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Jia F, Liu X, Liu Y. Bile acid signaling in skeletal muscle homeostasis: from molecular mechanisms to clinical applications. Front Endocrinol (Lausanne) 2025; 16:1551100. [PMID: 40144297 PMCID: PMC11936799 DOI: 10.3389/fendo.2025.1551100] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2024] [Accepted: 02/25/2025] [Indexed: 03/28/2025] Open
Abstract
The intricate relationship between bile acid metabolism and skeletal muscle function has emerged as a crucial area of research in metabolic health. This review synthesizes current evidence highlighting the fundamental role of bile acids as key signaling molecules in muscle homeostasis and their therapeutic potential in muscle-related disorders. Recent advances in molecular biology and metabolomics have revealed that bile acids, beyond their classical role in lipid absorption, function as essential regulators of muscle mass and function through multiple signaling pathways, particularly via the nuclear receptor FXR and membrane receptor TGR5. Clinical studies have demonstrated significant associations between altered bile acid profiles and muscle wasting conditions, while experimental evidence has elucidated the underlying mechanisms linking bile acid signaling to muscle protein synthesis, energy metabolism, and regeneration capacity. We critically examine the emerging therapeutic strategies targeting bile acid pathways, including receptor-specific agonists, microbiome modulators, and personalized interventions based on individual bile acid profiles. Additionally, we discuss novel diagnostic approaches utilizing bile acid-based biomarkers and their potential in early detection and monitoring of muscle disorders. This review also addresses current challenges in standardization and clinical translation while highlighting promising future directions in this rapidly evolving field. Understanding the bile acid-muscle axis may provide new opportunities for developing targeted therapies for age-related muscle loss and metabolic diseases.
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Affiliation(s)
- Feng Jia
- Department of Hepatobiliary and Pancreatic Surgery, General Surgery Center, The First Hospital of Jilin University, Changchun, China
| | - Xiangliang Liu
- Cancer Center, The First Hospital of Jilin University, Changchun, China
| | - Yahui Liu
- Department of Hepatobiliary and Pancreatic Surgery, General Surgery Center, The First Hospital of Jilin University, Changchun, China
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Hao J, Wang Z, Ren J, Cao S, Xie Z, Yang J, Li J, Ding W, Li J, Han Z, Yuan Y, Hai T, Ding S, Zhang MQ, Shi M. Single-cell multi-omics deciphers hepatocyte dedifferentiation and illuminates maintenance strategies. Cell Prolif 2025; 58:e13772. [PMID: 39810466 PMCID: PMC11882756 DOI: 10.1111/cpr.13772] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2024] [Revised: 10/09/2024] [Accepted: 10/29/2024] [Indexed: 01/16/2025] Open
Abstract
Due to the similarity to human hepatocytes, porcine hepatocytes play an important role in hepatic research and drug evaluation. However, once hepatocytes were cultured in vitro, it was often prone to dedifferentiate, resulting in the loss of their characteristic features and normal functions, which impede their application in liver transplantation and hepatotoxic drugs evaluation. Up to now, this process has yet to be thoroughly investigated from the single-cell resolution and multi-omics perspective. In this study, we utilized 10× multiome technology to dissect the heterogeneity of porcine hepatocytes at different time points (Days 0, 1, 3, 5 and 7) during dedifferentiation. We comprehensively investigated cell heterogeneity, cellular dynamics, signalling pathways, potential gene targets, enhancer-driven gene regulatory networks, cell-cell communications of these cells and the conservation of mechanisms across species. We found that a series of critical signalling pathways driven by ERK, PI3K, Src and TGF-β were activated during this process, especially in the early stage of dedifferentiation. Based on these discoveries, we constructed a chemical combination targeting these pathways, which effectively inhibited the dedifferentiation of porcine hepatocytes in vitro. To validate the effectiveness of this combination, we transplanted such treated hepatocytes into FRGN mice, and the results demonstrated that these cells could effectively repopulate the liver and improve the survival of mice.
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Affiliation(s)
- Jie Hao
- School of Pharmaceutical SciencesTsinghua UniversityBeijingChina
| | - Zhenyi Wang
- MOE Key Laboratory of Bioinformatics, Beijing National Research Center for Information Science and Technology, Bioinformatics DivisionTsinghua UniversityBeijingChina
- Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine (Shanghai)Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghaiChina
| | - Jilong Ren
- Key Laboratory of Organ Regeneration and ReconstructionState Key Laboratory of Stem Cell and Reproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijingChina
- Beijing Farm Animal Research Center, Institute of ZoologyChinese Academy of SciencesBeijingChina
| | - Shenghao Cao
- Institute of Image Processing and Pattern RecognitionShanghai Jiao Tong UniversityShanghaiChina
- Key Laboratory of System Control and Information ProcessingMinistry of Education of ChinaShanghaiChina
| | - Zhongchen Xie
- School of Life SciencesTsinghua UniversityBeijingChina
| | - Jinghuan Yang
- School of Pharmaceutical SciencesTsinghua UniversityBeijingChina
| | - Jiachen Li
- State Key Laboratory of Biopharmaceutical Preparation and DeliveryInstitute of Process EngineeringChinese Academy of SciencesBeijingChina
| | - Weizhe Ding
- School of Pharmaceutical SciencesTsinghua UniversityBeijingChina
| | - Jie Li
- Key Laboratory of Organ Regeneration and ReconstructionState Key Laboratory of Stem Cell and Reproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijingChina
- Beijing Farm Animal Research Center, Institute of ZoologyChinese Academy of SciencesBeijingChina
| | - Zhiqiang Han
- Key Laboratory of Organ Regeneration and ReconstructionState Key Laboratory of Stem Cell and Reproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijingChina
- Beijing Farm Animal Research Center, Institute of ZoologyChinese Academy of SciencesBeijingChina
| | - Ye Yuan
- State Key Laboratory of Biopharmaceutical Preparation and DeliveryInstitute of Process EngineeringChinese Academy of SciencesBeijingChina
| | - Tang Hai
- Key Laboratory of Organ Regeneration and ReconstructionState Key Laboratory of Stem Cell and Reproductive BiologyInstitute of ZoologyChinese Academy of SciencesBeijingChina
- Beijing Farm Animal Research Center, Institute of ZoologyChinese Academy of SciencesBeijingChina
| | - Sheng Ding
- School of Pharmaceutical SciencesTsinghua UniversityBeijingChina
| | - Michael Q. Zhang
- MOE Key Laboratory of Bioinformatics, Beijing National Research Center for Information Science and Technology, Bioinformatics DivisionTsinghua UniversityBeijingChina
- Department of Biological Sciences, Center for Systems BiologyThe University of TexasRichardsonTexasUSA
| | - Minglei Shi
- MOE Key Laboratory of Bioinformatics, Beijing National Research Center for Information Science and Technology, Bioinformatics DivisionTsinghua UniversityBeijingChina
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A bioartificial transgenic porcine whole liver expressing human proteins alleviates acute liver failure in pigs. Hepatobiliary Pancreat Dis Int 2022; 22:270-281. [PMID: 35835690 DOI: 10.1016/j.hbpd.2022.06.012] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/06/2021] [Accepted: 06/21/2022] [Indexed: 02/05/2023]
Abstract
BACKGROUND Preventing heterologous protein influx in patients is important when using xenogeneic bioartificial livers (BALs) to treat liver failure. The development of transgenic porcine livers synthesizing human proteins is a promising approach in this regard. Here, we evaluated the safety and efficacy of a transgenic porcine liver synthesizing human albumin (hALB) and coagulation factor VII (hFVII) within a bioartificial system. METHODS Tibetan miniature pigs were randomly subjected to different interventions after surgery-induced partially ischemic liver failure. Group A (n = 4) was subjected to basic treatment; group B (n = 4) was to standard medical treatment and wild-type porcine BAL perfusion, and group C (n = 2) was to standard medical treatment and transgenic BAL perfusion. Biochemical parameters, coagulation status, survival time, and pathological changes were determined. Expressions of hALB and hFVII were detected using immunohistochemistry and enzyme-linked immunosorbent assays. RESULTS The survival time in group A was 9.75 ± 1.26 days; this was shorter than that in both perfused groups, in which all animals reached an endpoint of 12 days (P = 0.006). Ammonia, bilirubin, and lactate levels were significantly decreased, whereas albumin and fibrinogen levels were increased after perfusion (all P < 0.05). hALB and hFVII were detected in transgenic BAL-perfused pig serum and ex vivo in the liver tissues. CONCLUSIONS The humanized transgenic pig livers could synthesize and secrete hALB and hFVII ex vivo in a whole organ-based bioartificial system, while maintaining their metabolism, detoxification, transformation, and excretion functions, which were comparable to those observed in wild-type porcine livers. Therefore, the use of transgenic bioartificial whole livers is expected to become a new approach in treating acute liver failure.
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Pluta KD, Ciezkowska M, Wisniewska M, Wencel A, Pijanowska DG. Cell-based clinical and experimental methods for assisting the function of impaired livers – Present and future of liver support systems. Biocybern Biomed Eng 2021. [DOI: 10.1016/j.bbe.2021.06.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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Catapano G, Unger JK, Zanetti EM, Fragomeni G, Gerlach JC. Kinetic Analysis of Lidocaine Elimination by Pig Liver Cells Cultured in 3D Multi-Compartment Hollow Fiber Membrane Network Perfusion Bioreactors. Bioengineering (Basel) 2021; 8:104. [PMID: 34436107 PMCID: PMC8389311 DOI: 10.3390/bioengineering8080104] [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: 06/14/2021] [Revised: 07/15/2021] [Accepted: 07/21/2021] [Indexed: 11/16/2022] Open
Abstract
Liver cells cultured in 3D bioreactors is an interesting option for temporary extracorporeal liver support in the treatment of acute liver failure and for animal models for preclinical drug screening. Bioreactor capacity to eliminate drugs is generally used for assessing cell metabolic competence in different bioreactors or to scale-up bioreactor design and performance for clinical or preclinical applications. However, drug adsorption and physical transport often disguise the intrinsic drug biotransformation kinetics and cell metabolic state. In this study, we characterized the intrinsic kinetics of lidocaine elimination and adsorption by porcine liver cells cultured in 3D four-compartment hollow fiber membrane network perfusion bioreactors. Models of lidocaine transport and biotransformation were used to extract intrinsic kinetic information from response to lidocaine bolus of bioreactor versus adhesion cultures. Different from 2D adhesion cultures, cells in the bioreactors are organized in liver-like aggregates. Adsorption on bioreactor constituents significantly affected lidocaine elimination and was effectively accounted for in kinetic analysis. Lidocaine elimination and cellular monoethylglicinexylidide biotransformation featured first-order kinetics with near-to-in vivo cell-specific capacity that was retained for times suitable for clinical assist and drug screening. Different from 2D cultures, cells in the 3D bioreactors challenged with lidocaine were exposed to close-to-physiological lidocaine and monoethylglicinexylidide concentration profiles. Kinetic analysis suggests bioreactor technology feasibility for preclinical drug screening and patient assist and that drug adsorption should be accounted for to assess cell state in different cultures and when laboratory bioreactor design and performance is scaled-up to clinical use or toxicological drug screening.
