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Saran C, Fu D, Ho H, Klein A, Fallon JK, Honkakoski P, Brouwer KLR. A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity. Sci Rep 2022; 12:14333. [PMID: 35995956 PMCID: PMC9395349 DOI: 10.1038/s41598-022-18174-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2022] [Accepted: 08/05/2022] [Indexed: 11/09/2022] Open
Abstract
Hepatic cell lines serve as economical and reproducible alternatives for primary human hepatocytes. However, the utility of hepatic cell lines to examine bile acid homeostasis and cholestatic toxicity is limited due to abnormal expression and function of bile acid-metabolizing enzymes, transporters, and the absence of canalicular formation. We discovered that culturing HuH-7 human hepatoma cells with dexamethasone (DEX) and 0.5% dimethyl sulfoxide (DMSO) for two weeks, with Matrigel overlay after one week, resulted in a shorter and improved differentiation process. These culture conditions increased the expression and function of the major bile acid uptake and efflux transporters, sodium taurocholate co-transporting polypeptide (NTCP) and the bile salt export pump (BSEP), respectively, in two-week cultures of HuH-7 cells. This in vitro model was further characterized for expression and function of bile acid-metabolizing enzymes, transporters, and cellular bile acids. Differentiated HuH-7 cells displayed a marked shift in bile acid composition and induction of cytochrome P450 (CYP) 7A1, CYP8B1, CYP3A4, and bile acid-CoA: amino acid N-acyltransferase (BAAT) mRNAs compared to control. Inhibition of taurocholate uptake and excretion after a 24-h treatment with prototypical cholestatic drugs suggests that differentiated HuH-7 cells are a suitable model to examine cholestatic hepatotoxicity.
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Affiliation(s)
- Chitra Saran
- Department of Pharmacology, UNC School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA
| | - Dong Fu
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA
| | - Henry Ho
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA
| | - Abigail Klein
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA
| | - John K Fallon
- Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA
| | - Paavo Honkakoski
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA
- School of Pharmacy, University of Eastern Finland, Kuopio, Finland
| | - Kim L R Brouwer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
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2
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Lu H, Lei X, Winkler R, John S, Kumar D, Li W, Alnouti Y. Crosstalk of hepatocyte nuclear factor 4a and glucocorticoid receptor in the regulation of lipid metabolism in mice fed a high-fat-high-sugar diet. Lipids Health Dis 2022; 21:46. [PMID: 35614477 PMCID: PMC9134643 DOI: 10.1186/s12944-022-01654-6] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Accepted: 05/06/2022] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Hepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study aimed to elucidate the role of down-regulation of HNF4α and GR in fatty liver and hyperlipidemia. METHODS Adult mice with liver-specific heterozygote (HET) and knockout (KO) of HNF4α or GR were fed a high-fat-high-sugar diet (HFHS) for 15 days. Alterations in hepatic and circulating lipids were determined with analytical kits, and changes in hepatic mRNA and protein expression in these mice were quantified by real-time PCR and Western blotting. Serum and hepatic levels of bile acids were quantified by LC-MS/MS. The roles of HNF4α and GR in regulating hepatic gene expression were determined using luciferase reporter assays. RESULTS Compared to HFHS-fed wildtype mice, HNF4α HET mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4α KO mice had fatty liver but mild hypolipidemia, down-regulation of lipid-efflux genes, and induction of genes for uptake, synthesis, and storage of lipids. Serum levels of chenodeoxycholic acid and deoxycholic acid tended to be decreased in the HNF4α HET mice but dramatically increased in the HNF4α KO mice, which was associated with marked down-regulation of cytochrome P450 7a1, the rate-limiting enzyme for bile acid synthesis. Hepatic mRNA and protein expression of sterol-regulatory-element-binding protein-1 (SREBP-1), a master lipogenic regulator, was induced in HFHS-fed HNF4α HET mice. In reporter assays, HNF4α cooperated with the corepressor small heterodimer partner to potently inhibit the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Hepatic nuclear GR proteins tended to be decreased in the HNF4α KO mice. HFHS-fed mice with liver-specific KO of GR had increased hepatic lipids and induction of SREBP-1C and PPARγ, which was associated with a marked decrease in hepatic levels of HNF4α proteins in these mice. In reporter assays, GR and HNF4α synergistically/additively induced lipid catabolic genes. CONCLUSIONS induction of lipid catabolic genes and suppression of lipogenic genes by HNF4α and GR may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.
