title: Novel cell-based reporter assay system using epitope-tagged protein for the identification of agonistic ligands of constitutive androstane receptor (CAR).
abstract: Constitutive androstane receptor (CAR) plays vital roles in multiple liver functions including xenobiotic and energy metabolisms, and also in hepatocarcinogenesis. CAR shows species-dependent ligand selectivity, leading to species differences in the actions of xenobiotics. Thus, to know whether a compound of interest activates human CAR or not is helpful for the chemical safety evaluation. However, it is very difficult to observe clear ligand response for CAR in cell-based assays, because of its high basal transcriptional activity in the absence of its ligands. In this study, we found that reporter assays using HepG2 cells human and mouse CAR fused with an epitope-tag at its C-terminus showed low basal activity and high response to the corresponding agonists but not to their indirect activator, phenobarbital. Using this system, 176 industrial chemicals were screened for their abilities to activate human and mouse CAR, and 6 compounds moderately or strongly activated human and/or mouse CAR. The CAR-mediated transcriptions induced by these compounds were blocked by clotrimazole or androstanol, the human and mouse CAR inverse agonists, respectively, suggesting that the newly identified CAR activators are agonistic ligands of CAR. Taken together, our reporter assay system may be a promising tool to assess chemicals' agonistic activities toward CAR.
authors.affiliation: Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Imai
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Yamazoe
authors.forename: Kouichi
authors.initials: K
authors.lastname: Yoshinari
mesh_terms: D000732:Androstanols / Q000494:pharmacology; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics; D001681:Biological Assay; D003022:Clotrimazole / Q000494:pharmacology; D000090702:Constitutive Androstane Receptor; D065702:Cytochrome P-450 CYP2B6; D000939:Epitopes / Q000737:chemistry; D056945:Hep G2 Cells; D006801:Humans; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D051379:Mice; D010634:Phenobarbital / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000819:agonists* / Q000187:drug effects; D013045:Species Specificity
chemical_list: D000732:Androstanols; D000090702:Constitutive Androstane Receptor; D000939:Epitopes; D008024:Ligands; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; C000658:androstan-3-ol; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D003022:Clotrimazole; D010634:Phenobarbital