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Affiliation(s)
- Gerardo Catapano
- Department of Mechanical, Energy and Management Engineering, University of Calabria, Via P. Bucci, I, 87030 Rende, CS, Italy;
| | - Juliane K. Unger
- Department of Experimental Medicine, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt Universität zu Berlin, 10117 Berlin, Germany;
| | | | - Gionata Fragomeni
- Department of Medical and Surgical Sciences, Magna Graecia University, 88100 Catanzaro, Italy;
| | - Jörg C. Gerlach
- Department of Surgery, School of Medicine, University of Pittsburgh, & McGowan Institute for Regenerative Medicine, Pittsburgh, PA 15213, USA; or
- Department of Bioengineering, School of Medicine, University of Pittsburgh, & McGowan Institute for Regenerative Medicine, Pittsburgh, PA 15213, USA
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Weng J, Han X, Zeng F, Zhang Y, Feng L, Cai L, Liang K, Liu S, Li S, Fu G, Zeng M, Gao Y. Fiber scaffold bioartificial liver therapy relieves acute liver failure and extrahepatic organ injury in pigs. Theranostics 2021; 11:7620-7639. [PMID: 34335954 PMCID: PMC8315066 DOI: 10.7150/thno.58515] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2021] [Accepted: 04/15/2021] [Indexed: 02/06/2023] Open
Abstract
Rationale: Acute liver failure (ALF) causes severe liver injury and a systemic inflammatory response, leading to multiorgan failure with a high short-term mortality. Bioartificial liver (BAL) therapy is a promising approach that is hampered by the lack of appropriate bioreactors and carriers to retain hepatic cell function and poor understanding of BAL treatment mechanisms in ALF and extrahepatic organ injury. Recently, we used a fiber scaffold bioreactor (FSB) for the high-density, three-dimensional (3D) culture of primary porcine hepatocytes (PPHs) combined with an absorption component to construct a BAL and verified its function in a D-galactosamine (D-gal)-induced ALF porcine model to evaluate its protective effects on the liver and extrahepatic organs. Methods: Male pigs were randomized into standard/supportive therapy (ST), ST+no-cell BAL (ST+Sham BAL) and ST+BAL groups and received treatment 48 h after receiving a D-gal injection. Changes in blood chemistry and clinical symptoms were monitored for 120 h. Tissues and plasma were collected for analysis by pathological examination, immunoblotting, quantitative PCR and immunoassays. Results: PPHs cultured in the FSB obtained sufficient aeration and nutrition for high-density, 3D culture and maintained superior viability and functionality (biosynthesis and detoxification) compared with those cultured in flasks. All the animals developed ALF, acute kidney injury (AKI) and hepatic encephalopathy (HE) 48 h after D-gal infusion and received corresponding therapies. Animals in the BAL group showed markedly improved survival (4/5; 80%) compared with those in the ST+Sham BAL (0/5; p < 0.001) and ST (0/5; p < 0.001) groups. The levels of blood ammonia and biochemical and inflammatory indices were alleviated after BAL treatment. Increased liver regeneration and attenuations in the occurrence and severity of ALF, AKI and HE were observed in the ST+BAL group compared with the ST (p = 0.0009; p = 0.038) and ST+Sham BAL (p = 0.011; p = 0.031) groups. Gut leakage, the plasma endotoxin level, bacterial translocation, and peripheral and neuroinflammation were alleviated in the ST+BAL group compared with those in the other groups. Conclusions: BAL treatment enhanced liver regeneration and alleviated the systemic inflammatory response and extrahepatic organ injury to prolong survival in the ALF model and has potential as a therapeutic approach for ALF patients.
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Affiliation(s)
- Jun Weng
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Xu Han
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Fanhong Zeng
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Yue Zhang
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Lei Feng
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Lei Cai
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Kangyan Liang
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Shusong Liu
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Shao Li
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
| | - Gongbo Fu
- Department of Medical Oncology, Jinling Hospital, First School of Clinical Medicine, Southern Medical University, Nanjing 210000, China
| | - Min Zeng
- Guangdong Qianhui Biotechnology Co., Ltd., Guangzhou 510285, China
| | - Yi Gao
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510515, China
- State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou 510515, China
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Gerbal-Chaloin S, Briolotti P, Daujat-Chavanieu M, Rasmussen MK. Primary hepatocytes isolated from human and porcine donors display similar patterns of cytochrome p450 expression following exposure to prototypical activators of AhR, CAR and PXR. Curr Res Toxicol 2021; 2:149-158. [PMID: 34345857 PMCID: PMC8320632 DOI: 10.1016/j.crtox.2021.03.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2021] [Revised: 03/01/2021] [Accepted: 03/02/2021] [Indexed: 01/13/2023] Open
Abstract
CYP mRNA induction were compared between human and porcine primary hepatocytes. Both human and porcine primary hepatocytes responded to prototypical CYP inducers. CYP mRNA induction displayed similar patterns in human and porcine primary hepatocytes. The hepatic cytochrome p450’s (CYP) are of major importance for the metabolism of xenobiotics and knowledge about their regulation is crucial. This knowledge often originates from cell models; primary human hepatocytes (PHH) being the gold standard. However, due to limited availability of high-quality human donor organs, basic knowledge on alternative models are needed. Primary porcine hepatocytes (PPH) have been suggested as an alternative to PHH. Unfortunately, data comparing the response in gene-transcription to standard CYP inducers between PHH and PPH are missing. In the present study we, cultured PHH and PPH under the same conditions, treated them with standard inducers of the CYP1-3 and determined the response in gene and protein expression. The results demonstrated that in both species TCDD and omeprazole caused an increase in CYP1A/B expression. In PPH, CITCO increased the content of CYP1A/B. For the CYP2B/C/D’s, phenobarbital and rifampicin caused increases in expression. For the CYP2D’s, TCDD and omeprazole caused increased gene expression in PPH, which were not the case for PHH. Both phenobarbital, rifampicin and omeprazole increased CYP3A expression in PHH and PPH. Moreover, TCDD increased the gene expression of CYP3A in PPH; this was not the case for PHH. Multivariate data analysis found no difference in gene expression between PHH and PPH for phenobarbital, rifampicin and CITCO. However, differential clustering was observed for TCDD and omeprazole. In conclusion, despite model specificity, there are a high number of similar responses, and experiments investigating mRNA regulation made in PPH permits for a reliable translation into human setting.
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Affiliation(s)
| | - Philippe Briolotti
- IRMB, University of Montpellier, INSERM, CHU Montpellier, Montpellier, France
| | | | - Martin Krøyer Rasmussen
- Department of Food Science, Aarhus University, Agro Food Park 48, DK-8200 Aarhus N, Denmark
- Corresponding author at: Department of Food Science, Aarhus University, Agro Food Park 48, DK-8200 Aarhus N, Denmark.
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Xenobiotica-metabolizing enzymes in the lung of experimental animals, man and in human lung models. Arch Toxicol 2019; 93:3419-3489. [PMID: 31673725 DOI: 10.1007/s00204-019-02602-7] [Citation(s) in RCA: 42] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2019] [Accepted: 10/15/2019] [Indexed: 12/15/2022]
Abstract
The xenobiotic metabolism in the lung, an organ of first entry of xenobiotics into the organism, is crucial for inhaled compounds entering this organ intentionally (e.g. drugs) and unintentionally (e.g. work place and environmental compounds). Additionally, local metabolism by enzymes preferentially or exclusively occurring in the lung is important for favorable or toxic effects of xenobiotics entering the organism also by routes other than by inhalation. The data collected in this review show that generally activities of cytochromes P450 are low in the lung of all investigated species and in vitro models. Other oxidoreductases may turn out to be more important, but are largely not investigated. Phase II enzymes are generally much higher with the exception of UGT glucuronosyltransferases which are generally very low. Insofar as data are available the xenobiotic metabolism in the lung of monkeys comes closed to that in the human lung; however, very few data are available for this comparison. Second best rate the mouse and rat lung, followed by the rabbit. Of the human in vitro model primary cells in culture, such as alveolar macrophages and alveolar type II cells as well as the A549 cell line appear quite acceptable. However, (1) this generalization represents a temporary oversimplification born from the lack of more comparable data; (2) the relative suitability of individual species/models is different for different enzymes; (3) when more data become available, the conclusions derived from these comparisons quite possibly may change.
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Gleich A, Kaiser B, Honscha W, Fuhrmann H, Schoeniger A. Evaluation of the hepatocyte-derived cell line BFH12 as an in vitro model for bovine biotransformation. Cytotechnology 2019; 71:231-244. [PMID: 30617848 DOI: 10.1007/s10616-018-0279-4] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2018] [Accepted: 11/08/2018] [Indexed: 12/17/2022] Open
Abstract
The knowledge of drug metabolising enzymes (DMEs) in cattle is rather limited. The capability of the bovine foetal hepatocyte-derived cell line BFH12 to serve as model for biotransformation was evaluated. Gene expression analysis of DMEs was performed by reverse transcription PCR (RT-PCR). The presence of efflux transporters was visualised by immunocytochemistry, and functional induction of cytochrome P450 (CYP) 1A was assessed by the ethoxyresorufin-O-deethylase (EROD) assay. The production of bile acids was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RT-PCR revealed the expression of cytochromes 1A1, 1A2, 3A4 and phase II enzymes UGT1A1, UGT1A6 and GSTM1. Immunofluorescence demonstrated efflux transporters ABCG2 and ABCC1. The EROD assay revealed a dose-dependent CYP1A induction after treatment with benzo[a]pyrene (BP). LC-MS/MS analysis of cell culture supernatants showed the production of bile acids including taurocholic acid, tauro-chenodeoxycholic acid, taurodeoxycholic acid and taurolithocholic acid. The results strongly suggest the applicability of the cell line BFH12 for subsequent experiments in the emerging field of bovine biotransformation.
