Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats
Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor in rats
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DOI：
10.1016/j.fct.2022.113510
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发表时间：
2022-11-17
影响因子：
4.3
通讯作者：
Yoshinari，Kouichi
中科院分区：
文献类型：
--
作者：
Sato，Takumi;Shizu，Ryota;Yoshinari，Kouichi
组成型雄甾烷受体（CAR）是一种在啮齿类动物体内药物代谢和处置以及肝脏肿瘤发生发展中起关键作用的核受体。CAR被配体和间接激活剂激活，这些配体和间接激活剂不与受体结合，而是通过细胞信号传导激活受体。在这项研究中，我们试图确定大鼠CAR（rCAR）的直接和间接激活剂。评估突变对rCAR转录活性的影响，鉴定了一种称为rCAR-3A-G354 Q的突变体，其显示出低组成型活性和高配体反应性。使用增加大鼠原代肝细胞中CAR靶基因Cyp 2b 1 mRNA水平的化合物进行了使用突变体的报告基因测定。几种化合物激活rCAR-3A-G354 Q并被认为是rCAR配体。由于间接CAR激活剂被认为表现出很小的种属差异，我们随后测定了用增加大鼠肝细胞中Cyp 2b 1 mRNA水平但不激活rCAR-3A-G354 Q的化合物处理后人肝细胞样HepaRG细胞中CYP 2B 6 mRNA水平。结果证明六种化合物是可能的rCAR间接激活剂。总之，大鼠原代肝细胞中Cyp 2b 1 mRNA水平和报告基因试验中rCAR-3A-G354 Q激活的联合测量可用于评价化学品对rCAR的激活。
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 ofCyp2b1, 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 determinedCYP2B6mRNA levels in human hepatocyte-like HepaRG cells after treatment with compounds that increasedCyp2b1mRNA 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 ofCyp2b1mRNA levels in rat primary hepatocytes and rCAR-3A-G354Q activation in reporter assays can be useful for evaluating rCAR activation by chemicals.