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.

中文翻译：

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苯巴比妥钠和两种其他组成型雄激素受体（CAR）激活剂对野生型和CAR剔除大鼠培养的肝细胞中细胞色素P450酶诱导和复制性DNA合成的影响的比较研究。

非遗传毒性化学物质可通过有丝分裂作用方式在大鼠和小鼠中产生肝脏肿瘤，该作用方式包括激活组成型雄甾烷受体（CAR）。这项研究的目的是评估正常（野生型； WT）和CAR敲除（KO）大鼠培养的肝细胞用于筛选化合物作为大鼠CAR的潜在激活剂的有效性，并验证该测试系统的有效性。使用100和1000μM苯巴比妥钠（NaPB），3-100μM氟喹康唑（FQZ）或3-300μM3-（二氟甲基）-1-甲基- N-（3′，4′，6-三氟[1,1′-联苯] -2-基）-1H-吡唑-4-羧酰胺（TI1）持续96 h。细胞色素P450（CYP）酶的诱导可通过测量7-戊氧基间苯二酚O-去戊基酶（PROD）进行监测，7-苄氧基间苯二酚O-脱苄基酶（BROD）和7-苄氧基喹啉O-脱苄基酶（BQ）的活性。通过向培养基中添加10μM5-溴-2′-脱氧尿苷标记进行复制性DNA合成（RDS）的肝细胞，以测定肝细胞标记指数。用NaPB，FQZ和TI1处理WT大鼠肝细胞而不是CAR KO，可增加肝细胞RDS并诱导CYP2B依赖性PROD活性。相反，在WT和CAR KO大鼠肝细胞中，所有这三种化合物均增加了CYP2B / 3A依赖性BROD和CYP3A依赖性BQ活性。通过用25 ng / ml表皮生长因子作为阳性对照处理，WT和CAR KO大鼠肝细胞中的肝细胞RDS均增加。总体，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酶诱导的影响均被消除。如该验证研究所示，CAR KO肝细胞模型是一种有用的体外机制工具，可用于快速筛选作为大鼠CAR潜在激活剂的化学物质。