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Affiliation(s)
- Hong Lu
- Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, 13210, USA.
| | - Xiaohong Lei
- Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, 13210, USA
| | - Rebecca Winkler
- Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY, 13210, USA
| | - Savio John
- Department of Medicine, SUNY Upstate Medical University, Syracuse, NY, 13210, USA
| | - Devendra Kumar
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, 68198, USA
| | - Wenkuan Li
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, 68198, USA
| | - Yazen Alnouti
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, 68198, USA
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3
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Alamoudi JA, Li W, Gautam N, Olivera M, Meza J, Mukherjee S, Alnouti Y. Bile acid indices as biomarkers for liver diseases I: Diagnostic markers. World J Hepatol 2021; 13:433-455. [PMID: 33959226 PMCID: PMC8080550 DOI: 10.4254/wjh.v13.i4.433] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Revised: 02/11/2021] [Accepted: 03/22/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Hepatobiliary diseases result in the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues which may contribute to an unfavorable prognosis. AIM To discover and validate diagnostic biomarkers of cholestatic liver diseases based on the urinary BA profile. METHODS We analyzed urine samples by liquid chromatography-tandem mass spectrometry and compared the urinary BA profile between 300 patients with hepatobiliary diseases vs 103 healthy controls by statistical analysis. The BA profile was characterized using BA indices, which quantifies the composition, metabolism, hydrophilicity, and toxicity of the BA profile. BA indices have much lower inter- and intra-individual variability compared to absolute concentrations of BA. In addition, BA indices demonstrate high area under the receiver operating characteristic curves, and changes of BA indices are associated with the risk of having a liver disease, which demonstrates their use as diagnostic biomarkers for cholestatic liver diseases. RESULTS Total and individual BA concentrations were higher in all patients. The percentage of secondary BA (lithocholic acid and deoxycholic acid) was significantly lower, while the percentage of primary BA (chenodeoxycholic acid, cholic acid, and hyocholic acid) was markedly higher in patients compared to controls. In addition, the percentage of taurine-amidation was higher in patients than controls. The increase in the non-12α-OH BA was more profound than 12α-OH BA (cholic acid and deoxycholic acid) causing a decrease in the 12α-OH/ non-12α-OH ratio in patients. This trend was stronger in patients with more advanced liver diseases as reflected by the model for end-stage liver disease score and the presence of hepatic decompensation. The percentage of sulfation was also higher in patients with more severe forms of liver diseases. CONCLUSION BA indices have much lower inter- and intra-individual variability compared to absolute BA concentrations and changes of BA indices are associated with the risk of developing liver diseases.
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Affiliation(s)
- Jawaher Abdullah Alamoudi
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Wenkuan Li
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Nagsen Gautam
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Marco Olivera
- Department of Internal Medicine, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Jane Meza
- Department of Biostatistics, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Sandeep Mukherjee
- Department of Internal Medicine, College of Medicine, Creighton University Medical Center, Omaha, NE 68124, United States
| | - Yazen Alnouti
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States.
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4
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Fang M, Zhang Q, Yu P, Ge C, Guo J, Zhang Y, Wang H. The effects, underlying mechanism and interactions of dexamethasone exposure during pregnancy on maternal bile acid metabolism. Toxicol Lett 2020; 332:97-106. [PMID: 32599024 DOI: 10.1016/j.toxlet.2020.06.011] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2020] [Revised: 06/07/2020] [Accepted: 06/16/2020] [Indexed: 12/12/2022]
Abstract
As important members in steroids related signal pathways, bile acids are very important in regulating substance metabolism and immune homeostasis. However, bile acids are highly cytotoxic, and the excessive accumulation can induce several abnormalities such as cholestatic liver injury. It is known that the bile acid metabolism alters during pregnancy and mostly will not result in pathologies. However, the effect of dexamethasone exposure during pregnancy on bile acid metabolism is still unknown. In this study, pregnant Wistar rats were subcutaneously administered dexamethasone (0.2 mg/kg.d) or saline from gestation day 9-21, while virgin rats were given the same treatment for 13 days. We found that, physiological pregnancy or dexamethasone exposure during non-pregnancy did not affect maternal serum TBA level and liver function. Nevertheless, dexamethasone exposure during pregnancy increased serum TBA level and accompanied with liver injury. Furthermore, we discovered that the conservation of bile acid homeostasis under pregnancy or dexamethasone exposure was maintained through compensatory pathways. However, dexamethasone exposure during pregnancy tipped the balance of liver bile acid homeostasis by increasing classical synthesis and decreasing efflux and uptake. In addition, dexamethasone exposure during pregnancy also increased serum estrogen level and nuclear receptors mRNA expression levels. Finally, two-way ANOVA analysis showed that dexamethasone exposure during pregnancy could induce or facilitate maternal cholestasis and liver injury by up-regulating ERα and CYP7A1 expression. This study confirmed that dexamethasone exposure during pregnancy was related to maternal intrahepatic cholestasis of pregnancy and should be carefully monitored in clinical settings.