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Affiliation(s)
- Alexander Gleich
- Institute of Biochemistry, University of Leipzig, An den Tierkliniken 1, 04103, Leipzig, Germany
| | - Bastian Kaiser
- Institute of Veterinary Physiology, University of Leipzig, An den Tierkliniken 7, 04103, Leipzig, Germany
| | - Walther Honscha
- Institute of Veterinary Pharmacology and Toxicology, University of Leipzig, An den Tierkliniken 15, 04103, Leipzig, Germany
| | - Herbert Fuhrmann
- Institute of Biochemistry, University of Leipzig, An den Tierkliniken 1, 04103, Leipzig, Germany
| | - Axel Schoeniger
- Institute of Biochemistry, University of Leipzig, An den Tierkliniken 1, 04103, Leipzig, Germany.
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10
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Wang T, Yao W, He Q, Shao Y, Zheng R, Huang F. L-leucine stimulates glutamate dehydrogenase activity and glutamate synthesis by regulating mTORC1/SIRT4 pathway in pig liver. ANIMAL NUTRITION (ZHONGGUO XU MU SHOU YI XUE HUI) 2018; 4:329-337. [PMID: 30175263 PMCID: PMC6116330 DOI: 10.1016/j.aninu.2017.12.002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/07/2017] [Accepted: 12/15/2017] [Indexed: 01/09/2023]
Abstract
The liver is the most essential organ for the metabolism of ammonia, in where most of ammonia is removed by urea and glutamine synthesis. Regulated by leucine, glutamate dehydrogenase (GDH) catalyzes the reversible inter-conversion of glutamate to ammonia. To determine the mechanism of leucine regulating GDH, pigs weighing 20 ± 1 kg were infused for 80 min with ammonium chloride or alanine in the presence or absence of leucine. Primary pig hepatocytes were incubated with or without leucine. In the in vivo experiments with either ammonium or alanine as the nitrogen source, addition of leucine significantly inhibited ureagenesis and promoted the production of glutamate and glutamine in the perfused pig liver (P < 0.05). Similarly, leucine stimulated GDH activity and inhibited sirtuin4 (SIRT4) gene expression (P < 0.01). Leucine could also activate mammalian target of rapamycin complex 1 (mTORC1) signaling (P < 0.05), as evidenced by the increased phosphorylation levels of ribosomal protein S6 kinase 1 (S6K1) and ribosomal protein S6 (S6). Interestingly, the leucine-induced mTORC1 pathway activation suitably correlated with increased GDH activity and decreased expression of SIRT4. Similar results were observed in primary cultured hepatocytes. Notably, leucine exerted no significant change in GDH activity in SIRT4-deficient hepatocytes (P > 0.05), while mTORC1 signaling was activated. Leucine exerted no significant changes in both GDH activity and SIRT4 gene expression in rapamycin treated hepatocytes (P > 0.05). In conclusion, L-leucine increases GDH activity and stimulates glutamate synthesis from different nitrogen sources by regulating mTORC1/SIRT4 pathway in the liver of pigs.
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Affiliation(s)
| | | | | | | | | | - Feiruo Huang
- Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China
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Jiang H, Wu J, Zhang F, Wen J, Jiang J, Deng Y. The critical role of porcine cytochrome P450 3A46 in the bioactivation of aflatoxin B 1. Biochem Pharmacol 2018; 156:177-185. [PMID: 30142320 DOI: 10.1016/j.bcp.2018.08.030] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2018] [Accepted: 08/20/2018] [Indexed: 11/19/2022]
Abstract
Aflatoxin B1 (AFB1) is bioactivated by cytochrome P450 (CYP) 3A isoforms in humans to generate the highly reactive epoxide intermediate AFB1-8,9-epoxide (AFBO), causing hepatotoxicity and hepatocarcinoma. Due to the unavoidable contamination in their feed, pigs are more likely to be exposed to AFB1 and indirectly harm human health. Therefore, identifying the porcine CYP3A isoforms involved in AFB1-8,9-epoxidation is critical. In this study, we used codon optimization and N-terminal coding sequence modification to modify a CYP3A46 recombinant protein that exhibits good structure and catalytic activities and revealed its strong AFB1-8,9-epoxidase activity for the first time. Site-directed mutagenesis, kinetics and docking analyses were performed and demonstrated that residues Phe-108, Ser-119, Phe-215, Phe-304 and Thr-309 play important roles in AFB1-8,9-epoxidation and its responsiveness to α-naphthoflavone. Interestingly, we uncovered the dual and reverse roles of Phe-304 in CYP3A46, CYP3A5 and CYP3A4 in AFB1 oxidation. Unlike the π-π interaction between the Phe-304 phenyl of CYP3A4 and the AFB1 aromatic ring, Phe-304 of CYP3A46 may function to provide steric hindrance to bind AFB1. Phe-108 and Phe-215 could stabilize AFB1 with a potentially productive orientation through van der Waals interactions with AFB1. Ser-119 and Thr-309 are likely to function to form H-bonds with AFB1. This study broadens our knowledge of AFB1 bioactivation in pigs and may contribute to reduce the deleterious effects of AFB1 in pigs and humans.
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Affiliation(s)
- Haoran Jiang
- Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, China; Key Laboratory of Zoonosis of the Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China
| | - Jun Wu
- Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, China; Key Laboratory of Zoonosis of the Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China
| | - Feiyong Zhang
- Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, China; Key Laboratory of Zoonosis of the Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China
| | - Jikai Wen
- Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, China; Key Laboratory of Zoonosis of the Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China
| | - Jun Jiang
- Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, China; Key Laboratory of Zoonosis of the Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.
| | - Yiqun Deng
- Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, China; Key Laboratory of Zoonosis of the Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, China.
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12
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Vilei M, Granato A, Ferraresso C, Neri D, Carraro P, Gerunda G, Muraca M. Comparison of Pig, Human and Rat Hepatocytes as a Source of Liver Specific Metabolic Functions in Culture Systems - Implications for Use in Bioartificial Liver Devices. Int J Artif Organs 2018. [DOI: 10.1177/039139880102400609] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The limited availability of human hepatocytes results in the use of animal cells in most bioartificial liver support devices. In the present work, clinically relevant liver specific metabolic activities were compared in rat, pig and human hepatocytes cultured on liver-derived biomatrix to optimize the expression of differentiated functions. Pig hepatocytes showed higher rates of diazepam metabolism (2.549±0.821 μg/h/million cells vs. 0.474±0.079 μg/h/million cells rats, p<0.005, and vs. 0.704±0.171 μg/h/million cells in man, p<0.005) and of bilirubin conjugation (21.60116±8.433237 μmoles/l/24 h vs. 6.786809±2.983758 in man, p<0.001 and vs. 9.956538±1.781016 μmoles/l/24 h in rats, p<0.005). Urea synthesis was similar in pig and in human hepatocytes (150±46.3 vs. 144.8±21.46 nmoles/h/million cells) and it was lower in rats (84.38±35.2; p<0.001 vs. man, p<0.02 vs. pig). High liver specific metabolic activities in cultured pig hepatocytes further support their use as a substitue for human cells in bioartificial liver devices
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Affiliation(s)
- M.T. Vilei
- Department of Medical and Surgical Sciences, University of Padova, Padova - Italy
| | - A. Granato
- Department of Medical and Surgical Sciences, University of Padova, Padova - Italy
| | - C. Ferraresso
- Department of Medical and Surgical Sciences, University of Padova, Padova - Italy
| | - D. Neri
- Department of Medical and Surgical Sciences, University of Padova, Padova - Italy
| | - P. Carraro
- Department of Clinical Chemistry, University of Padova, Padova - Italy
| | - G. Gerunda
- Department of Medical and Surgical Sciences, University of Padova, Padova - Italy
| | - M. Muraca
- Department of Medical and Surgical Sciences, University of Padova, Padova - Italy
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13
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Segatto NV, Remião MH, Schachtschneider KM, Seixas FK, Schook LB, Collares T. The Oncopig Cancer Model as a Complementary Tool for Phenotypic Drug Discovery. Front Pharmacol 2017; 8:894. [PMID: 29259556 PMCID: PMC5723300 DOI: 10.3389/fphar.2017.00894] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2017] [Accepted: 11/22/2017] [Indexed: 12/14/2022] Open
Abstract
The screening of potential therapeutic compounds using phenotypic drug discovery (PDD) is being embraced once again by researchers and pharmaceutical companies as an approach to enhance the development of new effective therapeutics. Before the genomics and molecular biology era and the consecutive emergence of targeted-drug discovery approaches, PDD was the most common platform used for drug discovery. PDD, also known as phenotypic screening, consists of screening potential compounds in either in vitro cellular or in vivo animal models to identify compounds resulting in a desirable phenotypic change. Using this approach, the biological targets of the compounds are not taken into consideration. Suitable animal models are crucial for the continued validation and discovery of new drugs, as compounds displaying promising results in phenotypic in vitro cell-based and in vivo small animal model screenings often fail in clinical trials. Indeed, this is mainly a result of differential anatomy, physiology, metabolism, immunology, and genetics between humans and currently used pre-clinical small animal models. In contrast, pigs are more predictive of therapeutic treatment outcomes in humans than rodents. In addition, pigs provide an ideal platform to study cancer due to their similarities with humans at the anatomical, physiological, metabolic, and genetic levels. Here we provide a mini-review on the reemergence of PDD in drug development, highlighting the potential of porcine cancer models for improving pre-clinical drug discovery and testing. We also present precision medicine based genetically defined swine cancer models developed to date and their potential as biomedical models.
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Affiliation(s)
- Natalia V. Segatto
- Biotechnology Graduate Program, Molecular and Cellular Oncology Research Group, Laboratory of Cancer Biotechnology, Technology Development Center, Federal University of Pelotas, Pelotas, Brazil
| | - Mariana H. Remião
- Biotechnology Graduate Program, Molecular and Cellular Oncology Research Group, Laboratory of Cancer Biotechnology, Technology Development Center, Federal University of Pelotas, Pelotas, Brazil
| | | | - Fabiana K. Seixas
- Biotechnology Graduate Program, Molecular and Cellular Oncology Research Group, Laboratory of Cancer Biotechnology, Technology Development Center, Federal University of Pelotas, Pelotas, Brazil
| | - Lawrence B. Schook
- Department of Radiology, University of Illinois at Chicago, Chicago, IL, United States
- Department of Animal Sciences, University of Illinois at Urbana–Champaign, Champaign, IL, United States
| | - Tiago Collares
- Biotechnology Graduate Program, Molecular and Cellular Oncology Research Group, Laboratory of Cancer Biotechnology, Technology Development Center, Federal University of Pelotas, Pelotas, Brazil
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14
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Dabos KJ, Nelson LJ, Hewage CH, Parkinson JA, Howie AF, Sadler IH, Hayes PC, Plevris JN. Comparison of Bioenergetic Activity of Primary Porcine Hepatocytes Cultured in Four Different Media. Cell Transplant 2017; 13:213-29. [PMID: 15191159 DOI: 10.3727/000000004783984007] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 × 1010; viability 87 ± 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 × 106 viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis, ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. William's E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.
