS analysis showed that the number of IL-6 producing and apoptotic microglia increased in those prepared from ERα KI brains. Times of ERα KI mice on rod were shortened in Rotarod tests. CONCLUSIONS Blocking of Ser216 phosphorylation aggravated microglia activation and inflammation of mouse brain, thus confirming that phosphorylated ERα exerts anti-inflammatory functions. ERα KI mice enable us to further investigate the mechanism by which phosphorylated ERα regulates brain immunity and inflammation and brain diseases. Video abstract.
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Affiliation(s)
- Sawako Shindo
- Pharmacogenetics, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
- Departments of Environmental Health, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Sendai, 981-8558 Japan
| | - Shih-Heng Chen
- Neurobiology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Saki Gotoh
- Pharmacogenetics, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Kosuke Yokobori
- Pharmacogenetics, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Hao Hu
- Pharmacogenetics, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Manas Ray
- Knockout Mouse Core, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Rick Moore
- Pharmacogenetics, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Kiyoshi Nagata
- Departments of Environmental Health, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, Sendai, 981-8558 Japan
| | - Jennifer Martinez
- Immunity, Inflammation and Disease Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Jau-Shyong Hong
- Neurobiology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
| | - Masahiko Negishi
- Pharmacogenetics, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 USA
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van der Mark VA, Adam AAA, Chang JC, Oude Elferink RP, Chamuleau RAFM, Hoekstra R. Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells. Toxicol Appl Pharmacol 2020; 399:115055. [PMID: 32428594 DOI: 10.1016/j.taap.2020.115055] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 05/11/2020] [Accepted: 05/13/2020] [Indexed: 02/06/2023]
Abstract
The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.
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Affiliation(s)
- Vincent A van der Mark
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands
| | - Aziza A A Adam
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Jung-Chin Chang
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Ronald P Oude Elferink
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Robert A F M Chamuleau
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Ruurdtje Hoekstra
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
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Hendriks DFG, Vorrink SU, Smutny T, Sim SC, Nordling Å, Ullah S, Kumondai M, Jones BC, Johansson I, Andersson TB, Lauschke VM, Ingelman-Sundberg M. Clinically Relevant Cytochrome P450 3A4 Induction Mechanisms and Drug Screening in Three-Dimensional Spheroid Cultures of Primary Human Hepatocytes. Clin Pharmacol Ther 2020; 108:844-855. [PMID: 32320483 DOI: 10.1002/cpt.1860] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2020] [Accepted: 03/29/2020] [Indexed: 12/15/2022]
Abstract
Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.
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Affiliation(s)
- Delilah F G Hendriks
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden.,Hubrecht Institute, KNAW (Royal Netherlands Academy of Arts and Sciences), Utrecht, The Netherlands
| | - Sabine U Vorrink
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
| | - Tomas Smutny
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
| | - Sarah C Sim
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
| | - Åsa Nordling
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
| | - Shahid Ullah
- Division of Clinical Pharmacology, Karolinska University Hospital Laboratory, Stockholm, Sweden
| | - Masaki Kumondai
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden.,Laboratory of Pharmacotherapy of Life-Style Related Diseases, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan
| | | | - Inger Johansson
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
| | - Tommy B Andersson
- DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
| | - Magnus Ingelman-Sundberg
- Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
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Shizu R, Yoshinari K. Nuclear receptor CAR-mediated liver cancer and its species differences. Expert Opin Drug Metab Toxicol 2020; 16:343-351. [PMID: 32202166 DOI: 10.1080/17425255.2020.1746268] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
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.