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Affiliation(s)
- Man Fang
- Department of Obstetrics and Gynaecology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuchang District, Wuhan, 430071, China
| | - Qi Zhang
- Department of Pharmacology, Basic Medical School of Wuhan University, 185 Donghu Road, Wuchang District, Wuhan, 430071, China
| | - Pengxia Yu
- Department of Pharmacology, Basic Medical School of Wuhan University, 185 Donghu Road, Wuchang District, Wuhan, 430071, China
| | - Caiyun Ge
- Department of Obstetrics and Gynaecology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuchang District, Wuhan, 430071, China
| | - Juanjuan Guo
- Department of Obstetrics and Gynaecology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuchang District, Wuhan, 430071, China
| | - Yuanzhen Zhang
- Department of Obstetrics and Gynaecology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuchang District, Wuhan, 430071, China; Hubei Provincial Key Laboratory of Developmentally Originated Diseases, 185 Donghu Road, Wuchang District, Wuhan, 430071, China.
| | - Hui Wang
- Department of Obstetrics and Gynaecology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuchang District, Wuhan, 430071, China; Department of Pharmacology, Basic Medical School of Wuhan University, 185 Donghu Road, Wuchang District, Wuhan, 430071, China; Hubei Provincial Key Laboratory of Developmentally Originated Diseases, 185 Donghu Road, Wuchang District, Wuhan, 430071, China.
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5
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Nikolaou N, Arvaniti A, Appanna N, Sharp A, Hughes BA, Digweed D, Whitaker MJ, Ross R, Arlt W, Penning TM, Morris K, George S, Keevil BG, Hodson L, Gathercole LL, Tomlinson JW. Glucocorticoids regulate AKR1D1 activity in human liver in vitro and in vivo. J Endocrinol 2020; 245:207-218. [PMID: 32106090 PMCID: PMC7182088 DOI: 10.1530/joe-19-0473] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Accepted: 02/27/2020] [Indexed: 12/14/2022]
Abstract
Steroid 5β-reductase (AKR1D1) is highly expressed in human liver where it inactivates endogenous glucocorticoids and catalyses an important step in bile acid synthesis. Endogenous and synthetic glucocorticoids are potent regulators of metabolic phenotype and play a crucial role in hepatic glucose metabolism. However, the potential of synthetic glucocorticoids to be metabolised by AKR1D1 as well as to regulate its expression and activity has not been investigated. The impact of glucocorticoids on AKR1D1 activity was assessed in human liver HepG2 and Huh7 cells; AKR1D1 expression was assessed by qPCR and Western blotting. Genetic manipulation of AKR1D1 expression was conducted in HepG2 and Huh7 cells and metabolic assessments were made using qPCR. Urinary steroid metabolite profiling in healthy volunteers was performed pre- and post-dexamethasone treatment, using gas chromatography-mass spectrometry. AKR1D1 metabolised endogenous cortisol, but cleared prednisolone and dexamethasone less efficiently. In vitro and in vivo, dexamethasone decreased AKR1D1 expression and activity, further limiting glucocorticoid clearance and augmenting action. Dexamethasone enhanced gluconeogenic and glycogen synthesis gene expression in liver cell models and these changes were mirrored by genetic knockdown of AKR1D1 expression. The effects of AKR1D1 knockdown were mediated through multiple nuclear hormone receptors, including the glucocorticoid, pregnane X and farnesoid X receptors. Glucocorticoids down-regulate AKR1D1 expression and activity and thereby reduce glucocorticoid clearance. In addition, AKR1D1 down-regulation alters the activation of multiple nuclear hormone receptors to drive changes in gluconeogenic and glycogen synthesis gene expression profiles, which may exacerbate the adverse impact of exogenous glucocorticoids.