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Affiliation(s)
- Konstantinos J Dabos
- Liver Cell Biology Laboratory, Department of Hepatology, University of Edinburgh, Edinburgh EH16 4SU, Scotland, UK.
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15
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Constitutive expression and activity of cytochrome P450 in conventional pigs. Res Vet Sci 2017; 111:75-80. [DOI: 10.1016/j.rvsc.2016.12.003] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2016] [Revised: 12/06/2016] [Accepted: 12/15/2016] [Indexed: 12/14/2022]
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16
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Baek S, Han NR, Yun JI, Hwang JY, Kim M, Park CK, Lee E, Lee ST. Effects of Culture Dimensions on Maintenance of Porcine Inner Cell Mass-Derived Cell Self-Renewal. Mol Cells 2017; 40:117-122. [PMID: 28196411 PMCID: PMC5339502 DOI: 10.14348/molcells.2017.2223] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2016] [Revised: 01/04/2017] [Accepted: 01/06/2017] [Indexed: 01/02/2023] Open
Abstract
Despite the fact that porcine embryonic stem cells (ESCs) are a practical study tool, in vitro long-term maintenance of these cells is difficult in a two-dimensional (2D) microenvironment using cellular niche or extracellular matrix proteins. However, a three-dimensional (3D) microenvironment, similar to that enclosing the inner cell mass of the blastocyst, may improve in vitro maintenance of self-renewal. Accordingly, as a first step toward constructing a 3D microenvironment optimized to maintain porcine ESC self-renewal, we investigated different culture dimensions for porcine ICM-derived cells to enhance the maintenance of self-renewal. Porcine ICM-derived cells were cultured in agarose-based 3D hydrogel with self-renewal-friendly mechanics and in 2D culture plates with or without feeder cells. Subsequently, the effects of the 3D microenvironment on maintenance of self-renewal were identified by analyzing colony formation and morphology, alkaline phosphatase (AP) activity, and transcriptional and translational regulation of self-renewal-related genes. The 3D microenvironment using a 1.5% (w/v) agarose-based 3D hydrogel resulted in significantly more colonies with stereoscopic morphology, significantly improved AP activity, and increased protein expression of self-renewal-related genes compared to those in the 2D microenvironment. These results demonstrate that self-renewal of porcine ICM-derived cells can be maintained more effectively in a 3D microenvironment than in a 2D microenvironment. These results will help develop novel culture systems for ICM-derived cells derived from diverse species, which will contribute to stimulating basic and applicable studies related to ESCs.
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Affiliation(s)
- Song Baek
- Department of Animal Life Science, Kangwon National University, Chuncheon 24341,
Korea
| | - Na Rae Han
- Department of Animal Life Science, Kangwon National University, Chuncheon 24341,
Korea
| | - Jung Im Yun
- Division of Animal Resource Science, Kangwon National University, Chuncheon 24341,
Korea
| | - Jae Yeon Hwang
- Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven 06510,
USA
| | - Minseok Kim
- Animal Nutrition and Physiology Team, National Institute of Animal Science, RDA, Wanju 55365,
Korea
| | - Choon Keun Park
- Department of Animal Life Science, Kangwon National University, Chuncheon 24341,
Korea
- Division of Applied Animal Science, Kangwon National University, Chuncheon 24341,
Korea
| | - Eunsong Lee
- College of Veterinary Medicine, Kangwon National University, Chuncheon 24341,
Korea
| | - Seung Tae Lee
- Department of Animal Life Science, Kangwon National University, Chuncheon 24341,
Korea
- Division of Applied Animal Science, Kangwon National University, Chuncheon 24341,
Korea
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17
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Burkina V, Rasmussen MK, Pilipenko N, Zamaratskaia G. Comparison of xenobiotic-metabolising human, porcine, rodent, and piscine cytochrome P450. Toxicology 2017; 375:10-27. [DOI: 10.1016/j.tox.2016.11.014] [Citation(s) in RCA: 58] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2016] [Revised: 11/16/2016] [Accepted: 11/20/2016] [Indexed: 12/25/2022]
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18
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Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B₁. Toxins (Basel) 2016; 8:toxins8090267. [PMID: 27626447 PMCID: PMC5037493 DOI: 10.3390/toxins8090267] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2016] [Accepted: 09/05/2016] [Indexed: 01/10/2023] Open
Abstract
Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1) and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.
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Salcido-Neyoy ME, Sánchez-Pérez Y, Osornio-Vargas AR, Gonsebatt ME, Meléndez-Zajgla J, Morales-Bárcenas R, Petrosyan P, Molina-Servin ED, Vega E, Manzano-León N, García-Cuellar CM. Induction of c-Jun by air particulate matter (PM₁₀) of Mexico city: Participation of polycyclic aromatic hydrocarbons. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2015; 203:175-182. [PMID: 25909326 DOI: 10.1016/j.envpol.2015.03.051] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/09/2015] [Revised: 03/29/2015] [Accepted: 03/30/2015] [Indexed: 06/04/2023]
Abstract
The carcinogenic potential of urban particulate matter (PM) has been partly attributed to polycyclic aromatic hydrocarbons (PAHs) content, which activates the aryl hydrocarbon receptor (AhR). Here we report the effect of PM with an aerodynamic size of 10 μm (PM10) on the induction of AhR pathway in A549 cells, evaluating its downstream targets CYP1B1, IL-6, IL-8 and c-Jun. Significant increases in CYP1B1 protein and enzyme activity; IL-6 and IL-8 secretion and c-Jun protein were found in response to PM10. The formation of PAH-DNA adducts was also detected. The involvement of AhR pathway was confirmed with Resveratrol as AhR antagonist, which reversed CYP1B1 and c-Jun induction. Nevertheless, in IL-6 and IL-8 secretion, the Resveratrol was ineffective, suggesting an effect independent of this pathway. Considering the role of c-Jun in oncogenesis, its induction by PM may be contributing to its carcinogenic potential through induction of AhR pathway by PAHs present in PM10.
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Affiliation(s)
- Martha Estela Salcido-Neyoy
- Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Col Sección XVI. C.P. 14080. Delegación Tlalpan, México, D.F., Mexico
| | - Yesennia Sánchez-Pérez
- Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Col Sección XVI. C.P. 14080. Delegación Tlalpan, México, D.F., Mexico
| | | | - María Eugenia Gonsebatt
- Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, A.P. 70228. Ciudad Universitaria. 04510, México, D.F., Mexico
| | - Jorge Meléndez-Zajgla
- Laboratorio de Genómica Funcional del Cáncer, Instituto Nacional de Medicina Genómica, Periférico Sur, No.4809. Col. Arenal Tepepan, C.P. 14610. Delegación Tlalpan, México, D.F., Mexico
| | - Rocío Morales-Bárcenas
- Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Col Sección XVI. C.P. 14080. Delegación Tlalpan, México, D.F., Mexico
| | - Pavel Petrosyan
- Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, A.P. 70228. Ciudad Universitaria. 04510, México, D.F., Mexico
| | - Edith Danny Molina-Servin
- Departamento de Medicina Genómica y Toxicología Ambiental, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, A.P. 70228. Ciudad Universitaria. 04510, México, D.F., Mexico
| | - Elizabeth Vega
- Instituto Mexicano del Petróleo, Eje Central Lázaro Cárdenas 152. San Bartolo Atepehuacan, C.P. 07730, México, D.F., Mexico
| | - Natalia Manzano-León
- Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Col Sección XVI. C.P. 14080. Delegación Tlalpan, México, D.F., Mexico
| | - Claudia M García-Cuellar
- Subdirección de Investigación Básica, Instituto Nacional de Cancerología, Av. San Fernando No. 22, Col Sección XVI. C.P. 14080. Delegación Tlalpan, México, D.F., Mexico.
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20
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Baldini E, Cursio R, Desousa G, Margara A, Honiger J, Saint-Paul MC, Bayer P, Raimondi V, Rahmani R, Mouiel J, Gugenheim J. Cryopreserved porcine hepatocytes: expression and induction of cytochrome P450, isoform CYP2E1. Transplant Proc 2014; 41:1367-9. [PMID: 19460561 DOI: 10.1016/j.transproceed.2009.02.061] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Cryopreservation of porcine hepatocytes for their use in bioartificial liver devices may result in reduced cytochrome P450 (CYP) enzyme activity. The aim of this study was to assess the effects of several CYP inducers on the isoform CYP2E1 protein expression in cryopreserved porcine hepatocytes. Isolated porcine hepatocytes were cryopreserved for 1 month, thawed, and cultured for 3 days. During medium culture, the hepatocytes were exposed to the following CYP inducers: dimethyl sulfoxide, rifampicin, phenobarbital, 3-methylcholanthrene, and dexamethasone. CYP2E1 protein expression was determined by immunoblotting. CYP2E1 protein levels were constantly detected in cryopreserved porcine hepatocytes. CYP inducers did not modify CYP2E1 protein levels. Long-term cryopreserved porcine hepatocytes preserved their capacity for CYP2E1 protein expression, although exposure of these hepatocytes to CYP inducers did not modify the CYP2E1 protein expression.
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Affiliation(s)
- E Baldini
- Laboratoire de Recherches Chirurgicales, IFR 50, Faculté de Médecine, Université de Nice Sophia Antipolis, Nice, France
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21
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Quesnot N, Bucher S, Fromenty B, Robin MA. Modulation of metabolizing enzymes by bisphenol a in human and animal models. Chem Res Toxicol 2014; 27:1463-73. [PMID: 25142872 DOI: 10.1021/tx500087p] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Xenobiotics, such as contaminants and drugs, can be converted to potentially toxic reactive metabolites by phase 1 oxidizing enzymes. These metabolites are further detoxified by phase 2 conjugating enzymes and eliminated from cells by phase 3 transporters. Moreover, many of these xenobiotics are also able to induce or inhibit these enzymes, potentially modulating their own toxicity or that of other chemicals. The present review is focused on bisphenol A, a synthetic monomer used for many industrial applications and exhibiting xenoestrogen properties. The impact of this contaminant on all major classes of metabolizing enzymes (i.e., cytochromes P450, glutathione-S-transferases, sulfotransferases, UDP-glucuronyltransferases, and transporters) was reviewed, with a highlight on the modulation of cytochromes P450 involved in steroid metabolism. Interestingly, most of the studies reported in this review show that BPA is able to induce or inhibit metabolizing enzymes at high doses but also at doses compatible with human exposure.