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Affiliation(s)
- Ryota Shizu
- Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
| | - Kouichi Yoshinari
- Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
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Fashe M, Hashiguchi T, Negishi M, Sueyoshi T. Ser100-Phosphorylated ROR α Orchestrates CAR and HNF4 α to Form Active Chromatin Complex in Response to Phenobarbital to Regulate Induction of CYP2B6. Mol Pharmacol 2020; 97:191-201. [PMID: 31924695 PMCID: PMC6978708 DOI: 10.1124/mol.119.118273] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2019] [Accepted: 12/16/2019] [Indexed: 01/11/2023] Open
Abstract
We have previously shown that the retinoid-related orphan receptor alpha (RORα) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated RORα orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). RORα knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of RORα in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that RORα in the form of phosphorylated (p-) S100 directly bound to a newly identified RORα response element (RORα response element on CYP2B6 promoter, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 RORα was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4α binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORα at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4α formed a complex with Ser 100-phosphorylated RORα, as shown by supershifted complexes with anti-p-Ser100 RORα and anti-HNF4α antibodies. Altogether, the results established that p-Ser100 RORα bridging the PBREM and OARE orchestrates CAR and HNF4α to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT: CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics, and it is prone to induction by xenobiotics, including phenobarbital via constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α). Here, we show that retinoid-related orphan receptor alpha (RORα), through phosphorylated S100 residue, orchestrated CAR-HNF4α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role of RORα in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation sites within the DNA-binding domain of the receptor.
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Affiliation(s)
- Muluneh Fashe
- Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
| | - Takuyu Hashiguchi
- Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
| | - Masahiko Negishi
- Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
| | - Tatsuya Sueyoshi
- Pharmacogenetics section, Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina
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Lin W, Bwayi M, Wu J, Li Y, Chai SC, Huber AD, Chen T. CITCO Directly Binds to and Activates Human Pregnane X Receptor. Mol Pharmacol 2020; 97:180-190. [PMID: 31882411 PMCID: PMC6978709 DOI: 10.1124/mol.119.118513] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2019] [Accepted: 12/23/2019] [Indexed: 12/12/2022] Open
Abstract
The xenobiotic receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are activated by structurally diverse chemicals to regulate the expression of target genes, and they have overlapping regulation in terms of ligands and target genes. Receptor-selective agonists are, therefore, critical for studying the overlapping function of PXR and CAR. An early effort identified 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) as a selective human CAR (hCAR) agonist, and this has since been widely used to distinguish the function of hCAR from that of human PXR (hPXR). The selectivity was demonstrated in a green monkey kidney cell line, CV-1, in which CITCO displayed >100-fold selectivity for hCAR over hPXR. However, whether the selectivity observed in CV-1 cells also represented CITCO activity in liver cell models was not hitherto investigated. In this study, we showed that CITCO: 1) binds directly to hPXR; 2) activates hPXR in HepG2 cells, with activation being blocked by an hPXR-specific antagonist, SPA70; 3) does not activate mouse PXR; 4) depends on tryptophan-299 to activate hPXR; 5) recruits steroid receptor coactivator 1 to hPXR; 6) activates hPXR in HepaRG cell lines even when hCAR is knocked out; and 7) activates hPXR in primary human hepatocytes. Together, these data indicate that CITCO binds directly to the hPXR ligand-binding domain to activate hPXR. As CITCO has been widely used, its confirmation as a dual agonist for hCAR and hPXR is important for appropriately interpreting existing data and designing future experiments to understand the regulation of hPXR and hCAR. SIGNIFICANCE STATEMENT: The results of this study demonstrate that 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is a dual agonist for human constitutive androstane receptor (hCAR) and human pregnane X receptor (hPXR). As CITCO has been widely used to activate hCAR, and hPXR and hCAR have distinct and overlapping biological functions, these results highlight the value of receptor-selective agonists and the importance of appropriately interpreting data in the context of receptor selectivity of such agonists.
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Affiliation(s)
- Wenwei Lin
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Monicah Bwayi
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Jing Wu
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Yongtao Li
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Sergio C Chai
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Andrew D Huber
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Taosheng Chen
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee
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Yoshinari K. Role of Nuclear Receptors PXR and CAR in Xenobiotic-Induced Hepatocyte Proliferation and Chemical Carcinogenesis. Biol Pharm Bull 2020; 42:1243-1252. [PMID: 31366862 DOI: 10.1248/bpb.b19-00267] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
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.