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Affiliation(s)
- Nikolaos Nikolaou
- Oxford Centre for Diabetes,
Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre,
University of Oxford, Churchill Hospital, Oxford, UK
| | - Anastasia Arvaniti
- Oxford Centre for Diabetes,
Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre,
University of Oxford, Churchill Hospital, Oxford, UK
- Department of Biological and Medical
Sciences, Oxford Brookes University, Oxford,
UK
| | - Nathan Appanna
- Oxford Centre for Diabetes,
Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre,
University of Oxford, Churchill Hospital, Oxford, UK
| | - Anna Sharp
- Oxford Centre for Diabetes,
Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre,
University of Oxford, Churchill Hospital, Oxford, UK
| | - Beverly A Hughes
- Institute of Metabolism and Systems
Research, University of Birmingham, Edgbaston, Birmingham,
UK
| | | | | | - Richard Ross
- Department of Oncology and
Metabolism, Faculty of Medicine, Dentistry and Health,
University of Sheffield, Sheffield, UK
| | - Wiebke Arlt
- Institute of Metabolism and Systems
Research, University of Birmingham, Edgbaston, Birmingham,
UK
- NIHR Birmingham Biomedical Research
Centre, University Hospitals Birmingham NHS Foundation Trust
and University of Birmingham, Birmingham, UK
| | - Trevor M Penning
- Department of Systems Pharmacology &
Translational Therapeutics, University of Pennsylvania Perelman
School of Medicine, Philadelphia, Pennsylvania, USA
| | - Karen Morris
- Biochemistry Department,
Manchester University NHS Trust, Manchester, UK
| | - Sherly George
- Biochemistry Department,
Manchester University NHS Trust, Manchester, UK
| | - Brian G Keevil
- Biochemistry Department,
Manchester University NHS Trust, Manchester, UK
| | - Leanne Hodson
- Oxford Centre for Diabetes,
Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre,
University of Oxford, Churchill Hospital, Oxford, UK
| | - Laura L Gathercole
- Oxford Centre for Diabetes,
Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre,
University of Oxford, Churchill Hospital, Oxford, UK
- Department of Biological and Medical
Sciences, Oxford Brookes University, Oxford,
UK
| | - Jeremy W Tomlinson
- Oxford Centre for Diabetes,
Endocrinology and Metabolism, NIHR Oxford Biomedical Research Centre,
University of Oxford, Churchill Hospital, Oxford, UK
- Correspondence should be addressed to J W Tomlinson:
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6
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Phelps T, Snyder E, Rodriguez E, Child H, Harvey P. The influence of biological sex and sex hormones on bile acid synthesis and cholesterol homeostasis. Biol Sex Differ 2019; 10:52. [PMID: 31775872 PMCID: PMC6880483 DOI: 10.1186/s13293-019-0265-3] [Citation(s) in RCA: 85] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/27/2019] [Accepted: 09/11/2019] [Indexed: 12/13/2022] Open
Abstract
Obesity and elevated serum lipids are associated with a threefold increase in the risk of developing atherosclerosis, a condition that underlies stroke, myocardial infarction, and sudden cardiac death. Strategies that aim to reduce serum cholesterol through modulation of liver enzymes have been successful in decreasing the risk of developing atherosclerosis and reducing mortality. Statins, which inhibit cholesterol biosynthesis in the liver, are considered among the most successful compounds developed for the treatment of cardiovascular disease. However, recent debate surrounding their effectiveness and safety prompts consideration of alternative cholesterol-lowering therapies, including increasing cholesterol catabolism through bile acid (BA) synthesis. Targeting the enzymes that convert cholesterol to BAs represents a promising alternative to other cholesterol-lowering approaches that treat atherosclerosis as well as fatty liver diseases and diabetes mellitus. Compounds that modify the activity of these pathways have been developed; however, there remains a lack of consideration of biological sex. This is necessary in light of strong evidence for sexual dimorphisms not only in the incidence and progression of the diseases they influence but also in the expression and activity of the proteins affected and in the manner in which men and women respond to drugs that modify lipid handling in the liver. A thorough understanding of the enzymes involved in cholesterol catabolism and modulation by biological sex is necessary to maximize their therapeutic potential.