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22
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Yu CB, Pan XP, Yu L, Yu XP, Du WB, Cao HC, Li J, Chen P, Li LJ. Evaluation of a novel choanoid fluidized bed bioreactor for future bioartificial livers. World J Gastroenterol 2014; 20:6869-77. [PMID: 24944477 PMCID: PMC4051926 DOI: 10.3748/wjg.v20.i22.6869] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2013] [Revised: 02/26/2014] [Accepted: 03/06/2014] [Indexed: 02/07/2023] Open
Abstract
AIM To construct and evaluate the functionality of a choanoid-fluidized bed bioreactor (CFBB) based on microencapsulated immortalized human hepatocytes. METHODS Encapsulated hepatocytes were placed in the constructed CFBB and circulated through Dulbecco's Modified Eagle's Medium (DMEM) for 12 h, and then through exchanged plasma for 6 h, and compared with encapsulated cells cultivated under static conditions in a spinner flask. Levels of alanine aminotransferase (ALT) and albumin were used to evaluate the CFBB during media circulation, whereas levels of ALT, total bilirubin (TBil), and albumin were used to evaluate it during plasma circulation. Mass transfer and hepatocyte injury were evaluated by comparing the results from the two experimental conditions. In addition, the viability and microstructure of encapsulated cells were observed in the different environments. RESULTS The bioartificial liver model based on a CFBB was verified by in vitro experiments. The viability of encapsulated cells accounting for 84.6% ± 3.7% in CFBB plasma perfusion was higher than the 74.8% ± 3.1% in the static culture group (P < 0.05) after 6 h. ALT release from cells was 29 ± 3.5 U/L vs 40.6 ± 3.2 U/L at 12 h (P < 0.01) in the CFBB medium circulation and static medium culture groups, respectively. Albumin secretion from cells was 234.2 ± 27.8 μg/1 × 10(7) cells vs 167.8 ± 29.3 μg/1 × 10(7) cells at 6 h (P < 0.01), 274.4 ± 34.6 μg/1 × 10(7) cells vs 208.4 ± 49.3 μg/1 × 10(7) cells (P < 0.05) at 12 h, in the two medium circulation/culture groups, respectively. Furthermore, ALT and TBil levels were 172.3 ± 24.1 U/L vs 236.3 ± 21.5 U/L (P < 0.05), 240.1 ± 23.9 μmol/L vs 241.9 ± 31.4 μmol/L (P > 0.05) at 6 h in the CFBB plasma perfusion and static plasma culture groups, respectively. There was no significant difference in albumin concentration between the two experimental plasma groups at any time point. The microstructure of the encapsulated hepatocytes remained healthier in the CFBB group compared with the static culture group after 6 h of plasma perfusion. CONCLUSION The CFBB can function as a bioartificial liver based on a bioreactor. The efficacy of this novel bioreactor is promising for the study of liver failure.
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23
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Lepers C, André V, Dergham M, Billet S, Verdin A, Garçon G, Dewaele D, Cazier F, Sichel F, Shirali P. Xenobiotic metabolism induction and bulky DNA adducts generated by particulate matter pollution in BEAS-2B cell line: geographical and seasonal influence. J Appl Toxicol 2013; 34:703-13. [DOI: 10.1002/jat.2931] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2013] [Revised: 07/25/2013] [Accepted: 08/13/2013] [Indexed: 11/07/2022]
Affiliation(s)
- Capucine Lepers
- Université Lille Nord de France; France
- ULCO, UCEIV EA4492; F-59140 Dunkerque France
| | - Véronique André
- Normandie Université; France
- UCBN, ABTE EA4651; F-14032 Caen France
- Centre François Baclesse; F-14076 Caen France
| | - Mona Dergham
- Université Lille Nord de France; France
- ULCO, UCEIV EA4492; F-59140 Dunkerque France
| | - Sylvain Billet
- Université Lille Nord de France; France
- ULCO, UCEIV EA4492; F-59140 Dunkerque France
| | - Anthony Verdin
- Université Lille Nord de France; France
- ULCO, UCEIV EA4492; F-59140 Dunkerque France
| | - Guillaume Garçon
- Université Lille Nord de France; France
- ULCO, UCEIV EA4492; F-59140 Dunkerque France
| | - Dorothée Dewaele
- Université Lille Nord de France; France
- ULCO, Centre Commun de Mesure; F-59140 Dunkerque France
| | - Fabrice Cazier
- Université Lille Nord de France; France
- ULCO, Centre Commun de Mesure; F-59140 Dunkerque France
| | - François Sichel
- Normandie Université; France
- UCBN, ABTE EA4651; F-14032 Caen France
- Centre François Baclesse; F-14076 Caen France
| | - Pirouz Shirali
- Université Lille Nord de France; France
- ULCO, UCEIV EA4492; F-59140 Dunkerque France
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Trp266 determines the binding specificity of a porcine aflatoxin B₁ aldehyde reductase for aflatoxin B₁-dialdehyde. Biochem Pharmacol 2013; 86:1357-65. [PMID: 24008121 DOI: 10.1016/j.bcp.2013.08.014] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2013] [Revised: 08/10/2013] [Accepted: 08/12/2013] [Indexed: 11/23/2022]
Abstract
Aflatoxin B₁ (AFB₁) is a severe threat to human and animal health. The aflatoxin B₁ aldehyde reductase (AFAR) family specifically catalyzes AFB₁-dialdehyde, a toxic metabolic intermediate of AFB₁, producing a nontoxic dialcohol. Although several AFARs have been found and characterized, the binding specificity of the family for AFB₁-dialdehyde remains unclear. Herein, according to the published sequence, we cloned a porcine AFAR gene. Recombinant porcine AFAR was expressed and purified from Escherichia coli as hexa-histidine tagged fusion protein. Using the cloned porcine AFAR as a model, site-directed mutagenesis combined with high performance liquid chromatography studies revealed that the substitution of Trp266 with Ala resulted in almost complete loss of catalytic activity for AFB₁-dialdehyde. Interestingly, the substitution of Met86 with Ala exhibited an obviously increased activity to the dialdehyde. Based on these results and by using molecular docking simulations, this work provides a structural explanation for why the AFAR family exhibits high specificity for AFB₁-dialdehyde. The Trp266 residue in porcine AFAR plays a critical role in stabilizing the binding of AFB₁-dialdehyde in the active pocket through the hydrophobic interaction of the side-chain indole ring of Trp266 with the fused coumarin rings of the dialdehyde molecule. The enhanced activity of M86A may be attributed to the formed π-π stacking interaction between Trp266 and the dialdehyde. In addition, other hydrophobic residues (e.g. Phe and Trp) around the dialdehyde molecule also stabilize the substrate binding. The findings may contribute to understanding the substrate specificity of the AFAR family for AFB₁-dialdehyde.
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Tolosa L, Pareja-Ibars E, Donato MT, Cortés M, López S, Jiménez N, Mir J, Castell JV, Gómez-Lechón MJ. Neonatal livers: a source for the isolation of good-performing hepatocytes for cell transplantation. Cell Transplant 2013; 23:1229-42. [PMID: 23803290 DOI: 10.3727/096368913x669743] [Citation(s) in RCA: 32] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. However, the supply of hepatocytes is limited given the shortage of organs available to isolate good-functioning quality cells. Neonatal livers may be a potential source alternative to adult livers to obtain good-performing hepatic cells for hepatocyte transplantation, which has not yet been explored profoundly. High-yield preparations of viable hepatocytes were isolated from 1- to 23-day-old liver donors, cryopreserved, and banked. Cell integrity and functional quality assessment were performed after thawing. Neonatal hepatocytes showed better postthawing recovery compared with adult hepatocytes, as shown by the viability values that did not differ significantly from freshly isolated cells, a higher expression of adhesion molecules (β1-integrin, β-catenin, and E-cadherin), better attachment efficiency, cell survival, and a lower number of apoptotic cells. The metabolic performance of thawed hepatocytes has been assessed by ureogenesis and drug-metabolizing capability (cytochrome P450 and UDP-glucuronosyltransferase enzymes). CYP2A6, CYP2C9, CYP2E1, and CYP3A4 activities were found in all cell preparations, while CYP1A2, CYP2B6, CYP2C19, and CYP2D6 activities were detected only in hepatocytes from a few neonatal donors. The expression of UGT1A1 and UGT1A9 (transcripts and protein) was detected in all hepatocyte preparations, while activity was measured only in some preparations, probably due to lack of maturity of the enzymes. However, isoforms UGT1A6 and UGT2B7 showed considerable activity in all preparations. Compared to adult liver, the hepatocyte isolation procedure in neonatal livers also provides thawed cell suspensions with a higher proportion of hepatic progenitor cells (EpCAM(+) staining), which could also participate in regeneration of liver parenchyma after transplantation. These results could imply important advantages of neonatal hepatocytes as a source of high-quality cells to improve human hepatocyte transplantation applicability.
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Affiliation(s)
- Laia Tolosa
- Unidad de Hepatología Experimental, Centro de Investigación, Instituto de Investigación Sanitaria La Fe, Valencia, Spain
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26
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Ginai M, Elsby R, Hewitt CJ, Surry D, Fenner K, Coopman K. The use of bioreactors as in vitro models in pharmaceutical research. Drug Discov Today 2013; 18:922-35. [PMID: 23748137 DOI: 10.1016/j.drudis.2013.05.016] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2012] [Revised: 04/24/2013] [Accepted: 05/22/2013] [Indexed: 12/23/2022]
Abstract
Bringing a new drug to market is costly in terms of capital and time investments, and any development issues encountered during late-stage clinical trials can often be the result of in vitro-in vivo extrapolations (IVIVE) not accurately reflecting clinical outcome. In the discipline of drug metabolism and pharmacokinetics (DMPK), current in vitro cellular methods do not provide the 3D structure and function of organs found in vivo; therefore, new dynamic methods need to be established to aid improvement of IVIVE. In this review, we highlight the importance of model progression into dynamic systems for use within drug development, focusing on devices developed currently in the areas of the liver and blood-brain barrier (BBB), and the potential to develop models for other organ systems, such as the kidney. We discuss the development of dynamic 3D bioreactor-based systems as in vitro models for use in DMPK studies.
