Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells
Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive andro...
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Veröffentlicht in: | Drug metabolism and disposition 2017-01, Vol.45 (1), p.56-67 |
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container_title | Drug metabolism and disposition |
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description | Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression. |
doi_str_mv | 10.1124/dmd.116.072603 |
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Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression.</description><identifier>ISSN: 0090-9556</identifier><identifier>EISSN: 1521-009X</identifier><identifier>DOI: 10.1124/dmd.116.072603</identifier><identifier>PMID: 27780834</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Cell Culture Techniques ; Cell Differentiation - drug effects ; Cell Line, Tumor ; Culture Media - chemistry ; Dimethyl Sulfoxide - pharmacology ; Drug Evaluation, Preclinical ; Genetic Vectors ; Humans ; Lentivirus - genetics ; Liver - metabolism ; Metabolic Detoxication, Phase I ; Metabolic Detoxication, Phase II ; Models, Biological ; Pharmaceutical Preparations - metabolism ; Receptors, Cytoplasmic and Nuclear - genetics</subject><ispartof>Drug metabolism and disposition, 2017-01, Vol.45 (1), p.56-67</ispartof><rights>2017 American Society for Pharmacology and Experimental Therapeutics</rights><rights>Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c384t-ac18e88c76406cbc6f823a549b494e2e19d13da3dc6c68a61cf7fc25d8ac18c23</citedby><cites>FETCH-LOGICAL-c384t-ac18e88c76406cbc6f823a549b494e2e19d13da3dc6c68a61cf7fc25d8ac18c23</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>315,781,785,27929,27930</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27780834$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>van der Mark, Vincent A.</creatorcontrib><creatorcontrib>Rudi de Waart, D.</creatorcontrib><creatorcontrib>Shevchenko, Valery</creatorcontrib><creatorcontrib>Elferink, Ronald P.J. Oude</creatorcontrib><creatorcontrib>Chamuleau, Robert A.F.M.</creatorcontrib><creatorcontrib>Hoekstra, Ruurdtje</creatorcontrib><title>Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells</title><title>Drug metabolism and disposition</title><addtitle>Drug Metab Dispos</addtitle><description>Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression.</description><subject>Cell Culture Techniques</subject><subject>Cell Differentiation - drug effects</subject><subject>Cell Line, Tumor</subject><subject>Culture Media - chemistry</subject><subject>Dimethyl Sulfoxide - pharmacology</subject><subject>Drug Evaluation, Preclinical</subject><subject>Genetic Vectors</subject><subject>Humans</subject><subject>Lentivirus - genetics</subject><subject>Liver - metabolism</subject><subject>Metabolic Detoxication, Phase I</subject><subject>Metabolic Detoxication, Phase II</subject><subject>Models, Biological</subject><subject>Pharmaceutical Preparations - metabolism</subject><subject>Receptors, Cytoplasmic and Nuclear - genetics</subject><issn>0090-9556</issn><issn>1521-009X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1v1DAQhi0EokvhyhH5yCVbf8VxjlWAbqWiSluQuEVee9I1SuLUH0v7d_iluN3CjdO80jzzamZehN5TsqaUiTM72SLkmjRMEv4CrWjNaEVI--MlWpVCqrau5Ql6E-NPQqgQvH2NTljTKKK4WKHfN0nvRsDXBwhwvwSI0fkZ-wGnPeDOzzG5lJM7AD6fbfAx6RnwFgwsyYcibDYQn-At3GUXYII54aH0ujymHAD_cmmPP7kJ0v5hxDd5HPy9s_DEbNxt6YV8i79CWcSPLk7YzXgDi95e4A7GMb5FrwY9Rnj3XE_R9y-fv3Wb6ur64rI7v6oMVyJV2lAFSplGCiLNzshBMa5r0e5EK4ABbS3lVnNrpJFKS2qGZjCstupx0jB-ij4efZfg7zLE1E8umrJBudjn2FPFa6laRmRB10fUlI_EAEO_BDfp8NBT0j_m0pdcipD9MZcy8OHZO-8msP_wv0EUQB0BKBceHIQ-GgezAVtealJvvfuf9x9tN5-B</recordid><startdate>201701</startdate><enddate>201701</enddate><creator>van der Mark, Vincent A.</creator><creator>Rudi de Waart, D.</creator><creator>Shevchenko, Valery</creator><creator>Elferink, Ronald P.J. Oude</creator><creator>Chamuleau, Robert A.F.M.</creator><creator>Hoekstra, Ruurdtje</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>201701</creationdate><title>Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells</title><author>van der Mark, Vincent A. ; Rudi de Waart, D. ; Shevchenko, Valery ; Elferink, Ronald P.J. 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Oude</creatorcontrib><creatorcontrib>Chamuleau, Robert A.F.M.</creatorcontrib><creatorcontrib>Hoekstra, Ruurdtje</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Drug metabolism and disposition</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>van der Mark, Vincent A.</au><au>Rudi de Waart, D.</au><au>Shevchenko, Valery</au><au>Elferink, Ronald P.J. Oude</au><au>Chamuleau, Robert A.F.M.</au><au>Hoekstra, Ruurdtje</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells</atitle><jtitle>Drug metabolism and disposition</jtitle><addtitle>Drug Metab Dispos</addtitle><date>2017-01</date><risdate>2017</risdate><volume>45</volume><issue>1</issue><spage>56</spage><epage>67</epage><pages>56-67</pages><issn>0090-9556</issn><eissn>1521-009X</eissn><abstract>Dimethylsulfoxide (DMSO) induces cellular differentiation and expression of drug metabolic enzymes in the human liver cell line HepaRG; however, DMSO also induces cell death and interferes with cellular activities. The aim of this study was to examine whether overexpression of the constitutive androstane receptor (CAR, NR1I3), the nuclear receptor controlling various drug metabolism genes, would sufficiently promote differentiation and drug metabolism in HepaRG cells, optionally without using DMSO. By stable lentiviral overexpression of CAR, HepaRG cultures were less affected by DMSO in total protein content and obtained increased resistance to acetaminophen- and amiodarone-induced cell death. Transcript levels of CAR target genes were significantly increased in HepaRG-CAR cultures without DMSO, resulting in increased activities of cytochrome P450 (P450) enzymes and bilirubin conjugation to levels equal or surpassing those of HepaRG cells cultured with DMSO. Unexpectedly, CAR overexpression also increased the activities of non-CAR target P450s, as well as albumin production. In combination with DMSO treatment, CAR overexpression further increased transcript levels and activities of CAR targets. Induction of CYP1A2 and CYP2B6 remained unchanged, whereas CYP3A4 was reduced. Moreover, the metabolism of low-clearance compounds warfarin and prednisolone was increased. In conclusion, CAR overexpression creates a more physiologically relevant environment for studies on hepatic (drug) metabolism and differentiation in HepaRG cells without the utilization of DMSO. DMSO still may be applied to accomplish higher drug metabolism, required for sensitive assays, such as low-clearance studies and identification of (rare) metabolites, whereas reduced total protein content after DMSO culture is diminished by CAR overexpression.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>27780834</pmid><doi>10.1124/dmd.116.072603</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Cell Culture Techniques Cell Differentiation - drug effects Cell Line, Tumor Culture Media - chemistry Dimethyl Sulfoxide - pharmacology Drug Evaluation, Preclinical Genetic Vectors Humans Lentivirus - genetics Liver - metabolism Metabolic Detoxication, Phase I Metabolic Detoxication, Phase II Models, Biological Pharmaceutical Preparations - metabolism Receptors, Cytoplasmic and Nuclear - genetics |
title | Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells |
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