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Affiliation(s)
- Taylor Phelps
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA
| | - Erin Snyder
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA
| | - Erin Rodriguez
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA
| | - Hailey Child
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA
| | - Pamela Harvey
- Department of Molecular, Cellular, and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA.
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7
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Prenatal dexamethasone exposure-induced a gender-difference and sustainable multi-organ damage in offspring rats via serum metabolic profile analysis. Toxicol Lett 2019; 316:136-146. [DOI: 10.1016/j.toxlet.2019.09.007] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Revised: 08/01/2019] [Accepted: 09/08/2019] [Indexed: 11/19/2022]
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8
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Rendic SP, Peter Guengerich F. Human cytochrome P450 enzymes 5-51 as targets of drugs and natural and environmental compounds: mechanisms, induction, and inhibition - toxic effects and benefits. Drug Metab Rev 2019; 50:256-342. [PMID: 30717606 DOI: 10.1080/03602532.2018.1483401] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Cytochrome P450 (P450, CYP) enzymes have long been of interest due to their roles in the metabolism of drugs, pesticides, pro-carcinogens, and other xenobiotic chemicals. They have also been of interest due to their very critical roles in the biosynthesis and metabolism of steroids, vitamins, and certain eicosanoids. This review covers the 22 (of the total of 57) human P450s in Families 5-51 and their substrate selectivity. Furthermore, included is information and references regarding inducibility, inhibition, and (in some cases) stimulation by chemicals. We update and discuss important aspects of each of these 22 P450s and questions that remain open.
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Affiliation(s)
| | - F Peter Guengerich
- b Department of Biochemistry , Vanderbilt University School of Medicine , Nashville , TN , USA
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9
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Improving Energy Metabolism of Deproteinized Extract of Calf Blood Through Regulation of Hmgcs2, Cpt1a, Angptl4, Cyp8b1, and Ehhadh Genes in Mice. Chem Res Chin Univ 2019. [DOI: 10.1007/s40242-019-9021-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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10
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Hua C, Geng Y, Chen Q, Niu L, Cai L, Tao S, Ni Y, Zhao R. Effects of chronic dexamethasone exposure on bile acid metabolism and cecal epithelia function in goats. Domest Anim Endocrinol 2018; 65:9-16. [PMID: 29803110 DOI: 10.1016/j.domaniend.2018.05.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/24/2017] [Revised: 05/08/2018] [Accepted: 05/08/2018] [Indexed: 01/20/2023]
Abstract
Bile acids (BAs) are synthesized in the liver via the oxidation of cholesterol and further metabolized by microbiota in the gut, where they simultaneously impact gut function. In the present study, 10 goats were randomly divided into 2 groups; 1 group was injected with dexamethasone (Dex; 0.2 mg/kg), and the other group was injected with saline as the control (Con) for 21 d. Expression levels of key genes and proteins in the liver and gut mucosa were analyzed and compared to investigate the impact of chronic stress on BA metabolism and related functions in ruminants. The results revealed that Dex decreased plasma total BAs (TBAs) concentration (P < 0.05) but increased TBA concentration in the cecal digesta (P < 0.05). Total cholesterol in the liver decreased moderately in response to Dex. The protein expression of cytochrome P450 family 7 subfamily A member 1 and cytochrome P450 family 27 subfamily A member 1, 2 enzymes that control BA synthesis in the liver, remained unchanged by Dex administration (P > 0.05). The expression of several genes in the cecal mucosa encoding epithelial tight junction proteins, including occludin (P < 0.05), tight junction protein 1 (P < 0.01), and claudin 1 (P < 0.05), increased significantly in response to Dex, and expression of defensin beta 1, which can strengthen the innate immune system, was also upregulated (P < 0.05). In addition, BAs increased the expression of the Solute Carrier family 9 member A 2 (P < 0.01) that encodes a sodium hydrogen exchanger. These results suggest that the Dex-induced disruption of BA homeostasis might be mediated through a liver-independent pathway in goats, and the Dex-induced accumulation of TBAs in the cecal digesta may improve volatile fatty acid transportation and mucosal defense.
