title: Stable Overexpression of the Constitutive Androstane Receptor Reduces the Requirement for Culture with Dimethyl Sulfoxide for High Drug Metabolism in HepaRG Cells.
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.
authors: Department of Experimental Surgery (V.A.M., R.A.F.M.C., R.H.), and the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (V.A.M., D.R.W., R.P.J.O.E., R.A.F.M.C., R.H.), Amsterdam, the Netherlands; and Biopredic International, Saint-Grégoire, France (V.S.).
authors: Vincent A
authors: VA
authors: van der Mark
authors: Department of Experimental Surgery (V.A.M., R.A.F.M.C., R.H.), and the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (V.A.M., D.R.W., R.P.J.O.E., R.A.F.M.C., R.H.), Amsterdam, the Netherlands; and Biopredic International, Saint-Grégoire, France (V.S.).
authors: D
authors: D
authors: Rudi de Waart
authors: Department of Experimental Surgery (V.A.M., R.A.F.M.C., R.H.), and the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (V.A.M., D.R.W., R.P.J.O.E., R.A.F.M.C., R.H.), Amsterdam, the Netherlands; and Biopredic International, Saint-Grégoire, France (V.S.).
authors: Valery
authors: V
authors: Shevchenko
authors: Department of Experimental Surgery (V.A.M., R.A.F.M.C., R.H.), and the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (V.A.M., D.R.W., R.P.J.O.E., R.A.F.M.C., R.H.), Amsterdam, the Netherlands; and Biopredic International, Saint-Grégoire, France (V.S.).
authors: Ronald P J Oude
authors: RP
authors: Elferink
authors: Department of Experimental Surgery (V.A.M., R.A.F.M.C., R.H.), and the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (V.A.M., D.R.W., R.P.J.O.E., R.A.F.M.C., R.H.), Amsterdam, the Netherlands; and Biopredic International, Saint-Grégoire, France (V.S.).
authors: Robert A F M
authors: RA
authors: Chamuleau
authors: Department of Experimental Surgery (V.A.M., R.A.F.M.C., R.H.), and the Tytgat Institute for Liver and Intestinal Research, Academic Medical Center (V.A.M., D.R.W., R.P.J.O.E., R.A.F.M.C., R.H.), Amsterdam, the Netherlands; and Biopredic International, Saint-Grégoire, France (V.S.) r.hoekstra@amc.uva.nl.
authors: Ruurdtje
authors: R
authors: Hoekstra
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D000090702:Constitutive Androstane Receptor; D003470:Culture Media / Q000737:chemistry*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008099:Liver / Q000378:metabolism*; D050216:Metabolic Detoxication, Phase I; D050217:Metabolic Detoxication, Phase II; D008954:Models, Biological; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*
chemical_list: D000090702:Constitutive Androstane Receptor; D003470:Culture Media; C000716327:NR1I3 protein, human; D004364:Pharmaceutical Preparations; D018160:Receptors, Cytoplasmic and Nuclear; D004121:Dimethyl Sulfoxide