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Affiliation(s)
- Maaria Ginai
- Centre for Biological Engineering, Department of Chemical Engineering, Loughborough University, Loughborough LE11 3TU, UK
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27
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Palakkan AA, Hay DC, Anil Kumar PR, Kumary TV, Ross JA. Liver tissue engineering and cell sources: issues and challenges. Liver Int 2013; 33:666-76. [PMID: 23490085 DOI: 10.1111/liv.12134] [Citation(s) in RCA: 73] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/11/2012] [Accepted: 01/27/2013] [Indexed: 02/13/2023]
Abstract
Liver diseases are of major concern as they now account for millions of deaths annually. As a result of the increased incidence of liver disease, many patients die on the transplant waiting list, before a donor organ becomes available. To meet the huge demand for donor liver, alternative approaches using liver tissue engineering principles are being actively pursued. Even though adult hepatocytes, the primary cells of the liver are most preferred for tissue engineering of liver, their limited availability, isolation from diseased organs, lack of in vitro propagation and deterioration of function acts as a major drawback to their use. Various approaches have been taken to prevent the functional deterioration of hepatocytes including the provision of an adequate extracellular matrix and co-culture with non-parenchymal cells of liver. Great progress has also been made to differentiate human stem cells to hepatocytes and to use them for liver tissue engineering applications. This review provides an overview of recent challenges, issues and cell sources with regard to liver tissue engineering.
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Affiliation(s)
- Anwar A Palakkan
- Tissue Injury and Repair Group, University of Edinburgh - MRC Centre for Regenerative Medicine, Edinburgh, UK
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28
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Nelson LJ, Treskes P, Howie AF, Walker SW, Hayes PC, Plevris JN. Profiling the impact of medium formulation on morphology and functionality of primary hepatocytes in vitro. Sci Rep 2013; 3:2735. [PMID: 24061220 PMCID: PMC3781401 DOI: 10.1038/srep02735] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2013] [Accepted: 09/03/2013] [Indexed: 12/28/2022] Open
Abstract
The characterization of fully-defined in vitro hepatic culture systems requires testing of functional and morphological variables to obtain the optimal trophic support, particularly for cell therapeutics including bioartificial liver systems (BALs). Using serum-free fully-defined culture medium formulations, we measured synthetic, detoxification and metabolic variables of primary porcine hepatocytes (PPHs)--integrated these datasets using a defined scoring system and correlated this hepatocyte biological activity index (HBAI) with morphological parameters. Hepatic-specific functions exceeded those of both primary human hepatocytes (PHHs) and HepaRG cells, whilst retaining biotransformation potential and in vivo-like ultrastructural morphology, suggesting PPHs as a potential surrogate for PHHs in various biotech applications. The HBAI permits assessment of global functional capacity allowing the rational choice of optimal trophic support for a defined operational task (including BALs, hepatocellular transplantation, and cytochrome P450 (CYP450) drug metabolism studies), mitigates risk associated with sub-optimal culture systems, and reduces time and cost of research and therapeutic applications.
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Affiliation(s)
- Leonard J. Nelson
- Hepatology Laboratory, University of Edinburgh, Chancellor's Building, Royal Infirmary of Edinburgh, EH16 4SB, Scotland, UK
| | - Philipp Treskes
- Hepatology Laboratory, University of Edinburgh, Chancellor's Building, Royal Infirmary of Edinburgh, EH16 4SB, Scotland, UK
| | - A. Forbes Howie
- Dept of Clinical Biochemistry, University of Edinburgh, Chancellor's Building, Royal Infirmary of Edinburgh, EH16 4SB, Scotland, UK
| | - Simon W. Walker
- Dept of Clinical Biochemistry, University of Edinburgh, Chancellor's Building, Royal Infirmary of Edinburgh, EH16 4SB, Scotland, UK
| | - Peter C. Hayes
- Hepatology Laboratory, University of Edinburgh, Chancellor's Building, Royal Infirmary of Edinburgh, EH16 4SB, Scotland, UK
| | - John N. Plevris
- Hepatology Laboratory, University of Edinburgh, Chancellor's Building, Royal Infirmary of Edinburgh, EH16 4SB, Scotland, UK
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29
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Helke KL, Swindle MM. Animal models of toxicology testing: the role of pigs. Expert Opin Drug Metab Toxicol 2012; 9:127-39. [PMID: 23216131 DOI: 10.1517/17425255.2013.739607] [Citation(s) in RCA: 87] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
INTRODUCTION In regulatory toxicological testing, both a rodent and non-rodent species are required. Historically, dogs and non-human primates (NHP) have been the species of choice of the non-rodent portion of testing. The pig is an appropriate option for these tests based on metabolic pathways utilized in xenobiotic biotransformation. AREAS COVERED This review focuses on the Phase I and Phase II biotransformation pathways in humans and pigs and highlights the similarities and differences of these models. This is a growing field and references are sparse. Numerous breeds of pigs are discussed along with specific breed differences in these enzymes that are known. While much available data are presented, it is grossly incomplete and sometimes contradictory based on methods used. EXPERT OPINION There is no ideal species to use in toxicology. The use of dogs and NHP in xenobiotic testing continues to be the norm. Pigs present a viable and perhaps more reliable model of non-rodent testing.
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Affiliation(s)
- Kristi L Helke
- Medical University South Carolina, Comparative Medicine, 114 Doughty St, Ste 648, MSC777, Charleston, SC 29425, USA.
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30
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Talbot NC, Caperna TJ, Garrett WM. Growth and Development Symposium: Development, characterization, and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19. J Anim Sci 2012; 91:66-77. [PMID: 23148238 DOI: 10.2527/jas.2012-5748] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
Totipotent embryonic stem cell lines have not been established from ungulates; however, we have developed a somatic stem cell line from the in vitro culture of pig epiblast cells. The cell line, ARS-PICM-19, was isolated via colony cloning and was found to spontaneously differentiate into hepatic parenchymal epithelial cell types, namely hepatocytes and bile duct cells. Hepatocytes form as monolayers and bile duct cells as 3-dimensional bile ductules. Transmission electron microscopy revealed that the ductules were composed of radially arranged, monociliated cells with their cilia projecting into the lumen of the ductule whereas hepatocytes were arranged in monolayers with lateral canalicular structures containing numerous microvilli and connected by tight junctions and desmosomes. Extensive Golgi and rough endoplasmic reticulum networks were also present, indicative of active protein synthesis. Analysis of conditioned medium by 2-dimensional electrophoresis and mass spectrometry indicated a spectrum of serum-protein secretion by the hepatocytes. The PICM-19 cell line maintains a range of inducible cytochrome P450 activities and, most notably, is the only nontransformed cell line that synthesizes urea in response to ammonia challenge. The PICM-19 cell line has been used for several biomedical- and agricultural-related purposes, such as the in vitro replication of hepatitis E virus, a zoonotic virus of pigs, and a spaceflight experiment to evaluate somatic stem cell differentiation and liver cell function in microgravity. The cell line was also evaluated as a platform for toxicity testing and has been used in a commercial artificial liver rescue device bioreactor. A PICM-19 subclone, PICM-19H, which only differentiates into hepatocytes, was isolated and methods are currently under development to grow PICM-19 cells without feeder cells. Feeder-cell-independent growth will facilitate the study of mesenchymal-parenchymal interactions that influence the divergent differentiation of the PICM-19 cells, enhance our ability to genetically modify the cells, and provide a better model system to investigate porcine hepatic metabolism.
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Affiliation(s)
- N C Talbot
- USDA, ARS, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA.
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31
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Zhao LF, Pan XP, Li LJ. Key challenges to the development of extracorporeal bioartificial liver support systems. Hepatobiliary Pancreat Dis Int 2012; 11:243-9. [PMID: 22672816 DOI: 10.1016/s1499-3872(12)60155-6] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
BACKGROUND For nearly three decades, extracorporeal bioartificial liver (BAL) support systems have been anticipated as promising tools for the treatment of liver failure. However, these systems are still far from clinical application. This review aimed to analyze the key challenges to the development of BALs. DATA SOURCE We carried out a PubMed search of English-language articles relevant to extracorporeal BAL support systems and liver failure. RESULTS Extracorporeal BALs face a series of challenges. First, an appropriate cell source for BAL is not readily available. Second, existing bioreactors do not provide in vivo-like oxygenation and bile secretion. Third, emergency needs cannot be met by current BALs. Finally, the effectiveness of BALs, either in animals or in patients, has been difficult to document. CONCLUSIONS Extracorporeal BAL support systems are mainly challenged by incompetent cell sources and flawed bioreactors. To advance this technology, future research is needed to provide more insights into interpreting the conditions for hepatocyte differentiation and liver microstructure formation.
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Affiliation(s)
- Li-Fu Zhao
- Zhejiang University School of Medicine, Hangzhou, China
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32
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Alexandre E, Baze A, Parmentier C, Desbans C, Pekthong D, Gerin B, Wack C, Bachellier P, Heyd B, Weber JC, Richert L. Plateable cryopreserved human hepatocytes for the assessment of cytochrome P450 inducibility: experimental condition-related variables affecting their response to inducers. Xenobiotica 2012; 42:968-79. [DOI: 10.3109/00498254.2012.676693] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
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Giantin M, Zancanella V, Lopparelli RM, Granato A, Carletti M, Vilei MT, Muraca M, Baratto C, Dacasto M. Effects of time culture and prototypical cytochrome P450 3A (CYP3A) inducers on CYP2B22, CYP2C, CYP3A and nuclear receptor (NR) mRNAs in long-term cryopreserved pig hepatocytes (CPHs). Drug Metab Pharmacokinet 2012; 27:495-505. [PMID: 22447117 DOI: 10.2133/dmpk.dmpk-11-rg-146] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
In the present study, transcriptional and post-translational effects of culturing time and prototypical cytochrome P450 3A (CYP3A) inducers on principal nuclear receptors (NRs), CYP2B22, 2C and 3A were investigated in long-term stored (~10 years) cryopreserved pig hepatocytes (CPHs). In the time-course study, a crush and rise effect was observed for pregnane X receptor (NR1I2) and constitutive androstane receptor (NR1I3) mRNAs, while a time-dependent increase of retinoid X receptor alpha (NR2B1) was noticed. Cytochrome P450 gene expression profiles were down-regulated as a function of time. In the induction study, an increase of NR1I2, NR1I3 and NR2B1 mRNAs was observed in dexamethasone-exposed CPHs. About CYPs, an overall up-regulation was seen in CPHs exposed to phenobarbital, while dexamethasone and rifampicin up-regulated only CYP3A. In both studies, transcriptional CYP results were confirmed at the post-translational level (immunoblotting and enzyme activities), except for CYP2B immunoblotting in the induction study. The present data demonstrate that long-term stored CPHs may be used to investigate mechanisms involved in CYPs regulation, expression and function; provide further info about NR regulation of CYPs, and confirm species-differences in these mechanisms of regulation; finally, they suggest the usefulness and relevance of gene expression profiling to early detect any modulation of CYP expression and bioactivity.