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Affiliation(s)
- C Hua
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China
| | - Y Geng
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China
| | - Q Chen
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China
| | - L Niu
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China
| | - L Cai
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China
| | - S Tao
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China
| | - Y Ni
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China.
| | - R Zhao
- Key Laboratory of Animal Physiology & Biochemistry, Nanjing Agricultural University, Nanjing, P.R. China
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11
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Gabbia D, Pozzo L, Zigiotto G, Roverso M, Sacchi D, Dalla Pozza A, Carrara M, Bogialli S, Floreani A, Guido M, De Martin S. Dexamethasone counteracts hepatic inflammation and oxidative stress in cholestatic rats via CAR activation. PLoS One 2018; 13:e0204336. [PMID: 30252871 PMCID: PMC6155538 DOI: 10.1371/journal.pone.0204336] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2018] [Accepted: 09/06/2018] [Indexed: 12/19/2022] Open
Abstract
Glucocorticoids (GCs) are currently used for the therapeutic management of cholestatic diseases, but their use and molecular mechanism remain controversial. The aims of this study were 1) to assess the therapeutic effect of a 2-week treatment with the GC dexamethasone on hepatic damage in bile duct-ligated rats; 2) to investigate its effect on the activation of the nuclear receptors (NRs) pregnane X receptor (PXR), constitutive androstane receptor (CAR) and GC receptor (GR), and NF-kB, as well as on oxidative stress and bile acid (BA) hepatic composition. Cholestasis was induced by ligation of bile duct (BDL animals) in 16 male Wistar-Kyoto rats, and eight of them were daily treated by oral gavage with 0.125 mg/ml/kg DEX for 14 days. Eight Sham-operated rats were used as controls. Severity of cholestasis was assessed histologically and on plasma biochemical parameters. The nuclear expression of NF-kB (p65), GR, PXR and CAR was measured in hepatic tissue by Western Blot. Oxidative stress was evaluated by measuring malondialdehyde, carbonylated proteins, GHS and ROS content in rat livers. LC-MS was used to measure the plasma and liver concentration of 7 BAs. Histological findings and a significant drop in several markers of inflammation (p65 nuclear translocation, mRNA expressions of TNF-α, IL-1β, IL-6) showed that DEX treatment reversed cholestasis-induced inflammation, and similar results have been obtained with oxidative stress markers. The nuclear expression of p65 and CAR were inversely correlated, with the latter increasing significantly after DEX treatment (p<0.01 vs vehicle). Hepatic BA levels tended to drop in the untreated cholestatic rats, whereas they were similar to those of healthy rats in DEX-treated animals. Plasma BAs decreased significantly in DEX-treated animals with respect to untreated cholestatic rats. In conclusion, DEX reduces inflammation and oxidative stress in BDL rats, and probably CAR is responsible for this effect. Therefore, this NR represents a promising pharmacological target for managing cholestatic and inflammatory liver diseases.
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Affiliation(s)
- Daniela Gabbia
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Luisa Pozzo
- Institute of Agricultural Biology and Biotechnology, CNR, Pisa, Italy
| | - Giorgia Zigiotto
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Marco Roverso
- Department of Chemical Sciences, University of Padova, Padova, Italy
| | - Diana Sacchi
- Department of Medicine, General Pathology and Cytopathology Unit, University of Padova, Padova, Italy
| | - Arianna Dalla Pozza
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Maria Carrara
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
| | - Sara Bogialli
- Department of Chemical Sciences, University of Padova, Padova, Italy
| | - Annarosa Floreani
- Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy
| | - Maria Guido
- Department of Medicine, General Pathology and Cytopathology Unit, University of Padova, Padova, Italy
| | - Sara De Martin
- Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy
- * E-mail:
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Hassan HM, Yousef BA, Guo H, Xiaoxin L, Zhang L, Jiang Z. Investigating the CYP2E1 Potential Role in the Mechanisms Behind INH/LPS-Induced Hepatotoxicity. Front Pharmacol 2018; 9:198. [PMID: 29563874 PMCID: PMC5850051 DOI: 10.3389/fphar.2018.00198] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2017] [Accepted: 02/21/2018] [Indexed: 12/14/2022] Open
Abstract
