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Liu X, Fang C, Yu H, Huang L, Feng J, Luo S, Song L, Wu M, Tan Y, Dong J, Gong T, Xiao P. Chondroitin Sulfate-Based Imatinib Nanoparticles Targeting Activated Hepatic Stellate Cells Against Hepatic Fibrosis. Pharmaceutics 2025; 17:351. [PMID: 40143016 PMCID: PMC11944399 DOI: 10.3390/pharmaceutics17030351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2025] [Revised: 02/16/2025] [Accepted: 02/28/2025] [Indexed: 03/28/2025] Open
Abstract
Background: Activated hepatic stellate cells (aHSCs) play a significant role during the onset of hepatic fibrosis, ultimately leading to excessive deposition of extracellular matrix (ECM) and other typical pathological features, and thus have become a popular target for the treatment of hepatic fibrosis. However, current aHSC-centric therapy strategies achieve unsatisfactory results, mainly due to the lack of approved anti-fibrosis drugs and sufficiently efficient aHSC-targeted delivery systems. In this study, our aim was to develop an Imatinib-loaded nanoparticle delivery system based on a chondroitin sulfate derivative to enhance aHSC targeting efficiency, improve the therapeutic effect for hepatic fibrosis, and investigate the underlying mechanism. Methods: The carboxyl group of chondroitin sulfate and the amino group of 1-hexadecylamine were linked by an amide bond in this study to produce the amphiphilic carrier CS-HDA. Then, the Imatinib-loaded nanoparticles (IM-CS NPs) were designed to efficiently target aHSCs through CD44-mediated endocytosis and effectively inhibit HSC overactivation via PDGF and TGF-β signaling pathways. Results: Both in vitro cellular uptake experiments and in vivo distribution experiments demonstrated that CS-HDA-modified nanoparticles (IM-CS NPs) exhibited a better targeting ability for aHSCs, which were subsequently utilized to treat carbon tetrachloride-induced hepatic fibrosis mouse models. Finally, significant fibrosis resolution was observed in the carbon tetrachloride-induced hepatic fibrosis mouse models after tail vein injection of the IM-CS NPs, along with their outstanding biocompatibility and biological safety. Conclusions: IM-loaded NPs based on an amphiphilic CS derivative have remarkable antifibrotic effects, providing a promising avenue for the clinical treatment of advanced hepatic fibrosis.
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Affiliation(s)
- Xunzhi Liu
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Changlong Fang
- Department of Pharmacy, Chongqing University Fuling Hospital, Chongqing University, Chongqing 408099, China;
| | - Hongling Yu
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Lu Huang
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Jiaxing Feng
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Shiqin Luo
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Li Song
- Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu 610041, China;
| | - Mengying Wu
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Yulu Tan
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Jianxia Dong
- Department of Pharmacy, West China Hospital, Sichuan University, Chengdu 610041, China;
| | - Tao Gong
- Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry and Sichuan Province, Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for Drug Precision Industrial Technology, West China School of Pharmacy, Sichuan University, Chengdu 610041, China; (X.L.); (H.Y.); (L.H.); (J.F.); (S.L.); (M.W.); (Y.T.); (T.G.)
| | - Peihong Xiao
- Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu 610041, China;
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Kaur I, Vasudevan A, Rawal P, Tripathi DM, Ramakrishna S, Kaur S, Sarin SK. Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements. Bioengineering (Basel) 2023; 10:131. [PMID: 36829625 PMCID: PMC9952008 DOI: 10.3390/bioengineering10020131] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2022] [Revised: 01/11/2023] [Accepted: 01/13/2023] [Indexed: 01/20/2023] Open
Abstract
Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the "sandwich cultures", were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, life-span and hepatic functions of these cells in vitro for several downstream applications. In the current review, we highlight the details, limitations and prospects of different technical strategies being used in primary hepatocyte cultures. We discuss the use of newer biomaterials as scaffolds for efficient culture of primary hepatocytes. We also describe the derivation of mature hepatocytes from other cellular sources such as induced pluripotent stem cells, bone marrow stem cells and 3D liver organoids. Finally, we also explain the use of perfusion-based bioreactor systems and bioengineering strategies to support the long-term function of hepatocytes in 3D conditions.
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Affiliation(s)
- Impreet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Ashwini Vasudevan
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Preety Rawal
- School of Biotechnology, Gautam Buddha University, Greater Noida 201312, India
| | - Dinesh M. Tripathi
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Seeram Ramakrishna
- Department of Mechanical Engineering, National University of Singapore, Singapore 117581, Singapore
| | - Savneet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Shiv K. Sarin
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
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Birkus G, Snyder C, Jordan R, Kobayashi T, Dick R, Puscau V, Li L, Ramirez R, Willkom M, Morikawa Y, Delaney Iv WE, Schmitz U. Anti-HBV activity of retinoid drugs in vitro versus in vivo. Antiviral Res 2019; 169:104538. [PMID: 31226346 DOI: 10.1016/j.antiviral.2019.104538] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2019] [Revised: 05/20/2019] [Accepted: 06/17/2019] [Indexed: 02/06/2023]
Abstract
We describe here the anti-HBV activity of natural and synthetic retinoids in primary human hepatocytes (PHHs). The most potent compounds inhibited HBsAg, HBeAg, viral RNA and DNA production by HBV infected cells with EC50 values ranging from 0.4 to 2.6 μM. The activity was independent of PHH donor and HBV genotype used in testing. 13-cis retinoic acid (Accutane) was selected for further evaluation in the PXB chimeric mouse model of HBV infection at doses allowing to achieve Accutane peak serum concentrations near its antiviral EC90 and exposures ∼5-fold higher than a typical clinical dose. While these supraclinical exposures of 100 mg/kg/day were well-tolerated by regular Balb/c mice, PXB mice were more sensitive and even a lower those of 60 mg/kg/day led to significant weight loss. Despite dosing at this maximal tolerated dose for 28 days, Accutane failed to show any anti-HBV activity. RAR target engagement was verified using transcriptome analysis of liver samples from treated versus vehicle groups. However, gene expression changes in PXB liver samples were vastly muted when compared to the in vitro PHH system. When comparing transcriptional changes associated with the conditioning of fresh hepatocytes toward enabling HBV infection, we also observed a large number of changes. Noticeably, a significant number of genes that were up- or down-regulated by the conditioning process were down- or up-regulated by HBV infected PHH treatment with Accutane, respectively. While the lack of efficacy in the PXB model may have many explanations, the observed, opposing transcriptional changes upon conditioning PHH and treating these cultured, HBV-infected PHH with Accutane allow for the possibility that the PHH system may yield artificial anti-HBV hits.
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Affiliation(s)
- Gabriel Birkus
- IOCB, Flemingovo nám. 542/2, 160 00, Praha 6, Czech Republic
| | - Chelsea Snyder
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Robert Jordan
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | | | - Ryan Dick
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Vlad Puscau
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Li Li
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | - Ricardo Ramirez
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA
| | | | - Yoshida Morikawa
- Phoenix Bio, 3-4-1, Kagamiyama, Higashi-Hiroshima City, 739-0046, Japan
| | | | - Uli Schmitz
- Gilead Sciences, 333 Lakeside Drive, Foster City, CA, 94494, USA.
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Foretz M, Viollet B. Measurement of AMPK-Induced Inhibition of Lipid Synthesis Flux in Cultured Cells. Methods Mol Biol 2018; 1732:363-371. [PMID: 29480487 DOI: 10.1007/978-1-4939-7598-3_23] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
AMP-activated protein kinase (AMPK) is a master regulator of multiple cellular metabolic pathways, including lipid metabolism. Some of the well-known substrates of AMPK are acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, regulatory enzymes of fatty acid and cholesterol synthesis, respectively. The discovery that both of them are inactivated by AMPK suggested the therapeutic potential of AMPK activation in the treatment of metabolic diseases associated with lipid disorders, such as nonalcoholic fatty liver disease (NAFLD). Here we describe a method to measure lipid synthesis flux in intact cells from the saponifiable (including fatty acids) and non-saponifiable (including sterols) fractions of lipid extracts.
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Affiliation(s)
- Marc Foretz
- U1016, Institut Cochin, Inserm, Paris, France.
- UMR8104, CNRS, Paris, France.