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Affiliation(s)
- Kouichi Yoshinari
- Laboratory of Molecular Toxicology, School of Pharmaceutical Sciences, University of Shizuoka
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Paszek M, Tukey RH. NRF2-Independent Regulation of Intestinal Constitutive Androstane Receptor by the Pro-Oxidants Cadmium and Isothiocyanate in hUGT1 Mice. Drug Metab Dispos 2020; 48:25-30. [PMID: 31704714 PMCID: PMC6904881 DOI: 10.1124/dmd.119.089508] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Accepted: 11/01/2019] [Indexed: 12/30/2022] Open
Abstract
Environmental toxicants such as heavy metals from contaminated water or soil and isothiocyanates (ITC) from dietary sources act as pro-oxidants by directly generating reactive oxygen species (ROS) or through depleting cellular antioxidants such as glutathione. Toxicants can alter drug metabolism, and it was reported that CYP2B10 and UGT1A1 are induced by phenethyl isothiocyanate (PEITC) through the constitutive androstane receptor (CAR). The possibility that nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of the antioxidant response, could coactivate CAR was investigated in neonatal hUGT1/Nrf2 -/- mice. Neonatal mice were treated with PEITC or cadmium (Cd2+) by oral gavage for 2 days. Both PEITC and Cd2+ induced UGT1A1 RNA and protein in intestinal tissues in both hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates, indicating NRF2-independent regulation of UGT1A1. Increases in CYP2B10 RNA in intestinal tissues were observed following PEITC or Cd2+ exposure. Activation of intestinal CAR by Cd2+ exposure was directly assessed by nuclear fractionation and Western blot analyses at 0.5, 1, 2, and 4 hours after treatment in hUGT1 neonates and after 48 hours in hUGT1/Nrf2 +/- and hUGT1/Nrf2 -/- neonates. CAR localized to the nucleus independently of NRF2 48 hours after exposure. Substantial CAR localization to the nucleus occurred at the 2- and 4-hour time points, coinciding with a decrease in phosphorylation of cytoplasmic extracellular signal-regulated kinases 1 and 2 and a nuclear increase in P38/p-P38 content. This suggests that a novel oxidative stress-MAPK-CAR axis exists with phenotypic consequences. SIGNIFICANCE STATEMENT: Pro-oxidant toxicants can alter drug metabolism through activation of CAR, independent of the NRF2-KEAP1 signaling pathway. Changes in proteins associated with drug metabolism and linked to increases in intestinal maturation are mediated through an oxidative stress-MAPK-CAR axis.
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Affiliation(s)
- Miles Paszek
- Laboratory of Environmental Toxicology, Departments of Chemistry and Biochemistry (M.P.) and Pharmacology (R.H.T.), University of California, San Diego, La Jolla, California
| | - Robert H Tukey
- Laboratory of Environmental Toxicology, Departments of Chemistry and Biochemistry (M.P.) and Pharmacology (R.H.T.), University of California, San Diego, La Jolla, California
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Kato H. Computational prediction of cytochrome P450 inhibition and induction. Drug Metab Pharmacokinet 2019; 35:30-44. [PMID: 31902468 DOI: 10.1016/j.dmpk.2019.11.006] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2019] [Revised: 10/27/2019] [Accepted: 11/17/2019] [Indexed: 12/14/2022]
Abstract
Cytochrome P450 (CYP) enzymes play an important role in the phase I metabolism of many xenobiotics. Most drug-drug interactions (DDIs) associated with CYP are caused by either CYP inhibition or induction. The early detection of potential DDIs is highly desirable in the pharmaceutical industry because DDIs can cause serious adverse events, which can lead to poor patient health and drug development failures. Recently, many computational studies predicting CYP inhibition and induction have been reported. The current computational modeling approaches for CYP metabolism are classified as ligand- and structure-based; various techniques, such as quantitative structure-activity relationships, machine learning, docking, and molecular dynamic simulation, are involved in both the approaches. Recently, combining these two approaches have resulted in improvements in the prediction accuracy of DDIs. In this review, we present important, recent developments in the computational prediction of the inhibition of four clinically crucial CYP isoforms (CYP1A2, 2C9, 2D6, and 3A4) and three nuclear receptors (aryl hydrocarbon receptor, constitutive androstane receptor, and pregnane X receptor) involved in the induction of CYP1A2, 2B6, and 3A4, respectively.
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Affiliation(s)
- Harutoshi Kato
- DMPK Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Aoba-ku, Yokohama-shi, 227-0033, Japan.
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