BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany.
BASF SE, Carl-Bosch-Strasse 38, 67056 Ludwigshafen, Germany.
Toxicology. 2020 Mar 30;433-434:152394. doi: 10.1016/j.tox.2020.152394. Epub 2020 Feb 3.
Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 μM sodium phenobarbital (NaPB), 3-100 μM fluquinconazole (FQZ), or 3-300 μM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-carboxamide (TI1) for 96 h. Induction of cytochrome P450 (CYP) enzymes was monitored by measurement of 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities. Hepatocytes undergoing replicative DNA synthesis (RDS) were labelled by adding 10 μM 5-bromo-2´-deoxyuridine to the culture medium for determination of the hepatocyte labelling index. The treatment of WT, but not of CAR KO, rat hepatocytes with NaPB, FQZ and TI1 increased hepatocyte RDS and induced CYP2B-dependent PROD activity. In contrast, all three compounds increased CYP2B/3A-dependent BROD and CYP3A-dependent BQ activities in both WT and CAR KO rat hepatocytes. Hepatocyte RDS was increased in both WT and CAR KO rat hepatocytes by treatment with 25 ng/ml epidermal growth factor as a positive control. Overall, these results demonstrate that the effects of three CAR activators on RDS and CYP2B enzyme induction are abolished in cultured CAR KO rat hepatocytes. As demonstrated by this validation study, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid screening of chemicals as potential activators of rat CAR.
非遗传毒性化学物质可以通过有丝分裂作用模式在大鼠和小鼠中产生肝肿瘤，该模式涉及组成型雄烷受体 (CAR) 的激活。本研究的目的是评估正常（野生型；WT）和 CAR 敲除 (KO) 大鼠培养的肝细胞在筛选化合物作为潜在的大鼠 CAR 激活剂方面的有用性，并验证该测试系统。用 100 和 1000 μM 苯巴比妥钠 (NaPB)、3-100 μM 氟康唑 (FQZ) 或 3-300 μM 3-(二氟甲基)-1-甲基-N-(3´,4´,6-三氟[1,1´-联苯]-2-基)-1H-吡唑-4-甲酰胺 (TI1) 处理雄性 Sprague-Dawley WT 和 CAR KO 大鼠培养的肝细胞 96 h。通过测量 7-戊氧基resorufin O-脱戊基酶 (PROD)、7-苄氧基resorufin O-脱苄基酶 (BROD) 和 7-苄氧基喹啉 O-脱苄基酶 (BQ) 活性来监测细胞色素 P450 (CYP) 酶的诱导。通过将 10 μM 5-溴-2´-脱氧尿苷添加到培养基中以确定肝细胞标记指数，来标记正在进行复制 DNA 合成 (RDS) 的肝细胞。用 NaPB、FQZ 和 TI1 处理 WT 大鼠肝细胞，但不能处理 CAR KO 大鼠肝细胞，可增加肝细胞 RDS 并诱导 CYP2B 依赖性 PROD 活性。相比之下，所有三种化合物均可增加 WT 和 CAR KO 大鼠肝细胞中 CYP2B/3A 依赖性 BROD 和 CYP3A 依赖性 BQ 活性。用 25 ng/ml 表皮生长因子作为阳性对照处理 WT 和 CAR KO 大鼠肝细胞均可增加肝细胞 RDS。总的来说，这些结果表明，在培养的 CAR KO 大鼠肝细胞中，三种 CAR 激活剂对 RDS 和 CYP2B 酶诱导的作用被消除。如本验证研究所示，CAR KO 肝细胞模型是一种有用的体外机制工具，可用于快速筛选潜在的大鼠 CAR 激活剂。