- Université Paris Descartes, Paris, France.
| | - Benoit Viollet
- UMR8104, CNRS, Paris, France
- Université Paris Descartes, Paris, France
- U1016, Department EMD, Institut Cochin, Inserm, Paris, France
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From in vivo to in vitro: Major metabolic alterations take place in hepatocytes during and following isolation. PLoS One 2017; 12:e0190366. [PMID: 29284039 PMCID: PMC5746264 DOI: 10.1371/journal.pone.0190366] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2017] [Accepted: 12/13/2017] [Indexed: 12/20/2022] Open
Abstract
The liver plays a key role in maintaining physiological homeostasis and hepatocytes are largely responsible for this. The use of isolated primary hepatocytes has become an essential tool for the study of nutrient physiology, xenobiotic metabolism and several liver pathologies. Since hepatocytes are removed from their normal environment, the isolation procedure and in vitro culture of primary hepatocytes is partially known to induce undesired metabolic changes. We aimed to perform a thorough metabolic profiling of primary cells before, during and after isolation using state-of-the-art techniques. Extensive metabolite measurements using HPLC were performed in situ in the liver, during hepatocyte isolation using the two-step collagenase perfusion method and during in vitro cell culture for up to 48 hours. Assessment of mitochondrial respiratory capacity and ATP-linked respiration of isolated primary hepatocytes was performed using extracellular flux analysis. Primary hepatocytes displayed a drastic decrease in antioxidative-related metabolites (NADPH, NADP, GSH and GSSG) during the isolation procedure when compared to the in situ liver (P<0.001). Parallel assessment of citric acid cycle activity showed a significant decrease of up to 95% in Acetyl-CoA, Isocitrate/Citrate ratio, Succinate, Fumarate and Malate in comparison to the in situ liver (P<0.001). While the levels of several cellular energetic metabolites such as Adenosine, AMP, ADP and ATP were found to be progressively reduced during the isolation procedure and cell culture (P<0.001), higher ATP/ADP ratio and energy charge level were observed when primary cells were cultured in vitro compared to the in situ liver (P<0.05). In addition, a significant decrease in the respiratory capacity occurred after 24 hours in culture. Interestingly, this was not associated with a significant modification of ATP-linked respiration. In conclusion, major metabolic alterations occur immediately after hepatocytes are removed from the liver. These changes persist or increase during in vitro culture. These observations need to be taken into account when using primary hepatocytes for the study of metabolism or liver physiopathology.
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Physiological oxygen tension reduces hepatocyte dedifferentiation in in vitro culture. Sci Rep 2017; 7:5923. [PMID: 28724942 PMCID: PMC5517567 DOI: 10.1038/s41598-017-06433-3] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2017] [Accepted: 06/12/2017] [Indexed: 12/31/2022] Open
Abstract
Primary hepatocytes cultured in vitro are a powerful tool to study the functions of hepatocytes and to evaluate the metabolism and toxicity of new drugs. However, in vitro culture of hepatocytes has proven to be very difficult. Ordinary culture conditions lead to dedifferentiation of hepatocytes, resulting in rapid change in cell morphology and significant reduction in specific cell functions. In the current study, we show that hepatocyte dedifferentiation is a rapid process under 21% O2 conditions. Hepatocytes cultured in 21% O2 undergo epithelial-to-mesenchymal transition (EMT), obtain fibroblast-like morphology, and show decreased hepatic functions. In contrast, 5% O2 is very effective in maintaining the epithelial morphology and many functions of the primary hepatocytes cultured in vitro for up to five days. These functions include albumin production, glycogen storage, LDL-uptake and CYP450-mediated drug metabolism. Furthermore, we find that 5% O2 can relieve the production of reactive oxygen species (ROS) and decrease the level of DNA damage in primary cultured hepatocytes. In addition, we also show that blocking the ERK and GSK-3β pathways can inhibit the dedifferentiation of hepatocytes to a certain extent. Lowering the oxygen tension in cell culture is easily achievable, we believe it could be combined with other methods, such as the use of small molecule cocktails and 3D culture, to maintain proliferation and functions of primary hepatocytes in vitro.
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WATANABE KAZUTO, HASEGAWA KAORU, KOGA MUTUYOSI. ROLE OF PLASMIN INHIBITORS IN GROWTH OF ADULT RAT HEPATOCYTES IN SERUM-FREE PRIMARY CULTURE . Biomed Res 2017. [DOI: 10.2220/biomedres.8.377] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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Mörk LM, Strom SC, Mode A, Ellis EC. Addition of Dexamethasone Alters the Bile Acid Composition by Inducing CYP8B1 in Primary Cultures of Human Hepatocytes. J Clin Exp Hepatol 2016; 6:87-93. [PMID: 27493455 PMCID: PMC4963319 DOI: 10.1016/j.jceh.2016.01.007] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/04/2015] [Accepted: 01/22/2016] [Indexed: 12/12/2022] Open
Abstract
BACKGROUND Primary human hepatocytes offer the best human in vitro model for studies on human liver cell metabolism. Investigators use a variety of different media supplements and matrix biocoatings and the type of culture system used may influence the outcome. OBJECTIVES To optimize in vitro conditions for primary human hepatocytes with regard to bile acid synthesis. METHODS Human hepatocytes were isolated and cultured on collagen type I or EHS matrigel in cell media with or without dexamethasone. The glucocorticoid receptor (GR) antagonist RU486 was used to elucidate the involvement of GR. RESULTS Hepatocytes cultured on EHS matrigel produced more bile acids and expressed higher levels of cholesterol 7α-hydroxylase (CYP7A1) than cells cultured on rat tail collagen. Supplementation with dexamethasone increased the formation of cholic acid (CA) and decreased chenodeoxycholic acid formation. In line with these results, the mRNA expression of sterol 12α-hydroxylase (CYP8B1) increased following dexamethasone treatment. Surprisingly, the mRNA expression of CYP7A1 and CYP27A1 was not increased to the same extent. By using the GR antagonist RU486, we concluded that CYP8B1 induction is mediated via a GR-independent pathway. An altered expression of retinoid-related orphan receptor (ROR) α and ROR α target gene Glucose-6-phosphatase (G6Pase) suggests that ROR α signaling may regulate CYP8B1 expression. CONCLUSION Primary human hepatocytes have an increased bile acid synthesis rate when cultured on matrigel as compared to collagen. Exposure to glucocorticoid hormones stimulates the expression of CYP8B1, leading to an increased formation of CA and alteration of the bile acid composition. The effect is most likely mediated through a GR-independent pathway, possibly through ROR α.
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Key Words
- BSEP, bile salt export pump
- CA, cholic acid
- CDCA, chenodeoxycholic acid
- CYP27A1, sterol 27α-hydroxylase
- CYP7A1, cholesterol 7α-hydroxylase
- CYP8B1, sterol 12α-hydroxylase
- FXR, farnesoid X receptor
- G6Pase, glucose-6-phosphatase
- GR, glucocorticoid receptor
- NTCP, Na+-taurocholate cotransporting polypeptide
- PXR, pregnane X receptor
- ROR, retinoid-related orphan receptor
- chenodeoxycholic acid
- cholic acid
- dexamethasone
- matrigel
- primary hepatocytes
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Affiliation(s)
- Lisa-Mari Mörk
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden,Address for correspondence: Lisa-Mari Mörk, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, F82, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. Tel.: +46 8 585 83062; fax: +46 8 585 82912.
| | - Stephen C. Strom
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institute, Sweden
| | - Agneta Mode
- Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden
| | - Ewa C.S. Ellis
- Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden
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Farooq M, Kelly EJ, Unadkat JD. CYP2D6 Is Inducible by Endogenous and Exogenous Corticosteroids. Drug Metab Dispos 2016; 44:750-7. [PMID: 26965986 PMCID: PMC4851303 DOI: 10.1124/dmd.115.069229] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2015] [Accepted: 03/09/2016] [Indexed: 12/21/2022] Open
Abstract
Although cytochrome P450 (CYP) 2D6 has been widely considered to be noninducible on the basis of human hepatocyte studies, in vivo data suggests that it is inducible by endo- and xenobiotics. Therefore, we investigated if the experimental conditions routinely used in human hepatocyte studies may be a confounding factor in the lack of in vitro induction of CYP2D6. Sandwich cultured human hepatocytes (SCHH) were preincubated with or without dexamethasone (100 nM) for 72 hours before incubation with 1μM endogenous (cortisol or corticosterone) or exogenous (dexamethasone or prednisolone) corticosteroids. At 72 hours, CYP2D6 mRNA, protein, and activity were quantified by real-time quantitative polymerase chain reaction, quantitative proteomics, and formation of dextrorphan from dextromethorphan, respectively. In the absence of supplemental dexamethasone, CYP2D6 activity, mRNA, and protein were significantly and robustly (>10-fold) induced by all four corticosteroids. However, this CYP2D6 induction was abolished in cells preincubated with supplemental dexamethasone. These data show, for the first time, that CYP2D6 is inducible in vitro but the routine presence of 100 nM dexamethasone in the culture medium masks this induction. Our cortisol data are in agreement with the clinical observation that CYP2D6 is inducible during the third trimester of pregnancy when the plasma concentrations of cortisol increase to ∼1μM. These findings, if confirmed in vivo, have implications for predicting CYP2D6-mediated drug-drug interactions and call for re-evaluation of regulatory guidelines on screening for CYP2D6 induction by xenobiotics. Our findings also suggest that cortisol may be a causative factor in the in vivo induction of CYP2D6 during pregnancy.
