Abstract
Constitutive androstane receptor (CAR) is highly expressed in the liver and plays central roles in the xenobiotic-induced expression of drug-metabolizing enzymes. CAR is also involved in hepatocyte proliferation and hepatocarcinogenesis, at least in rodents, and it remains unknown if CAR shows similar influences in human became clear species differences are often observed on the action of CAR activators. CAR is retained in cytoplasm and CAR activators induce its nuclear translocation to enhance target gene expression. As CAR is barely expressed in culture cells such as human hepatoma HepG2 cells, it is necessary to overexpress CAR in these cells through transfecting its expression plasmid, in order to analyze CAR functions and/or assess CAR activators. However, it is known that overexpressed CAR tends to stimulate the gene transcription even in the absence of CAR activators because of its automatic translocation into nucleus. In this study, we have sought to establish a new cell-based reporter assay system for the identification of species-selective activators of hCAR using tag-fused CAR proteins. We found that in reporter assays with HepG2 cells hCAR fused with V5 and His tag showed low basal activity and considerable response to a hCAR agonist CITCO treatment but not to phenobarbital. One hundred and seventy-six industrial chemicals were thus subjected to this reporter assay system to assess their ability to activate hCAR. As results, four compounds activated hCAR were identified as agonistic hCAR activators. In conclusion, our reporter assay system may be a promising tool to assess chemicals’ agonistic activities toward hCAR.