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Affiliation(s)
- Mery Giantin
- Dipartimento di Biomedicina Comparata e Alimentazione, Università degli Studi di Padova, Padova, Italy.
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Zamaratskaia G, Zlabek V, Ropstad E, Andresen Ø. In vitro and in vivo association of porcine hepatic cytochrome P450 3A and 2C activities with testicular steroids. Reprod Domest Anim 2012; 47:891-8. [PMID: 22276943 DOI: 10.1111/j.1439-0531.2012.01986.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
The aim of this study was to screen the inhibitory potential of several testicular steroids on cytochrome P450 3A (CYP3A) and 2C (CYP2C) activities in porcine liver microsomes. The microsomes used in this study were obtained from pubertal male pigs of two breeds, Landrace and Duroc. For the in vitro inhibition study, porcine microsomes were incubated in the presence of 17β-estradiol, 17α-estradiol, androstenone, dehydroepiandrosterone and dihydrotestosterone. Both reversible and mechanism-based inhibitions were examined. 7-benzyloxyresorufin (BR) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC) were used as substrates for CYP3A, and diclofenac and tolbutamide (TB) as substrates for CYP2C. 7-benzyloxyresorufin O-dealkylase (BROD) activity was inhibited by all tested steroids in the microsomes from Landrace pigs via mechanism-based mode, but in the microsomes from Duroc pigs, BROD activities were inhibited only in the presence of 17β-oestradiol. Mechanism-based inhibition of BFC metabolism by the tested steroids was observed in the microsomes from both breeds, but this inhibition was weak and did not exceed 20%. TB hydroxylase (TBOH) activity in the microsomes from Duroc pigs was inhibited by 17α-oestradiol through the mechanism-based mode of inhibition. None of the investigated steroids inhibited TBOH activity in Landrace pigs. For the in vivo study, male pigs were injected with a single dose of human chorionic gonadotropin (hCG) to stimulate testicular steroid production by the Leydig cells. In vivo stimulation with hGC did not alter BROD activity either in Landrace or in Duroc pigs. BFC metabolism was significantly induced by hCG stimulation in both breeds and TBOH activity only in Duroc pigs. Activity of diclofenac hydroxylase was not detected in either Landrace or Duroc pigs. Breed significantly affected BROD and TBOH activity with BROD being higher in Landrace and TBOH in Duroc pigs. This study improved our understanding of the role of testicular steroids in the regulation of porcine CYP450 activity.
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Affiliation(s)
- G Zamaratskaia
- Department of Food Science, BioCenter, Swedish University of Agricultural Sciences, Uppsala, Sweden.
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35
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Sevior DK, Pelkonen O, Ahokas JT. Hepatocytes: the powerhouse of biotransformation. Int J Biochem Cell Biol 2011; 44:257-61. [PMID: 22123318 DOI: 10.1016/j.biocel.2011.11.011] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2010] [Revised: 10/12/2011] [Accepted: 11/14/2011] [Indexed: 01/10/2023]
Abstract
Liver is the most important organ involved in biotransformation of xenobiotics. Within the main organisational unit, the hepatocyte, is an assembly of enzymes commonly classified as phase I and phase II enzymes. The phase I enzymes principally cytochrome P450 catalyse both oxidative and reductive reactions of a bewildering number of xenobiotics. Many of the products of phase I enzymes become substrates for the phase II enzymes, which catalyse conjugation reactions making use of endogenous cofactors. As xenobiotic metabolising enzymes are responsible for the toxicity of many chemicals and drugs, testing the role of the biotransformation enzymes and the transporters within the hepatocyte is critical. New methodologies may be able to provide information to allow for better in vitro to in vivo extrapolation of data.
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Cytochrome P450 expression profile of the PICM-19H pig liver cell line: potential application to rapid liver toxicity assays. In Vitro Cell Dev Biol Anim 2011; 46:11-9. [PMID: 19915937 DOI: 10.1007/s11626-009-9244-z] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2009] [Accepted: 09/30/2009] [Indexed: 10/20/2022]
Abstract
Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM- 19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with β-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6β-OH-, 2α-OH- and 2β-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD50 was calculated to be 14.9±0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrenetreated cultures and untreated controls; CD50 were 1.59 μM and 31 μM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.
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37
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Wang J, Jiang J, Zhang H, Wang J, Cai H, Li C, Li K, Liu J, Guo X, Zou G, Wang D, Deng Y, Dai J. Integrated transcriptional and proteomic analysis with in vitro biochemical assay reveal the important role of CYP3A46 in T-2 toxin hydroxylation in porcine primary hepatocytes. Mol Cell Proteomics 2011; 10:M111.008748. [PMID: 21685020 DOI: 10.1074/mcp.m111.008748] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Both T-2 toxin and its metabolites are highly potent mycotoxins that can cause severe human and animal diseases upon exposure. Understanding the toxic mechanism and biotransformation process of T-2 toxin at a cellular level is essential for the development of counter-measures. We investigated the effect of T-2 toxin in porcine primary hepatocytes using porcine genome array and two-dimensional difference gel electrophoresis with matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry. Integrated transcriptional and proteomic analysis demonstrated that T-2 toxin adversely affected porcine hepatocytes by initiating lipid metabolism disorder, oxidative stress response, and apoptosis. In addition, xenobiotic metabolism genes, including cytochrome P450 3As (CYP3A46 and CYP3A39), carboxylesterase 1Cs (CES1C4 and CES1C5), and epoxide hydrolase (EPHX1), increased in T-2 toxin treatment cells. Using HepG2 cells to over-express the recombinant xenobiotic metabolism genes above and rapid resolution liquid chromatography/tandem mass spectrometry to detect metabolites of T-2 toxin, we determined that porcine CYP3A46 mainly catalyzed T-2 to form 3'-hydroxy-T-2, which was further confirmed by purified CYP3A46 protein. However, recombinant porcine CES1C5 and EPHX1 did not enhance hydrolysis and de-epoxidation of T-2 implying that other esterases and epoxide hydrolases may play dominant roles in those reactions.
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Affiliation(s)
- Jianshe Wang
- Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
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Comparison of the substrate kinetics of pig CYP3A29 with pig liver microsomes and human CYP3A4. Biosci Rep 2011; 31:211-20. [DOI: 10.1042/bsr20100084] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022] Open
Abstract
CYP (cytochrome P450) 3A29 in pigs could be an important candidate gene responsible for xenobiotic metabolism, similar to CYP3A4 in humans. Accordingly, the tissue expression of CYP3A29 mRNA in domestic pigs has been determined by a real-time PCR. The enzymatic properties of CYP3A29, CYP3A4 and PLM (pig liver microsomes) were compared by kinetic analysis of TST (testosterone) 6β-hydroxylation and NIF (nifedipine) oxidation. CYP3A29 mRNA was highly expressed in the liver and small intestines of domestic pigs. The CYP3A29 enzyme expressed in Sf9 cells had the same TST-metabolizing activity as human CYP3A4 based on their roughly equal in vitro intrinsic clearance values. The affinity of CYP3A29 for NIF was lower than that of CYP3A4 but higher than that of PLM. KET (ketoconazole) was a more potent inhibitor of TST 6β-hydroxylation and NIF oxidation activities of CYP3A29 than TAO (troleandomycin). These findings indicate that pig CYP3A29 is similar to human CYP3A4 in both extent of expression and activity. The results reported in this paper provide a basis for future in vitro toxicity and metabolism studies.
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39
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Puccinelli E, Gervasi PG, La Marca M, Beffy P, Longo V. Expression and inducibility by phenobarbital of CYP2C33, CYP2C42, CYP2C49, CYP2B22, and CYP3As in porcine liver, kidney, small intestine, and nasal tissues. Xenobiotica 2010; 40:525-35. [PMID: 20509749 DOI: 10.3109/00498254.2010.489125] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
In this study, the expression and inducibility of CYP2C33, CYP2C42, CYP2C49, CYP2B22, CYP3A22, CYP3A29, and CYP3A46 were investigated at activity and/or transcriptional level in liver, kidney, small intestine, respiratory, and olfactory nasal mucosa of control and phenobarbital (PB)-treated pigs. PB treatment resulted in an up-regulation of mRNA levels of all analyzed CYPs in liver, of CYP2C42 and CYP2C49 in kidney, of CYP2C42, CYP2C49, CYP2B22, and CYP3As in small intestine. In liver microsomes from PB-treated pigs, these transcriptional activations were accompanied by an increase of various marker activities of human CYP2B6, CYP3As, CYP2C9, CYP2C19. Among the extrahepatic tissues, a significant induction by PB was observed only in kidney for the marker activities of CYP2C9. Taken together, our results demonstrated that the PB administration in pigs induced at least in liver, in addition to CYP2B22 and CYP3As, the expression of CYP2C33, CYP2C42, and CYP2C49 at transcriptional and activity levels. Furthermore our findings showed that the catalytic activities of porcine CYP2Cs are different amongst those observed and with respect to the human counterparts. Thus, the use of pigs as a model for humans in studies using drugs as substrates and/or inducers of CYP2Cs should be considered carefully.
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Cabaret O, Puel O, Botterel F, Pean M, Khoufache K, Costa JM, Delaforge M, Bretagne S. Metabolic Detoxication Pathways for Sterigmatocystin in Primary Tracheal Epithelial Cells. Chem Res Toxicol 2010; 23:1673-81. [DOI: 10.1021/tx100127b] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Affiliation(s)
- Odile Cabaret
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
| | - Olivier Puel
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
| | - Françoise Botterel
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
| | - Michel Pean
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
| | - Khaled Khoufache
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
| | - Jean-Marc Costa
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
| | - Marcel Delaforge
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
| | - Stéphane Bretagne
- UMR BIPAR, U-PEC, AFSSA, ENVA, Faculté de Médecine, Créteil Cedex F-94010, France, AP-HP, Groupe hospitalier Henri Mondor-Albert Chenevier, Laboratoire de Parasitologie-Mycologie, Créteil Cedex F-94010, France, INRA, UR 66, Laboratoire de Pharmacologie-Toxicologie, Toulouse F-31027, France, CEA, DSV, IBEB, Group Rech Appl Phytotechnol, Saint-Paul-lez-Durance F-13108, France, CNRS, UMR Biol Veget & Microbiol Environ, Saint-Paul-lez-Durance F-13108, France, Aix-Marseille Université, Saint-Paul-lez-Durance
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Donato MT, Hallifax D, Picazo L, Castell JV, Houston JB, Gomez-Lechón MJ, Lahoz A. Metabolite formation kinetics and intrinsic clearance of phenacetin, tolbutamide, alprazolam, and midazolam in adenoviral cytochrome P450-transfected HepG2 cells and comparison with hepatocytes and in vivo. Drug Metab Dispos 2010; 38:1449-55. [PMID: 20501911 DOI: 10.1124/dmd.110.033605] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the K(m) or S(50) values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CL(int) (or CL(max)), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CL(int) values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1'-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CL(int) for the substrates studied (in a range of 20-200% in vivo observed CL(int)). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CL(int)) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.