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Affiliation(s)
- Muhammad Farooq
- Department of Pharmaceutics, University of Washington, Seattle, Washington
| | - Edward J Kelly
- Department of Pharmaceutics, University of Washington, Seattle, Washington
| | - Jashvant D Unadkat
- Department of Pharmaceutics, University of Washington, Seattle, Washington
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Abstract
Despite the tremendous hurdles presented by the complexity of the liver's structure and function, advances in liver physiology, stem cell biology and reprogramming, and the engineering of tissues and devices are accelerating the development of cell-based therapies for treating liver disease and liver failure. This State of the Art Review discusses both the near- and long-term prospects for such cell-based therapies and the unique challenges for clinical translation.
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Affiliation(s)
- Sangeeta N Bhatia
- Institute for Medical Engineering & Science at MIT, Department of Electrical Engineering and Computer Science, David H. Koch Institute at MIT, and the Howard Hughes Medical Institute, Cambridge, MA 02139, USA. Division of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
| | - Gregory H Underhill
- Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
| | - Kenneth S Zaret
- Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Ira J Fox
- Department of Surgery, Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine, and McGowan Institute for Regenerative Medicine, Pittsburgh, PA 15224, USA
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Fraczek J, Bolleyn J, Vanhaecke T, Rogiers V, Vinken M. Primary hepatocyte cultures for pharmaco-toxicological studies: at the busy crossroad of various anti-dedifferentiation strategies. Arch Toxicol 2012; 87:577-610. [PMID: 23242478 DOI: 10.1007/s00204-012-0983-3] [Citation(s) in RCA: 91] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2012] [Accepted: 11/19/2012] [Indexed: 01/24/2023]
Abstract
Continuously increasing understanding of the molecular triggers responsible for the onset of diseases, paralleled by an equally dynamic evolution of chemical synthesis and screening methods, offers an abundance of pharmacological agents with a potential to become new successful drugs. However, before patients can benefit of newly developed pharmaceuticals, stringent safety filters need to be applied to weed out unfavourable drug candidates. Cost effectiveness and the need to identify compound liabilities, without exposing humans to unnecessary risks, has stimulated the shift of the safety studies to the earliest stages of drug discovery and development. In this regard, in vivo relevant organotypic in vitro models have high potential to revolutionize the preclinical safety testing. They can enable automation of the process, to match the requirements of high-throughput screening approaches, while satisfying ethical considerations. Cultures of primary hepatocytes became already an inherent part of the preclinical pharmaco-toxicological testing battery, yet their routine use, particularly for long-term assays, is limited by the progressive deterioration of liver-specific features. The availability of suitable hepatic and other organ-specific in vitro models is, however, of paramount importance in the light of changing European legal regulations in the field of chemical compounds of different origin, which gradually restrict the use of animal studies for safety assessment, as currently witnessed in cosmetic industry. Fortunately, research groups worldwide spare no effort to establish hepatic in vitro systems. In the present review, both classical and innovative methodologies to stabilize the in vivo-like hepatocyte phenotype in culture of primary hepatocytes are presented and discussed.
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Affiliation(s)
- J Fraczek
- Department of Toxicology, Faculty of Medicine and Pharmacy, Centre for Pharmaceutical Research, Vrije Universiteit Brussel, Belgium.
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Swift B, Pfeifer ND, Brouwer KLR. Sandwich-cultured hepatocytes: an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev 2010; 42:446-71. [PMID: 20109035 PMCID: PMC3097390 DOI: 10.3109/03602530903491881] [Citation(s) in RCA: 290] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Sandwich-cultured hepatocytes (SCH) are a powerful in vitro tool that can be utilized to study hepatobiliary drug transport, species differences in drug transport, transport protein regulation, drug-drug interactions, and hepatotoxicity. This review provides an up-to-date summary of the SCH model, including a brief history of, and introduction to, the use of SCH, as well as methodology to evaluate hepatobiliary drug disposition. A summary of the literature that has utilized this model to examine the interplay between drug-metabolizing enzymes and transport proteins, drug-drug interactions at the transport level, and hepatotoxicity as a result of altered hepatic transport also is provided.
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Affiliation(s)
- Brandon Swift
- University of North Carolina at Chapel Hill, 27599-7569, USA
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Hewitt NJ, Lechón MJG, Houston JB, Hallifax D, Brown HS, Maurel P, Kenna JG, Gustavsson L, Lohmann C, Skonberg C, Guillouzo A, Tuschl G, Li AP, LeCluyse E, Groothuis GMM, Hengstler JG. Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies. Drug Metab Rev 2007; 39:159-234. [PMID: 17364884 DOI: 10.1080/03602530601093489] [Citation(s) in RCA: 533] [Impact Index Per Article: 29.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.
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Affiliation(s)
- Nicola J Hewitt
- Scientific Writing Services, Wingertstrasse, Erzhausen, Germany.
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14
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Watanabe N, Kagawa T, Kojima S, Takashimizu S, Nagata N, Nishizaki Y, Mine T. Taurolithocholate impairs bile canalicular motility and canalicular bile secretion in isolated rat hepatocyte couplets. World J Gastroenterol 2006; 12:5320-5. [PMID: 16981261 PMCID: PMC4088198 DOI: 10.3748/wjg.v12.i33.5320] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/29/2006] [Revised: 05/28/2006] [Accepted: 06/15/2006] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the effects of taurolithocholate (TLC) on the canalicular motility in isolated rat hepatocyte couplets (IRHC). METHODS TLC was added to IRHC at concentrations of 10 and 50 mumol/L, respectively. In each group, five time-lapse movies containing 3 representative bile canaliculi were taken under phase-contrast microscopy for 12 h. The number of bile canalicular contractions and the intervals between consecutive canalicular contractions were calculated. Furthermore, the effects of TLC on IRHC were examined by transmission electron microscopy. RESULTS The bile canalicular contractions were spontaneous and forceful in the controls. Active vesicular movement was observed in the pericanalicular region. Immediately after the addition of TLC, the bile canaliculi were deformed, and canalicular bile was incorporated into the vacuoles. The canaliculi were gradually dilated, and canalicular contractions were markedly inhibited by TLC. The vesicular movements became extremely slow in the pericanalicular region. The number of canalicular contractions significantly decreased in the TLC-treated groups, as compared with that in the controls. The time intervals were prolonged, as the TLC dosage increased, indicating that bile secretion into the canaliculi was impaired with TLC. Transmission electron microscopy revealed the lamellar transformation of the canalicular membranes in IRHC treated with TLC. CONCLUSION TLC impairs both the bile canalicular contractions and the canalicular bile secretion, possibly by acting directly on the canalicular membranes in TLC-induced cholestasis.
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Affiliation(s)
- Norihito Watanabe
- Division of Gastroenterology, Department of Internal Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193, Japan.
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15
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Turncliff RZ, Tian X, Brouwer KLR. Effect of culture conditions on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured rat hepatocytes. Biochem Pharmacol 2006; 71:1520-9. [PMID: 16542640 DOI: 10.1016/j.bcp.2006.02.004] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2005] [Revised: 02/02/2006] [Accepted: 02/03/2006] [Indexed: 11/22/2022]
Abstract
Rat hepatocytes cultured in a sandwich configuration form functional canalicular networks. The influence of extracellular matrix configuration, medium composition, and confluency on the expression and function of Bsep, Mrp2, and Mdr1a/b in sandwich-cultured (SC) rat hepatocytes was examined. Primary rat hepatocytes were: (1) maintained in various extracellular matrix sandwich configurations, (2) cultured in Dulbecco's modified Eagle's medium (DMEM), Modified Chee's medium (MCM) or Williams' E medium (WME), and/or (3) plated at decreasing cell density. Bsep, Mrp2, and Mrdr1a/b expression in day 4 SC rat hepatocytes was assessed by Western blot; function was measured by accumulation of taurocholate, 5(and 6)-carboxy-2',7'-dichlorofluorescein, and rhodamine 123, respectively, in canalicular networks. In general, the extracellular matrix conditions examined resulted in similar protein expression and function. Function of Bsep, Mrp2, and Mdr1a/b was higher in SC rat hepatocytes maintained in DMEM or WME. Mrp2 and Mdr1a/b expression, representative of total cellular content, did not always correlate directly with function, which should be reflective of canalicular membrane expression. Mrp2 expression decreased significantly as cell density decreased in SC hepatocytes. Low plating density in Biocoat plates resulted in poor canalicular network formation and reduced function of Mrp2 and Mdr1a/b. Expression and/or function of Mrp2 and Mdr1a/b in rat hepatocytes cultured in a sandwich configuration may be influenced by plating density and media type.