Tuberculosis (TB) is one of the oldest infectious diseases that affected humankind and remains one of the world's deadliest communicable diseases that could be considered as global emergency, but the discovery and development of isoniazid (INH) in the 1950s paved the way to an effective single and/or combined first-line anti-TB therapy. However, administration of INH induces severe hepatic toxicity in some patients. Previously, we establish a rat model of INH hepatotoxicity utilizing the inflammatory stress theory, in which bacterial lipopolysaccharide (LPS) potentially enhanced INH toxicity. These enhancing activities ranged between augmenting the inflammatory stress, oxidative stress, alteration of bile acid homeostasis, and CYP2E1 over-expression. Although pre-treatment with dexamethasone (DEX) helped overcome both inflammatory and oxidative stress which ended-up in alleviation of LPS augmenting effects, but still minor toxicities were being detected, alongside with CYP2E1 over expression. This finding positively indicated the corner-stone role played by CYP2E1 in the pathogenesis of INH/LPS-induced liver damage. Therefore, we examined whether INH/LPS co-treatment with CYP2E1 inhibitor diallyl sulfide (DAS) and DEX can protect against the INH/LPS-induced hepatotoxicity. Our results showed that pre-administration of both DAS and DEX caused significant reduction in serum TBA, TBil, and gamma-glutamyl transferase levels. Furthermore, the histopathological analysis showed that DAS and DEX could effectively reverse the liver lesions seen following INH/LPS treatment and protect against hepatic steatosis as indicated by absence of lipid accumulation. Pre-treatment with DAS alone could not completely block the CYP2E1 protein expression following INH/LPS treatment, as appeared in the immunoblotting and immunohistochemistry results. This is probably due to the fact that the combined enhancement activities of both INH and LPS on CYP2E1 protein expression levels might resist the blocking probabilities of DAS. In the meantime, addition of DEX to the DAS/INH/LPS combination caused a significant reduction in CYP2E1 protein expression as revealed by the immunoblotting and fading coloration in immunohistochemistry results. Thus, addition of DEX and DAS together caused strong protection against INH/LPS-induced hepatic damage. These findings reveal the potential therapeutic value of combining DAS and DEX with INH in TB management for reducing the potential risk and incidences of hepatotoxicity.
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Affiliation(s)
- Hozeifa M Hassan
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China.,Department of Pharmacology, Faculty of Pharmacy, University of Gezira, Wad Medani, Sudan
| | - Bashir A Yousef
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China.,Department of Pharmacology, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan
| | - Hongli Guo
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China
| | - Liu Xiaoxin
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China
| | - Luyong Zhang
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China.,Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing, China.,Center for Drug Screening and Pharmacodynamics Evaluation, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou, China
| | - Zhenzhou Jiang
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, China.,Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing, China
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Sarkar U, Ravindra KC, Large E, Young CL, Rivera-Burgos D, Yu J, Cirit M, Hughes DJ, Wishnok JS, Lauffenburger DA, Griffith LG, Tannenbaum SR. Integrated Assessment of Diclofenac Biotransformation, Pharmacokinetics, and Omics-Based Toxicity in a Three-Dimensional Human Liver-Immunocompetent Coculture System. Drug Metab Dispos 2017; 45:855-866. [PMID: 28450578 PMCID: PMC5469400 DOI: 10.1124/dmd.116.074005] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2016] [Accepted: 04/21/2017] [Indexed: 12/13/2022] Open
Abstract
In vitro hepatocyte culture systems have inherent limitations in capturing known human drug toxicities that arise from complex immune responses. Therefore, we established and characterized a liver immunocompetent coculture model and evaluated diclofenac (DCF) metabolic profiles, in vitro-in vivo clearance correlations, toxicological responses, and acute phase responses using liquid chromatography-tandem mass spectrometry. DCF biotransformation was assessed after 48 hours of culture, and the major phase I and II metabolites were similar to the in vivo DCF metabolism profile in humans. Further characterization of secreted bile acids in the medium revealed that a glycine-conjugated bile acid was a sensitive marker of dose-dependent toxicity in this three-dimensional liver microphysiological system. Protein markers were significantly elevated in the culture medium at high micromolar doses of DCF, which were also observed previously for acute drug-induced toxicity in humans. In this immunocompetent model, lipopolysaccharide treatment evoked an inflammatory response that resulted in a marked increase in the overall number of acute phase proteins. Kupffer cell-mediated cytokine release recapitulated an in vivo proinflammatory response exemplified by a cohort of 11 cytokines that were differentially regulated after lipopolysaccharide induction, including interleukin (IL)-1β, IL-1Ra, IL-6, IL-8, IP-10, tumor necrosis factor-α, RANTES (regulated on activation normal T cell expressed and secreted), granulocyte colony-stimulating factor, macrophage colony-stimulating factor, macrophage inflammatory protein-1β, and IL-5. In summary, our findings indicate that three-dimensional liver microphysiological systems may serve as preclinical investigational platforms from the perspective of the discovery of a set of clinically relevant biomarkers including potential reactive metabolites, endogenous bile acids, excreted proteins, and cytokines to predict early drug-induced liver toxicity in humans.
