Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats

• Published in: Food and chemical toxicology 2022-12, Vol.170, p.113510, Article 113510
• Main Authors: Sato, Takumi, Shizu, Ryota, Miura, Yoshie, Hosaka, Takuomi, Kanno, Yuichiro, Sasaki, Takamitsu, Yoshinari, Kouichi
• Format: Article
• Language: English
• Subjects: androstanes; Constitutive androstane receptor; Drug-metabolizing enzyme; genes; Hepatocarcinogenesis; hepatocytes; humans; Ligands; liver; mutants; pharmacokinetics; Primary hepatocyte; rats; Reporter assay; species; transcription (genetics)
• ISSN: 0278-6915; 1873-6351
• DOI: 10.1016/j.fct.2022.113510

Constitutive androstane receptor (CAR) is a nuclear receptor that plays a key role in drug metabolism and disposition and in the development of liver tumors in rodents. CAR is activated by ligands and indirect activators, which do not bind to the receptor but activate it through cellular signaling. In this study, we sought to identify direct and indirect activators of rat CAR (rCAR). Assessment of the influence of mutations on the transcriptional activity of rCAR identified a mutant termed rCAR-3A-G354Q that displays low constitutive activity and high ligand responsiveness. Reporter assays using the mutant were performed with compounds that increased the mRNA levels of Cyp2b1, a CAR target gene, in rat primary hepatocytes. Several compounds activated rCAR-3A-G354Q and were implicated as rCAR ligands. Since indirect CAR activators are considered to display little species differences, we then determined CYP2B6 mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increased Cyp2b1 mRNA levels in rat hepatocytes but did not activate rCAR-3A-G354Q. The results demonstrated six compounds as possible rCAR indirect activators. Taken together, the combined measurement of Cyp2b1 mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals. •The insertion of 3 alanine (3A) before Helix 12 reduced basal activity of rat CAR.•Additional G354Q mutation in Helix 12 increased the ligand sensitivity of rat CAR.•Using a mutant with 3A and G354Q, several rat CAR ligands were identified.•Rat CAR activators can be identified using the mutant and rat primary hepatocytes.