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Affiliation(s)
- Ryan Z Turncliff
- School of Pharmacy, University of North Carolina at Chapel Hill, 27599-7360, Unites States
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16
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Turncliff RZ, Meier PJ, Brouwer KLR. Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes. Drug Metab Dispos 2004; 32:834-9. [PMID: 15258109 DOI: 10.1124/dmd.32.8.834] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Dexamethasone (DEX) is a well established inducer of CYP3A. These studies examined the influence of DEX treatment on transport protein expression and function in sandwich-cultured (SC) rat hepatocytes. Freshly isolated hepatocytes were cultured between two layers of gelled collagen and maintained in Dulbecco's modified Eagle's medium supplemented with DEX (0.1 microM, 0-48 h and 0.1-100 microM, 48-96 h). The expression of sinusoidal [(organic anion transporting polypeptide 1a1 (Oatp1a1), Oatp1a4, multidrug resistance-associated protein 3 (Mrp3), and Na(+)-dependent taurocholate cotransporting polypeptide (Ntcp)] and canalicular [bile salt export pump (Bsep), multidrug resistance protein 1a/b (Mdr1a/b), and Mrp2] transport proteins was determined by Western blot analysis. The accumulation and biliary excretion index (BEI; percentage of accumulated substrate in canalicular networks) of the probe substrates taurocholate (TC; 1 microM, 10 min), rhodamine 123 (Rh123; 10 microM, 30 min), and carboxy-2',7'-dichlorofluorescein (CDF; 10 microM, 10 min) were employed as measures of canalicular transport protein function in SC rat hepatocytes. DEX treatment increased CYP3A1/2, Oatp1a4, and Mrp2 expression, decreased the expression of Ntcp, and did not seem to alter the expression of Oatp1a1, Mrp3, Mdr1a/b, or Bsep. The BEI of CDF, an Mrp2 substrate, increased from 18 to 37% after DEX treatment (100 microM). The accumulation of TC, an Ntcp substrate, was reduced (<50% of control), whereas the BEI of TC, also a Bsep substrate, was unchanged. Treatment of SC rat hepatocytes with DEX resulted in alterations in the expression of CYP3A1/2 and some hepatic transport proteins. Modest alterations in hepatic transport protein function were consistent with changes in protein expression.
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Affiliation(s)
- Ryan Z Turncliff
- Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA
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17
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Thangavel C, Garcia MC, Shapiro BH. Intrinsic sex differences determine expression of growth hormone-regulated female cytochrome P450s. Mol Cell Endocrinol 2004; 220:31-9. [PMID: 15196697 DOI: 10.1016/j.mce.2004.04.002] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/02/2004] [Revised: 04/05/2004] [Accepted: 04/08/2004] [Indexed: 11/15/2022]
Abstract
The masculine profile of cytochrome P450s found in male liver is determined by the episodic secretion of growth hormone characteristic of males. In turn, the female pattern of P450s observed in female rat liver is regulated by the continuous secretion of growth hormone characteristic of the female. In order to determine if intrinsic and possibly permanent sex differences exist in the response of hepatic P450s to growth hormone regulation, we compared the effects of the episodic and continuous growth hormone profiles on the expression of female-dependent isoforms in cultured hepatocytes isolated from both sexes. We observed that female-specific CYP2C12 as well as female-predominant CYP2A1, 3A1, and 2C6 could be induced by growth hormone concentrations equal to as little as 6, 0.6, and 0.06% of the mean circulating hormone profile found in normal females. Irrespective of sex, all four female-dependent isoforms were far more responsive to the continuous growth hormone profile than the episodic pattern. Lastly, female-derived hepatocytes in general responded with strikingly greater induction levels of P450s than male hepatocytes exposed to the same growth hormone profiles. The present findings demonstrate intrinsic, irreversible sex differences in growth hormone-regulated female-dependent P450s.
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Affiliation(s)
- Chellappagounder Thangavel
- Laboratories of Biochemistry, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6048, USA
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18
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Wang H, Faucette SR, Gilbert D, Jolley SL, Sueyoshi T, Negishi M, LeCluyse EL. Glucocorticoid receptor enhancement of pregnane X receptor-mediated CYP2B6 regulation in primary human hepatocytes. Drug Metab Dispos 2003; 31:620-30. [PMID: 12695351 DOI: 10.1124/dmd.31.5.620] [Citation(s) in RCA: 67] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Although the glucocorticoid receptor (GR) facilitates the xenobiotic-induced expression of CYP2B in rodents, its role in the regulation of human CYP2B6 is unclear. In this report, the role of human GR in the regulation of CYP2B6 was evaluated using primary human hepatocytes and transfection assays with Huh7 cells. CYP2B6 expression was not induced in primary hepatocytes treated with dexamethasone (DEX) concentrations (0.01-1 microM) known to activate GR. In contrast, treatment with 0.1 microM DEX enhanced CYP2B6 induction by different pregnane X receptor (PXR) activators, including rifampin, phenytoin, clotrimazole, and phenobarbital. In Huh7 cells, cotransfection of human (h)GR and hPXR with CYP2B6-phenobarbital-responsive enhancer module (PBREM) reporter constructs revealed that all hPXR ligands induce CYP2B6 reporter gene activity, and this ligand-dependent activation is greatly enhanced by activated hGR. CYP2B6 reporter gene expression was not induced in the presence of hPXR ligands when hGR alone was cotransfected with CYP2B6 reporter construct. In hGR and human constitutive androstane receptor (hCAR) cotransfection assays, activated hGR increased the constitutive activation of PBREM reporter constructs by hCAR in the absence of inducers. In the presence of activated hGR and known inducers of CYP2B6, only PB treatment caused a further 2-fold activation of hCAR compared with control. These studies show that hGR is involved synergistically in the xenobiotic-responsive regulation of human CYP2B6 by hPXR and hCAR. Moreover, the results suggest that the GR-enhanced expression of CYP2B6 is mediated through an indirect mechanism that does not require increased expression of nuclear receptor.
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MESH Headings
- Aryl Hydrocarbon Hydroxylases/biosynthesis
- Aryl Hydrocarbon Hydroxylases/genetics
- Cells, Cultured
- Clotrimazole/pharmacology
- Constitutive Androstane Receptor
- Cytochrome P-450 CYP2B6
- Cytochrome P-450 CYP3A
- Cytochrome P-450 Enzyme System/metabolism
- Dexamethasone/pharmacology
- Enzyme Induction/drug effects
- Gene Expression Regulation
- Hepatocytes/metabolism
- Humans
- Hydroxylation
- Immunoblotting
- Ligands
- Oxidoreductases, N-Demethylating/biosynthesis
- Oxidoreductases, N-Demethylating/genetics
- Phenytoin/pharmacology
- Polymerase Chain Reaction
- Pregnane X Receptor
- RNA, Messenger/biosynthesis
- Receptors, Cytoplasmic and Nuclear/agonists
- Receptors, Cytoplasmic and Nuclear/genetics
- Receptors, Cytoplasmic and Nuclear/metabolism
- Receptors, Glucocorticoid/genetics
- Receptors, Glucocorticoid/metabolism
- Receptors, Steroid/agonists
- Receptors, Steroid/genetics
- Receptors, Steroid/metabolism
- Rifampin/pharmacology
- Transcription Factors/agonists
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Affiliation(s)
- Hongbing Wang
- Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
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19
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Luttringer O, Theil FP, Lavé T, Wernli-Kuratli K, Guentert TW, de Saizieu A. Influence of isolation procedure, extracellular matrix and dexamethasone on the regulation of membrane transporters gene expression in rat hepatocytes. Biochem Pharmacol 2002; 64:1637-50. [PMID: 12429353 DOI: 10.1016/s0006-2952(02)01382-5] [Citation(s) in RCA: 65] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The influence of the isolation procedure of hepatocytes, extracellular matrix (ECM) configuration and incubation medium supplementation by dexamethasone (DEX) on the cell morphology and on the gene expression of membrane transporters was examined in rat hepatocytes. The mRNA levels were determined using oligonucleotide microarrays, in liver, in suspension and in primary culture in monolayer (CPC), and in collagen gels sandwich (SPC) in absence and presence of DEX (100 and 1000 nM). The results indicated pronounced morphological differences between CPC and SPC in response to DEX demonstrating that the hepatocytes re-formed, as in vivo, multicellular arrays with extensive bile canalicular network only in SPC in presence of DEX. The mRNA levels of membrane transporters were not affected significantly during isolation procedure. However, plating hepatocytes in CPC resulted in a decrease of major basolateral transporters mRNA level whereas mRNA levels of mdr1b and mrp3 were increased (>100-fold). Similar observations were made in SPC in the absence of DEX demonstrating that the ECM configuration alone did not play a critical role in the regulation of membrane transporters. However, adding DEX to the incubation medium in SPC resulted in an up-regulation of mdr2, oatp2 and mrp2 in a concentration-dependent way for the two latter genes, whereas mdr1b and mrp3 expression were maintained to their baseline liver levels. These data suggested therefore that the combination of ECM and DEX supplementation is essential for the formation of the bile canalicular network and is a determinant factor in the regulation of membrane transporters in cultured rat hepatocytes.
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20
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Hamilton GA, Westmorel C, George AE. Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers. In Vitro Cell Dev Biol Anim 2002. [PMID: 11776971 DOI: 10.1290/1071-2690(2001)037<0656:eomcot>2.0.co;2] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.