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Affiliation(s)
- M Teresa Donato
- Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Avenida Campanar 21, Valencia, Spain.
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Validated assay for studying activity profiles of human liver UGTs after drug exposure: inhibition and induction studies. Anal Bioanal Chem 2010; 396:2251-63. [DOI: 10.1007/s00216-009-3441-1] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2009] [Revised: 12/23/2009] [Accepted: 12/28/2009] [Indexed: 10/19/2022]
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Effect of β-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig. Toxicology 2009; 265:69-79. [DOI: 10.1016/j.tox.2009.09.010] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2009] [Revised: 09/16/2009] [Accepted: 09/17/2009] [Indexed: 12/30/2022]
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Sakai Y, Tanaka T, Fukuda J, Nakazawa K. Alkoxyresorufin O-dealkylase assay using a rat hepatocyte spheroid microarray. J Biosci Bioeng 2009; 109:395-9. [PMID: 20226384 DOI: 10.1016/j.jbiosc.2009.10.001] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2009] [Revised: 09/28/2009] [Accepted: 10/01/2009] [Indexed: 11/25/2022]
Abstract
Hepatocyte multicellular aggregates (spheroids), which maintain high expression of liver functions, have been advocated as a useful culture technique for various cell-based assays. In this study, we investigated the drug metabolic function of a hepatocyte spheroid microarray (HSM) chip, which contained an array of 672 spheroids of primary rat hepatocytes within a 100-mm(2) region in the center of a poly(methylmethacrylate) plate (24 x 24 mm) and used an alkoxyresorufin (ethoxy-, methoxy-, pentoxy- and benzyloxyresorufin) O-dealkylase assay system. Ethoxyresorufin O-dealkylase (EROD) activity of the HSM chip initiated by 3-methylcholanthrene (3-MC), an inducer of cytochrome P450 enzymes, was 5- to 10-fold higher than that of monolayer hepatocytes, with activity being maintained for at least 2 weeks. We also demonstrated that 3-MC induced EROD, methoxyresorufin O-dealkylase (MROD) and benzyloxyresorufin O-dealkylase (BROD) activities in the HSM chip, while sodium phenobarbital (P450 inducer) induced pentoxyresorufin O-dealkylase (PROD), BROD, EROD and MROD activities. Induction of these activities was confirmed by increased gene expression of the related P450 enzymes. These results showed that the HSM chip had a good response to P450 inducers and that function was maintained for long periods of time. The HSM chip therefore may be a promising cellular platform for drug metabolic assays using hepatocytes.
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Affiliation(s)
- Yusuke Sakai
- Department of Life and Environment Engineering, The University of Kitakyushu, 1-1 Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan
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Rodeiro I, Donato MT, Jimenez N, Garrido G, Molina-Torres J, Menendez R, Castell JV, Gómez-Lechón MJ. Inhibition of human P450 enzymes by natural extracts used in traditional medicine. Phytother Res 2009; 23:279-82. [PMID: 18844254 DOI: 10.1002/ptr.2613] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Different medicinal plants are widely used in Cuba and Mexico to treat several disorders. This paper reports in vitro inhibitory effects on the P450 system of herbal products commonly used by people in Cuba and Mexico in traditional medicine for decades. Experiments were conducted in human liver microsomes. The catalytic activities of CYP1A1/2, 2D6, and 3A4 were measured using specific probe substrates. The Heliopsis longipes extract exhibited a concentration-dependent inhibition of the three enzymes, and similar effects were produced by affinin (an alkamide isolated from the H. longipes extract) and two catalytically reduced alkamides. Mangifera indica L. and Thalassia testudinum extracts, two natural polyphenol-rich extracts, diminished CYP1A1/2 and 3A4 activities, but not the CYP2D6 activity. These results suggest that these herbs inhibit the major human P450 enzymes involved in drug metabolism and could induce potential herbal-drug interactions.
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Affiliation(s)
- Idania Rodeiro
- Laboratorio de Farmacología, Centro de Química Farmacéutica, 200 y 21, Atabey, Playa, Ciudad de La Habana, Cuba
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Messina A, Chirulli V, Gervasi PG, Longo V. Purification, molecular cloning, heterologous expression and characterization of pig CYP1A2. Xenobiotica 2009; 38:1453-70. [PMID: 18949657 DOI: 10.1080/00498250802474437] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Porcine cytochrome P450 (CYP) 1A2 was purified to electrophoretic homogeneity from the hepatic microsomes of beta-naphthoflavone-treated male pigs. In a reconstituted system, this enzyme showed a good catalytic activity towards caffeine, acetanilide, and methoxyresorufin, all known markers of mammalian CYP1A2. Using 3'- and 5'-rapid amplification of coding DNA (cDNA) ends (RACE), we amplified from the liver RNA of control pigs a full-length 1827 bp cDNA containing an open reading frame of 1548 bp which encoded a putative CYP1A2 protein of 516 amino acids and an estimated Mr of 58 380 Da. Reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that the messenger RNA (mRNA) of CYP1A2 was expressed in liver, heart and nasal mucosa but not in lung, small intestine, kidney and brain. Using the pCW vector containing a N-terminal modified cDNA, pig CYP1A2 was expressed in Escherichia coli. 3-[(3-Chloroamidopropyl)dimethylmmonio]-1-propane-sulfonate (CHAPS)-solubilized E. coli preparations expressing CYP1A2 produced a functionally isoform which, in a reconstituted system, was catalytically active toward ethoxyresorufin and methoxyresorufin showing K(m)'s similar to those obtained with CYP1A2 purified from pig liver or human recombinant CYP1A2. Taken together, these results demonstrate that domestic pigs have a functionally active CYP1A2 gene well expressed in the liver with biochemical properties quite similar to those corresponding to the human enzyme.
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Affiliation(s)
- A Messina
- Istituto di Fisiologia Clinica, Area della Ricerca CNR, Pisa, Italy
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Genotoxic potential of Polycyclic Aromatic Hydrocarbons-coated onto airborne Particulate Matter (PM2.5) in human lung epithelial A549 cells. Cancer Lett 2008; 270:144-55. [DOI: 10.1016/j.canlet.2008.04.044] [Citation(s) in RCA: 72] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2008] [Revised: 04/29/2008] [Accepted: 04/29/2008] [Indexed: 10/22/2022]
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Omasa T, Kim K, Hiramatsu S, Katakura Y, Kishimoto M, Enosawa S, Ohtake H. Construction and Evaluation of Drug-Metabolizing Cell Line for Bioartificial Liver Support System. Biotechnol Prog 2008; 21:161-7. [PMID: 15903254 DOI: 10.1021/bp049757a] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Focusing on drug metabolism in liver, we constructed and evaluated a drug-metabolizing bioartificial liver (BAL) support system. In a previous study, we constructed ammonia-metabolizing CHO and hepatoma-derived HepG2 cell lines by recombination of the glutamine synthetase (GS) gene. For further mimicking of liver metabolism, the human hepatoma-derived cell line HepG2 was transformed by the pBudCE-GS-CYP3A4 vector, which contains GS and drug-metabolizing CYP 3A4 genes. The constructed GS-3A4-HepG2 cell line showed 3A4 activity higher than that of human primary hepatocytes. The drug-metabolizing activity of BAL (BAL clearance) was evaluated using this cell line. The estimated clearance was higher than that of the human hepatocyte system.
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Affiliation(s)
- Takeshi Omasa
- Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita Osaka 565-0871, Japan.
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Nannelli A, Chirulli V, Longo V, Gervasi PG. Expression and induction by rifampicin of CAR- and PXR-regulated CYP2B and CYP3A in liver, kidney and airways of pig. Toxicology 2008; 252:105-12. [PMID: 18786598 DOI: 10.1016/j.tox.2008.08.004] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2008] [Revised: 07/23/2008] [Accepted: 08/13/2008] [Indexed: 10/21/2022]
Abstract
The transcript levels of CYP2B22, 3A22, 3A29, 3A46, CAR, PXR and HNF4alpha were investigated in liver, kidney and airways from control and rifampicin-treated male pigs. The presence and induction of CYP genes transcription were studied by RT-PCR, real-time PCR, Western blotting and enzymatic activity whereas the expression of receptors was studied by RT-PCR or real-time PCR. Pretreatment with rifampicin resulted in a transcriptional activation, although to different extents, of all the CYP3A genes in liver but not in kidney, lung, bronchi or trachea. In the hepatic microsomes, the induction of CYP3A genes was accompanied by an increase of CYP3As marker activities and of two protein bands immunoreactive with anti-human CYP3A4. The CYP2B22 transcript was found to be markedly induced only in liver and kidney. In parallel, a protein band immunoreactive with anti-rat CYP2B1 was elevated while enhanced CYP2B marker activities were observed in hepatic and renal microsomes. As expected, based on human data, the basal expression of CAR, PXR and HNF4alpha was found to be high in liver and low in airways and not susceptible to induction by rifampicin. A significant expression of these transcriptional factors was also demonstrated in kidney. Thus, it is likely that rifampicin induced CYP2B22 both in liver and kidney of pig, not via activation of CAR, but via PXR, through a cross-talk mechanism, as previously observed in human liver. Taken together, our results demonstrated a differential expression and regulation of three individual CYP3As, CYP2B22, CAR, PXR and HNF4alpha genes in liver, kidney and airways of pig.
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Affiliation(s)
- Annalisa Nannelli
- Istituto di Fisiologia Clinica, CNR, Area della Ricerca CNR, via Moruzzi, 1, 56100 Pisa, Italy
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50
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Implications of hepatic cytochrome P450-related biotransformation processes in veterinary sciences. Eur J Pharmacol 2008; 585:502-9. [DOI: 10.1016/j.ejphar.2008.03.013] [Citation(s) in RCA: 79] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2008] [Revised: 03/05/2008] [Accepted: 03/06/2008] [Indexed: 02/07/2023]
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