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Affiliation(s)
- Ujjal Sarkar
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Kodihalli C Ravindra
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Emma Large
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Carissa L Young
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Dinelia Rivera-Burgos
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Jiajie Yu
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Murat Cirit
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - David J Hughes
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - John S Wishnok
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Douglas A Lauffenburger
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Linda G Griffith
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
| | - Steven R Tannenbaum
- Departments of Biological Engineering (U.S., K.C.R., C.L.Y., D.R.-B., J.Y., M.C., J.S.W., D.A.L., L.G.G., S.R.T.) and Chemistry (S.R.T.), Massachusetts Institute of Technology, Cambridge, Massachusetts; and CN Bio Innovations Ltd., Welwyn Garden City, Hertfordshire, United Kingdom (E.L., D.J.H.)
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Hassan HM, Guo H, Yousef BA, Ping-Ping D, Zhang L, Jiang Z. Dexamethasone Pretreatment Alleviates Isoniazid/Lipopolysaccharide Hepatotoxicity: Inhibition of Inflammatory and Oxidative Stress. Front Pharmacol 2017; 8:133. [PMID: 28360859 PMCID: PMC5350150 DOI: 10.3389/fphar.2017.00133] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2016] [Accepted: 03/03/2017] [Indexed: 12/14/2022] Open
Abstract
Isoniazid (INH) remains a cornerstone key constitute of the current tuberculosis management strategy, but its hepatotoxic potentiality remains a significant clinical problem. Our previous findings succeed to establish a rat model of INH hepatotoxicity employing the inflammatory stress theory in which non-injurious doses of inflammatory-mediating agent bacterial lipopolysaccharides (LPS) augmented the toxicity of INH that assist to uncover the mechanisms behind INH hepatotoxicity. Following LPS exposure, several inflammatory cells are activated and it is likely that the consequences of this activation rather than direct hepatocellular effects of LPS underlie the ability of LPS to augment toxic responses. In this study, we investigated the potential protective role of the anti-inflammatory agent dexamethasone (DEX), a potent synthetic glucocorticoid, in INH/LPS hepatotoxic rat model. DEX pre-treatment successfully eliminates the components of the inflammatory stress as shown through analysis of blood biochemistry and liver histopathology. DEX potentiated hepatic anti-oxidant mechanisms while serum and hepatic lipid profiles were reduced. However, DEX administration was not able to revoke the principal effects of cytochrome P450 2E1 (CYP2E1) in INH/LPS-induced liver damage. In conclusion, this study illustrated the DEX-preventive capabilities on INH/LPS-induced hepatotoxicity model through DEX-induced potent anti-inflammatory activity whereas the partial toxicity seen in the model could be attributed to the expression of hepatic CYP2E1. These findings potentiate the clinical applications of DEX co-administration with INH therapy in order to reduce the potential incidences of hepatotoxicity.
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Affiliation(s)
- Hozeifa M Hassan
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical UniversityNanjing, China; Department of Pharmacology, Faculty of Pharmacy, University of GeziraWad-Medani, Sudan
| | - Hongli Guo
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University Nanjing, China
| | - Bashir A Yousef
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical UniversityNanjing, China; Department of Pharmacology, Faculty of Pharmacy, University of KhartoumKhartoum, Sudan
| | - Ding Ping-Ping
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University Nanjing, China
| | - Luyong Zhang
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical UniversityNanjing, China; Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical UniversityNanjing, China
| | - Zhenzhou Jiang
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical UniversityNanjing, China; Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of EducationNanjing, China
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