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Affiliation(s)
- G A Hamilton
- Division of Biosciences, University of Hertfordshire, Hatfield, United Kingdom
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21
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Hamilton GA, Westmorel C, George AE. Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers. In Vitro Cell Dev Biol Anim 2001; 37:656-67. [PMID: 11776971 DOI: 10.1290/1071-2690(2001)037<0656:eomcot>2.0.co;2] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.
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Affiliation(s)
- G A Hamilton
- Division of Biosciences, University of Hertfordshire, Hatfield, United Kingdom
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22
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Chandra P, Lecluyse EL, Brouwer KL. Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes. In Vitro Cell Dev Biol Anim 2001; 37:380-5. [PMID: 11515972 DOI: 10.1007/bf02577575] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagen-coated 60-mm dishes, overlaid with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were conducted in Dulbecco's modified Eagle's medium (DMEM) + 1.0 microM dexamethasone (DEX) + 5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2 ml collagen and incubated in DMEM + 0.1 microM DEX, DMEM +/- 0.1 microM DEX + 5% FBS, Williams' medium E + 0.1 microM DEX + 1% ITS+, DMEM + 1.0 microM DEX, DMEM + 1.0 microM DEX + 5% FBS, or modified Chee's medium (MCM) + 0.1 microM DEX + 1% ITS+. [3H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca2+-free HBSS was measured, and the biliary-exeretion index (BEI: percentage of accumulated TC localized in the canalicular compartment) was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI = 0.649 [0.1 ml collagen]; BEI = 0.659 [0.2 ml collagen]) compared with those overlaid at 0 h after seeding (BEI = 0.538 [0.1 ml collagen]; BEI = 0.517 [0.2 ml collagen]), although the differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI = 0.396). Differing DEX concentrations (0.1 microM versus 1.0 microM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC.
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Affiliation(s)
- P Chandra
- Division of Drug Delivery and Disposition, University of North Carolina at Chapel Hill, 27599, USA
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23
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CHANDRA PRIYAMVADA, LECLUYSE EDWARDL, BROUWER KIMLR. OPTIMIZATION OF CULTURE CONDITIONS FOR DETERMINING HEPATOBILIARY DISPOSITION OF TAUROCHOLATE IN SANDWICH-CULTURED RAT HEPATOCYTES. ACTA ACUST UNITED AC 2001. [DOI: 10.1290/1071-2690(2001)037<0380:ooccfd>2.0.co;2] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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24
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Fahrig R, Rupp M, Steinkamp-Zucht A, Bader A. Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells. Toxicol In Vitro 1998; 12:431-44. [DOI: 10.1016/s0887-2333(98)00005-8] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/21/1997] [Indexed: 10/17/2022]
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25
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LeCluyse EL, Bullock PL, Parkinson A. Strategies for restoration and maintenance of normal hepatic structure and function in long-term cultures of rat hepatocytes. Adv Drug Deliv Rev 1996. [DOI: 10.1016/s0169-409x(96)00418-8] [Citation(s) in RCA: 143] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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26
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LeCluyse EL, Bullock PL, Parkinson A, Hochman JH. Cultured rat hepatocytes. PHARMACEUTICAL BIOTECHNOLOGY 1996; 8:121-59. [PMID: 8791809 DOI: 10.1007/978-1-4899-1863-5_9] [Citation(s) in RCA: 82] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Affiliation(s)
- E L LeCluyse
- INTERx Research/Merck Research Laboratories, Lawrence, Kansas 66047, USA
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27
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Cristofalo VJ, Pignolo RJ. Cell Culture as a Model. Compr Physiol 1995. [DOI: 10.1002/cphy.cp110104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
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28
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Krämer K, Markwitan A, Pallauf J. Studies on the metabolism of metallothionein and alkaline phosphatase of adult rat primary hepatocyte cultures: role of fetal calf serum and agonists of the phosphoinositide cascade. ZEITSCHRIFT FUR ERNAHRUNGSWISSENSCHAFT 1993; 32:176-86. [PMID: 8237077 DOI: 10.1007/bf01610728] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Adult rat primary hepatocytes maintained in DMEM/F12 (Ham) media were used as a model system for studying the role of fetal calf serum (FCS) and agonists of the phosphoinositide cascade in the metabolism of metallothionein (MT) and alkaline phosphatase (ALP). Experiments were performed both after a 24 h preincubation with FCS and with bovine serum albumin (BSA). Hepatocytes were treated with dexamethasone (DEX), zinc (Zn) and with the agonists of the phosphoinositide cascade A23187, 1,2-dioctanoyl-sn-glycerol (DiC8), 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II (AT), platelet activating factor (PAF), Arg8-vasopressin (VP) and were analyzed for MT and ALP activity in cell homogenates. Cell viability was evaluated by lactate dehydrogenase (LDH) liberation into culture medium, induction of tyrosine aminotransferase (TAT) through DEX and by trypan blue exclusion. Overall, cell viability was improved by the FCS pretreatment and by DEX. Exposure of hepatocytes to the established direct inducers Zn and DEX of MT resulted in a manifold increase in MT, independent of whether the cultures were FCS pretreated or not. The FCS preincubation produced a moderate elevation of ALP activity by stimulating cell viability. However, ALP was unaltered in response to Zn and DEX. None of the experiments conducted with agonists of the phosphoinositide cascade led to an elevation of MT and ALP. Only the incubation of hepatocytes with A23187 resulted in a concentration dependent significant decrease of MT and ALP. This observation was due to a cytotoxic effect of A 23187, displayed by LDH leakage and an increase in the number of cells stained with trypan blue. In conclusion, in primary hepatocyte cultures agonists of the phosphoinositide did not have an effect on the metabolism of MT and ALP. Previous in vivo results indicating alterations of Zn metabolism in liver, therefore seem to be caused by indirect systemic responses.
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Affiliation(s)
- K Krämer
- Institut für Tierernährung, Justus-Liebig-Universität Giessen
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29
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Luo QJ, MacRae JC, Scislowski PW. Characterization of sheep hepatocytes in primary culture. THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY 1993; 25:719-23. [PMID: 8349013 DOI: 10.1016/0020-711x(93)90359-m] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
1. Primary cultures of isolated sheep hepatocytes were used to characterize metabolic functions of liver: gluconeogenesis, ureagenesis and protein synthesis. The rates of all three metabolic activities were linear over a 20 hr culture period. 2. Hepatocytes in the presence of glucagon increased the synthesis of urea by approx 30% (P < 0.05) and increased release of glucose into the medium by 60% (P < 0.05). 3. In the absence of insulin, significantly more (35%; P < 0.05) glucose was released in the medium than in the presence of insulin. 4. Results help evaluate the primary culture of sheep hepatocytes as an appropriate experimental model to study nutritional and hormonal regulation of liver in the ruminant species.
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Affiliation(s)
- Q J Luo
- Rowett Research Institute, Bucksburn, Aberdeen, Scotland
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30
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Saad B, Schawalder H, Maier P. Crude liver membrane fractions as substrate preserve liver-specific functions in long-term, serum-free rat hepatocyte cultures. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY : JOURNAL OF THE TISSUE CULTURE ASSOCIATION 1993; 29A:32-40. [PMID: 7680337 DOI: 10.1007/bf02634369] [Citation(s) in RCA: 28] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Over time, rat hepatocytes cultured on collagen lose the capacity to express liver-specific functions. The influence on this degradation process of an alternative substratum--crude membrane fractions prepared from the liver of the same rat strain--was investigated. Freshly isolated rat hepatocytes were cultured in serum-free Williams E medium supplemented with aprotinin, selenium, dexamethasone, and insulin in flasks coated with a mixture of rat liver crude membrane fractions:collagen type I (100:1). The cells adhered firmly, exhibiting minimal spreading and remaining grouped in columns or in cell islands, and retained their liver-specific functions for more than 1 wk. Hepatocytes secreted substantially higher amounts of albumin than cells cultured on collagen-coated dishes, and on Days 1 and 9 in culture the total P-450 content was 72 and 40%, respectively, of that of freshly isolated cells. On Day 6, the 7-ethoxyresorufin-O-deethylase and the aldrin epoxidase activities were still more than 50% that of freshly isolated hepatocytes. Exposure to phenobarbital on Days 3 to 6 increased the total cytochrome P-450 content twofold; exposure to 3-methylcholanthrene increased the activity of the corresponding cytochrome P-450 isoforms to 20 times that observed in untreated cultures and 6 times that observed in freshly isolated cells. Thus, given the ease with which they are prepared, the use of crude membrane fractions combined with culture medium supplemented with aprotinin and selenium can facilitate the preparation of reproducible cultures suitable for long-term in vitro pharmacotoxicologic studies using rat hepatocytes.
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Affiliation(s)
- B Saad
- Institute of Toxicology, Swiss Federal Institute of Technology, Schwerzenbach
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31
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McMillan JM, Shaddock JG, Casciano DA, Arlotto MP, Leakey JE. Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone. Mutat Res 1991; 249:81-92. [PMID: 2067545 DOI: 10.1016/0027-5107(91)90134-a] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b5 contents progressively decreased throughout the 72-h culture period to less than 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching less than 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6 beta-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50-60% of initial values at 72 h. UDP-glucuronyltransferase activity towards digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione-S-transferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 microM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6 beta-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione-S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation. Furthermore, the use of DEX in cell culture medium to enhance cell survival does not maintain total drug-metabolizing enzyme capability, but appears to transiently and selectively increase expression of certain isozymes at the expense of others.
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Affiliation(s)
- J M McMillan
- Division of Reproductive and Developmental Toxicology, National Center for Toxicological Research, Jefferson, AR 72079
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32
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Perraud F, Dalemans W, Gendrault JL, Dreyer D, Ali-Hadji D, Faure T, Pavirani A. Characterization of trans-immortalized hepatic cell lines established from transgenic mice. Exp Cell Res 1991; 195:59-65. [PMID: 1711473 DOI: 10.1016/0014-4827(91)90500-t] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Hepato-specific regulatory (promoter/enhancer) DNA sequences were used for targeting the expression of onc genes, such as murine c-myc and Simian Virus 40 T Antigen, to hepatocytes of transgenic mice which subsequently developed hepatocellular carcinomas after a variable period of time (depending on the type of onc gene employed). Several trans-immortalized cell lines were established and compared with respect to the expression of adult hepatic markers and response to growth factors. Despite the morphological differences observed between trans-hepatomas, owing to the expression of the two different onc genes, all tumor-derived cell lines behaved in a comparable fashion during long-term culture displaying an adult hepatic phenotype for at least 40 passages. They differed, however, in response to epidermal growth factor. When the gene coding for human alpha 1-antitrypsin was placed under the control of the same hepato-specific promoter/enhancer, high levels of the human recombinant protein could be harvested from the supernatants of trans-hepatoma-derived cell lines.
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33
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Gebhardt R, Fitzke H, Fausel M, Eisenmann-Tappe I, Mecke D. Influence of hormones and drugs on glutathione-S-transferase levels in primary culture of adult rat hepatocytes. Cell Biol Toxicol 1990; 6:365-78. [PMID: 2085792 DOI: 10.1007/bf00120803] [Citation(s) in RCA: 26] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
GST activities against 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were measured in isolated and cultured adult rat hepatocytes. Within 24 h in culture, both GST activities decreased to about 70% and either stabilized at this level (CDNB) or recovered (DCNB) to the initial level. Use of hyaluronidase in addition to collagenase during the isolation of the cells strongly reduced both activities and its stimulation by various drugs for up to 168 h. The hormones insulin, glucagon, triiodothyronine, estradiol, testosterone, and progesterone did not affect GST activity, while dexamethasone showed some interference. In the presence of dexamethasone the activity against CDNB was mainly stimulated by the combination of methylcholanthrene (MC) and phenobarbital (PB) to about 260% within 168 h. The activity against DCNB was stimulated predominantly by MC alone reaching 170% after 168 h. Quantification of the GST subunits Ya, Yb1 and Yp by an ELISA technique revealed a strong decrease of Ya, a transient increase of Yb1 after 24 h followed by a moderate decrease, and a stable low level of the transformation marker Yp during cultivation. The level of Ya was markedly induced by PB, particularly in combination with MC. The level of Yb1 was equally induced by MC or PB with no synergistic effect. Yp was not affected by these drugs. None of the hormones affected the level of these GST subunits. These results indicate that the physiological type of regulation of the GSTs is maintained during primary culture and no signs of dedifferentiation or transformation are observed. Furthermore, they demonstrate that the interaction of drugs and hormones and their inducing potential can be efficiently studied in the cultured hepatocytes.
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Affiliation(s)
- R Gebhardt
- Physiologisch-chemisches Institut, Universität Tübingen, FRG
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34
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Miyazaki M, Suzuki Y, Oda M, Kawai A, Bai LY, Sato J. Improved maintenance of adult rat hepatocytes in a new serum-free medium in the presence or absence of barbiturates. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY : JOURNAL OF THE TISSUE CULTURE ASSOCIATION 1989; 25:839-48. [PMID: 2793782 DOI: 10.1007/bf02623668] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
For serum-free primary culture of adult rat hepatocytes, a synthetic medium DM-160 and rat-tail collagen were selected for the basal medium and for the culture substratum, respectively. Barbiturates, such as phenobarbital and 1-ethyl-5-isobutylbarbiturate, efficiently supported survival of hepatocytes and maintained their morphologic features at lower concentrations under the serum-free conditions than under the serum-supplemented conditions. However, the hepatocyte survival rates under the serum-free conditions were lower than those under the serum-supplemented conditions in the presence or absence of barbiturates. Supplementation of the basal medium with a combination of five groups of factors (5Fs), such as eight amino acids (Ala, Arg, Gly, Ile, Met, Phe, Pro, and Trp), two unsaturated fatty acids (linoleate and oleate), a protease inhibitor (aprotinin), three vitamins (A, C, and E), and five trace elements (Mn, Fe, Cu, Zn, and Se), improved the hepatocyte survival under the serum-free conditions in the presence or absence of barbiturates. In other words, the serum could be completely substituted by the 5Fs. Hepatocyte cultures maintained in the 5Fs-supplemented basal medium showed excellent induction of tyrosine aminotransferase activity in response to dexamethasone in the presence or absence of barbiturates. The efficiency of the 5Fs-supplemented basal medium for maintaining hepatocytes was not inferior to those of other media in common use with hepatocytes, such as Williams' medium E and Waymouth's medium MB-752/1. In conclusion, maintenance of functional hepatocytes in serum-free primary culture could be improved by use of the new medium preparation (the 5Fs-supplemented DM-160) in the presence of barbiturates.
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Affiliation(s)
- M Miyazaki
- Division of Pathology, Okayama University Medical School, Japan
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35
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Miyazaki M, Utsumi K, Sato J. Mechanisms responsible for long-term survival of adult rat hepatocytes in the presence of phenobarbital in primary culture. Exp Cell Res 1989; 182:415-24. [PMID: 2721587 DOI: 10.1016/0014-4827(89)90246-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
The mechanisms, by which phenobarbital (PB) supports the survival of adult rat hepatocytes in primary culture, were investigated. PB altered the shape of rat erythrocytes to produce cup-formed cells and protected them from hypotonic hemolysis. Anesthetics (ketamine, lidocaine, mepivacaine, and bupivacaine) and an anti-inflammatory agent (indomethacin), which are also known to protect erythrocytes from hypotonic hemolysis by stabilizing their membranes, efficiently supported the survival of hepatocytes in primary culture. Furthermore, the well-known biological membrane stabilizers, such as cholesterol and vitamin E, also showed the maintenance effect on primary cultured hepatocytes. PB effectively reduced the leakage of lactate dehydrogenase from hepatocytes caused by chenodeoxycholic acid in primary culture. Rotenone and amobarbital, which act repressively on the PB-sensitive site in the respiratory chain and are known to inhibit the mitochondrial formation of active oxygen species with NAD-linked substances, effectively prolonged the hepatocyte survival in primary culture. Elevation of oxygen tension in primary culture remarkably decreased the hepatocyte survival rate, which was preserved by addition of antioxidant substances, such as vitamin C, vitamin E, bifemelane, selenite, and superoxide dismutase. On the other hand, in the presence of PB, the hepatocyte survival rate hardly changed with the elevation of oxygen tension. From these findings, it seems that PB stabilizes the hepatocyte membranes and reduces the mitochondrial formation of active oxygen species and that the stabilized functions of membrane and the reduction of oxidative stress result in the prolonged survival of hepatocytes in primary culture.
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Affiliation(s)
- M Miyazaki
- Division of Pathology, Okayama University Medical School, Japan
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36
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Abramovitz M, Ishigaki S, Listowsky I. Differential regulation of glutathione S-transferases in cultured hepatocytes. Hepatology 1989; 9:235-9. [PMID: 2912828 DOI: 10.1002/hep.1840090212] [Citation(s) in RCA: 34] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Specific cDNA probes were used to determine steady-state mRNA levels for the multiple glutathione S-transferases in primary hepatocyte cultures. In the first 24 hr of culture, gene transcripts for the Ya family decreased sharply, Yb3 disappeared completely, but changes in levels of mRNA for Yb1 and Yb2 were smaller. These results suggest that the isoenzymes are regulated independently. Yp mRNA, which is present at greatly elevated levels in hyperplastic nodules and hepatocellular carcinomas but not in normal adult livers, was hardly detectable in freshly isolated hepatocytes, but Yp transcripts rapidly accumulated in the first 24 hr in culture and continued to increase for 72 hr. Decreased levels in Ya and Yc and increases in Yp were detected by immunoblotting methods, indicating that translation products changed together with mRNA levels in the cultured cells. The appearance of Yp transcripts in hepatocytes was effectively blocked by addition of dexamethasone to the culture medium. Elevations of Yp levels are characteristic of the cell culture system and factors regulating Yp transcription in nodules and carcinomas may also be operative in cultured hepatocytes.
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Affiliation(s)
- M Abramovitz
- Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
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37
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McGowan JA. Reciprocal regulation of adult rat hepatocyte growth and functional activities in culture by dimethyl sulfoxide. J Cell Physiol 1988; 137:497-504. [PMID: 3263972 DOI: 10.1002/jcp.1041370315] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Hepatocyte DNA synthesis, initiated by epidermal growth factor (EGF), is reversibly inhibited by 2% dimethyl sulfoxide (DMSO). At that concentration, both the survival of the cells in culture and the expression of differentiated functions are prolonged. DMSO does not affect thymidine uptake or EGF receptor binding. Moreover, EGF receptor binding is maintained at 84% of initial 12 hr binding when cells are cultured for several days in the presence of DMSO, whereas specific receptor binding declines to 49% of initial binding under standard culture conditions without DMSO. Studies of hepatocyte functional activity indicate that, during early culture, total cellular export protein synthesis, specific albumin synthesis, and glycogen synthesis are enhanced in the presence of DMSO. Dexamethasone is required for the effect of DMSO on survival, and although dexamethasone alone enhances hepatocyte DNA synthesis in the presence of EGF, it does not reverse the inhibitory effect of 2% DMSO on DNA replication. The correlation of prolonged survival with growth inhibition supports the hypothesis that hepatic growth and differentiated functional activity may be reciprocally regulated.
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Affiliation(s)
- J A McGowan
- Shriners Burns Institute, Childrens Service, Massachusetts General Hospital, Boston
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38
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Mezey E, Potter JJ, Litt MR, Rhodes D. Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture. Biochem Pharmacol 1988; 37:2993-3000. [PMID: 3395374 DOI: 10.1016/0006-2952(88)90287-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.
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Affiliation(s)
- E Mezey
- Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205
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39
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Gallo G, Mazzei M, Voci A, Fugassa E. Effects of insulin and dexamethasone on adenine nucleotide levels in cultured hepatocytes from adult rat. Cell Biochem Funct 1988; 6:101-5. [PMID: 3288371 DOI: 10.1002/cbf.290060204] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Insulin and dexamethasone, usually added to culture media, play an important role in maintaining the survival of functional hepatocytes. Adenine nucleotide concentrations and energy charge values of cultured hepatocytes were determined to investigate the relationship between the beneficial effects of these hormones and the energy status of the cells. The results indicate that insulin and dexamethasone are essential in maintaining the metabolic competence of cultured hepatocytes and that this correlates with the absolute concentration of ATP rather than with the energy charge.
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Affiliation(s)
- G Gallo
- Instituto di Fiologia Generale, Università di Genova, Italy
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40
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Vind C, Dich J, Grunnet N. The content and activity of cytochrome P-450 in long-term culture of hepatocytes from male and female rats. Biochem Pharmacol 1988; 37:1371-5. [PMID: 3355606 DOI: 10.1016/0006-2952(88)90796-4] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The content of cytochrome P-450 and the capacity for O-demethylation have been measured in cultures of hepatocytes from male and female rats for a period of 21 days. The effect of dexamethasone, insulin, glucagon, phenobarbital and hemin was investigated. In hepatocytes from female rats the content of cytochrome P-450 was unchanged after one day of culture. From day 1 to day 3 the content of cytochrome P-450 decreased by 65% and only the combined addition of dexamethasone, phenobarbital and hemin diminished the fall. After the initial fall, addition of 0.1 microM dexamethasone resulted in a stable value. Addition of 1 microM dexamethasone or 1 mM phenobarbital gave rise to an induction of cytochrome P-450 (285%). The high level of cytochrome P-450 was maintained for 3 weeks. In hepatocytes from male rats the content of cytochrome P-450 decreased by 40% after one day of culture. From day 1 to day 3 the content decreased by 45% and the decrease continued irrespective of the presence of hormones and/or phenobarbital. The O-demethylase activity in cultures of hepatocytes from female rats correlated to the cytochrome P-450 content independent of medium composition and age of the cultures, whereas no correlation was found in cultures from male rats. The present study demonstrates that hepatocytes from female rats in cultures retain O-demethylase activity for at least 3 weeks and that, with the experimental conditions used, the response to the hormones and inducers is different for hepatocytes from male and female rats.
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Affiliation(s)
- C Vind
- Department of Biochemistry A, Panum Institute, University of Copenhagen, Denmark
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41
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Dich J, Vind C, Grunnet N. Long-term culture of hepatocytes: effect of hormones on enzyme activities and metabolic capacity. Hepatology 1988; 8:39-45. [PMID: 3276589 DOI: 10.1002/hep.1840080109] [Citation(s) in RCA: 83] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
(i) Hepatocytes isolated from adult rats were cultured for 2 to 3 weeks on collagen in a modified, serum-free Waymouth medium containing fatty acids and varying concentrations of glucocorticoid, insulin and glucagon. (ii) In the presence of all three hormones, it was possible to maintain the content of DNA, the activity of glucokinase, pyruvate kinase, hexokinase and lactate dehydrogenase at initial levels for 2 to 3 weeks. The activity of glucokinase and pyruvate kinase was affected by the concentration of insulin. (iii) The activity of alcohol dehydrogenase was stable for 3 days and declined to about 25% of the initial level after 2 weeks of culture, irrespective of the presence of hormones. (iv) Maintenance of albumin secretion was dependent on the presence of glucocorticoid, and glucocorticoid and insulin showed an additive or, at some time points, a synergistic effect on its secretion. (v) The content of cytochrome P-450 could be kept at 65% of the initial level, provided that a relatively high concentration of dexamethasone was present (10(-6) M). (vi) In the absence of hormones, urea synthesis was 70% of initial levels throughout the experimental period. With insulin and glucocorticoid present, a high concentration of glucagon (10(-8) M) was required to maintain the synthesis of urea at this level. (vii) It is concluded that hepatocyte cultures as described in the present study may be a useful, well-defined system for long-term metabolic, pharmacologic and toxicologic studies.
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Affiliation(s)
- J Dich
- Department of Biochemistry A, Panum Institute, University of Copenhagen, Denmark
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42
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Tokiwa T, Taketa K, Sato J. Production of albumin and alpha-fetoprotein in primary culture of fetal human liver cells on collagenous substrata in the presence of hydrocortisone. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY : JOURNAL OF THE TISSUE CULTURE ASSOCIATION 1987; 23:830-6. [PMID: 2447057 DOI: 10.1007/bf02620961] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, alpha-fetoprotein (AFP), of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin and AFP is facilitated by synergies between HC and collagenous substrata.
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Affiliation(s)
- T Tokiwa
- Division of Pathology, Okayama University Medical School, Japan
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43
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Meredith MJ. Cystathionase activity and glutathione metabolism in redifferentiating rat hepatocyte primary cultures. Cell Biol Toxicol 1987; 3:361-77. [PMID: 3507263 DOI: 10.1007/bf00119910] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Capacity to incorporate methionine sulfur into glutathione as well as cystathionase activity were lost in cultured hepatocytes in a biphasic manner with 75% of the total capacity disappearing with a half-life of about 10.6 hr, the remainder with a half-life of greater than 20 hr. Nicotinamide, 25 mM, produced a single phase loss with a t 1/2 of approximately 21 hr for both transsulfuration and cystathionase activity. Loss of both methionine sulfur incorporation and cystathionase activity occurred in transferrin/sodium selenite-supplemented Williams Medium E (TS-HWME) with a t 1/2 of about 96 hr through 72 hr in culture. Addition of the cystathionase inhibitor, propargylglycine, blocked glutathione synthesis in TS-HWME cells through 48 hr in culture, while propargylglycine blocked glutathione synthesis only at 4 hr in HWME cultured cells. Further, the accumulation of gamma-glutamyl transpeptidase was delayed by 48 hr in TS-HWME versus unsupplemented medium. Variation in the transport of sulfur amino acids was also found to occur with culture age. The Km values for cysteine and methionine transport were found to be approximately 150 and 100 microM, respectively, and were unaffected by culture age or the presence of TS-HWME. However, the Vmax for transport of methionine declined from 0.29 to 0.012 nmol/min/mg protein over 48 hr in culture. In TS medium, the Vmax at 48 hr for methionine transport had only decreased to 0.20 nmol/min/mg protein and increased for cysteine transport to 0.17 nmol/min/mg protein. These data suggest that during the redifferentiation of hepatocytes in culture, transsulfuration is regulated by control of the flow of substrate through cystathionase and that cystathionase is regulated by alteration of enzyme activity or content. Variations in the rate of transport of precursor sulfur amino acids are also an important component of the regulation of the net glutathione status of the redifferentiating hepatocyte.
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Affiliation(s)
- M J Meredith
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
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44
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Muakkassah-Kelly SF, Bieri F, Waechter F, Bentley P, Stäubli W. Long-term maintenance of hepatocytes in primary culture in the presence of DMSO: further characterization and effect of nafenopin, a peroxisome proliferator. Exp Cell Res 1987; 171:37-51. [PMID: 3622636 DOI: 10.1016/0014-4827(87)90249-7] [Citation(s) in RCA: 52] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
The addition of 2% dimethyl sulfoxide to adult rat hepatocytes cultured in a chemically defined medium at Day 1 after cell plating resulted in maintenance of the cytochrome P-450 content and the cyanide-insensitive palmitoyl-CoA beta-oxidation activity at 66 and 70% of the initial Day 1 values. The addition of phenobarbital, 3-methylcholanthrene, or nafenopin from Day 3 to Day 6 increased the contents of cytochrome P-450 to 128, 239, and 251%, respectively, compared to untreated controls at Day 3. In addition, nafenopin also caused a pronounced and time-dependent increase in palmitoyl-CoA beta-oxidation activity but was found to have only a weak stimulating effect on replicative DNA synthesis (2-fold) when compared to that of epidermal growth factor (6.5-fold). In the presence of dimethyl sulfoxide the hepatocyte cultures could be kept alive for more than 1 month. Exposure of such cultures to nafenopin from Day 1 do Day 37 resulted in survival which was even better than that of their untreated counterparts. This effect was accompanied by the appearance of abundant endoplasmic reticulum membranes and an increased number of peroxisomes.
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45
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Affiliation(s)
- D A Casciano
- Division of Genetic Toxicology, National Center for Toxicological Research, Jefferson, AR 72079
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46
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Hsia MT. Use of scintillometric quantitation of unscheduled DNA synthesis in isolated rat hepatocytes for the screening of genotoxic agents. Cell Biol Toxicol 1987; 3:127-42. [PMID: 3507251 DOI: 10.1007/bf00058452] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
The induction of unscheduled DNA synthesis has been considered as a suitable endpoint for the screening of genotoxic agents. Experimentally, unscheduled DNA synthesis is most frequently measured by autoradiography. The purpose of this report was to examine the usefulness of the liquid scintillation counting technique in measuring unscheduled DNA synthesis response in isolated rat hepatocytes. The various liquid scintillation counting-based unscheduled DNA synthesis assay procedures were examined according to the following groupings: (1) procedures based on the acid precipitation of cellular macromolecules, (2) procedures based on isopycnic gradient centrifugation of solubilized cells, (3) procedures based on nuclei isolation in conjunction with other DNA purification methods, and (4) procedures based on the selective retention of hepatocellular DNA. Limited cases in which test chemicals gave positive unscheduled DNA synthesis response in liquid scintillation counting-based assays and negative unscheduled DNA synthesis response in autoradiography-based assays are presented. It is concluded that liquid scintillation counting-based unscheduled DNA synthesis assays represent an appropriate system for inclusion in carcinogenicity and mutagenicity testing programs.
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Affiliation(s)
- M T Hsia
- Department of Entomology, University of Wisconsin, Madison 53706
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47
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Furukawa K, Shimada T, England P, Mochizuki Y, Williams GM. Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY : JOURNAL OF THE TISSUE CULTURE ASSOCIATION 1987; 23:339-48. [PMID: 3294781 DOI: 10.1007/bf02620990] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
A highly efficient method is described for obtaining proliferative epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10- to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50 X g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 X g for 1 min, whereas many non-hepatocytic cells remained in the supernatant and could be sedimented by a second centrifugation at 50 X g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for gamma-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.
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Manos P, Holten D. Primary cultures of hepatocytes in serum and hormone-free medium: identification of conditions which stimulate an in vivo-like induction of G6PD. IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY : JOURNAL OF THE TISSUE CULTURE ASSOCIATION 1987; 23:367-73. [PMID: 3294782 DOI: 10.1007/bf02620994] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Recent results from several laboratories suggest that complex interactions between hormones and dietary carbohydrate may be responsible for regulating the induction of several hepatic lipogenic enzymes. Elucidation of these interactions requires the ability to culture hepatocytes for several days in serum-free medium where the hormones or carbohydrate or both present is strictly controlled. The functional response of primary adult rat hepatocytes was examined in a medium without exposure to serum, hormones, or carbohydrates and on three substrata commonly used to culture cells in a defined medium. Hepatocytes cultured on a floating collagen gel in which is embedded a nylon mesh possess cell attachment and morphologic characteristics superior to either cells cultured on a collagen-coated or fibronectin(Fn)-coated substratum. Cells cultured on the gel-mesh system retain insulin responsivity, as measured by protein synthesis rates and glucose-6-phosphate dehydrogenase (G6PD) induction, for at least 6 d in culture. Under these conditions, insulin, dexamethasone, and fructose increase G6PD specific activity to levels comparable to that seen in an induced animal. Hepatocytes cultured on the gel-mesh system tolerate restricted medium conditions better than cells cultured on collagen or Fn-coated substratum, and remain viable for sufficient times to allow, for the first time, full expression and maximal induction (i.e. like in vivo) of G6PD in cultured cells. This system represents a satisfactory model for in vivo liver metabolism and a superior system for studying the effects of hormones and metabolites on G6PD levels, as well as other nutritional-hormonally regulated enzymes.
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Edwards AM, Baddams HM, Lucas CM. Two distinct mechanisms for regulation of gamma-glutamyl transpeptidase in cultured rat hepatocytes by glucocorticoid-like steroids. Biochem Pharmacol 1987; 36:1223-30. [PMID: 2885003 DOI: 10.1016/0006-2952(87)90074-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Adult rat hepatocytes maintained in primary monolayer culture with defined medium were used to characterise two effects of glucocorticoid-like steroids in regulating gamma-glutamyltranspeptidase (GGT). Low concentrations of glucocorticoids alone had little effect on GGT but synergistically enhanced induction of the enzyme by liver tumor-promoting xenobiotics such as 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)-ethane (DDT) and hexachlorocyclohexane. The enhancing effect appears to be mediated by the classical glucocorticoid hormone receptor since structural requirements and concentration-dependence for enhancement were similar to those for induction of tyrosine aminotransferase in parallel cultures. Higher concentrations (1-100 microM) of various glucocorticoids alone increased GGT activity. Most glucocorticoids induced GGT but their order of potency did not parallel that for induction of tyrosine aminotransferase under similar culture conditions. Among the most potent glucocorticoids, triamcinolone was a weak GGT inducer and cortivazol appeared to act as an antagonist of GGT induction by steroids. Some non-glucocorticoids including pregnenolone 16 alpha-carbonitrile, and some progestins, also induced but required addition of 30 nM dexamethasone for maximal effect. Some specific steroid structural features were identified which increased (presence of a 16 alpha methyl group) or impaired GGT-inducing activity. Although interpretation is complicated by differential metabolism of individual steroids in culture, the results suggest that GGT induction by pharmacological levels of steroids may be mediated, directly or indirectly, by one or more relatively specific receptors distinct from the classical glucocorticoid receptor.
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Miyazaki M, Handa Y, Suzuki Y, Sato J. Effect of various barbituric acid derivatives on survival of functional hepatocytes from adult rats in primary culture. RESEARCH IN EXPERIMENTAL MEDICINE. ZEITSCHRIFT FUR DIE GESAMTE EXPERIMENTELLE MEDIZIN EINSCHLIESSLICH EXPERIMENTELLER CHIRURGIE 1987; 187:105-17. [PMID: 2884703 DOI: 10.1007/bf01851972] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Eighteen barbituric acid (BA) derivatives and three structurally related chemicals (non-BA-derivatives) were tested for their potency in supporting survival of functional hepatocytes from adult rats in primary culture. Of the 18 BA derivatives, nine drugs showed excellent maintenance effect on hepatocyte survival and function. Although four BA derivatives were also effective, their potency was relatively lower. The remaining five BA derivatives and three structurally related chemicals exhibited no maintenance effect. Thus, a correlation was found between the BA derivative structure and the potency for supporting hepatocyte survival in primary culture. The dose response curves of hepatocyte survival were generally biphasic in shape, as a function of BA derivative concentration. The optimum concentrations for observing the morphological and biochemical effects of the BA derivatives differed from each other. The maintenance of hepatocytes was attained only in the continuous presence of the BA derivatives in the medium. The nine excellent BA derivatives efficiently prevented hepatocytes from morphological degeneration which was observed in the control cultures. The surviving hepatocytes in the presence of these BA derivatives showed higher albumin secretion and retained higher basal levels of tyrosine aminotransferase (TAT) activity for at least 2 weeks in primary culture, as compared with control. Furthermore, the addition of dexamethasone (10 microM) caused a 2- to 4-fold induction of TAT activity for at least 2-weeks in primary culture.
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