Induction of drug metabolism enzymes and MDR1 using a novel human hepatocyte cell line
Induction of drug metabolism enzymes and MDR1 using a novel human hepatocyte cell line
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DOI：
10.1124/jpet.103.061713
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发表时间：
2004-04-01
影响因子：
3.5
通讯作者：
de Morais, SMF
中科院分区：
文献类型：
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作者：
Mills, JB;Rose, KA;de Morais, SMF
药物代谢酶和转运蛋白的诱导可能导致药物间相互作用和功效丧失。体外诱导研究传统上使用原代肝细胞培养物和具有选定标记化合物的酶活性。我们研究了使用新型人类肝细胞克隆 Fa2N-4 细胞系作为替代试剂，该试剂易于获得并提供一致、可重复的系统。我们使用 Invader 检测来监测这些细胞中的基因表达。该测定是一种强大而简单的高通量系统，用于 mRNA 转录本的定量。从用一组已知诱导剂处理的 Fa2N-4 细胞的总 RNA 提取物中对 CYP1A2、CYP3A4、CYP2C9、UGT1A 和 MDR1 转录本进行定量，并与载体对照进行比较。此外，我们使用酶活性测定来监测 CYP1A2、CYP2C9 和 CYP3A4 的诱导。 Fa2N-4 细胞的反应方式与原代人肝细胞类似。用 10 muM 利福平治疗导致 CYP3A4 mRNA 增加（17 倍）和活性（6-β-羟基睾酮形成，9 倍）；以及 CYP2C9 mRNA（4 倍）和活性（4'-羟基双氯芬酸形成，2 倍）。用 50 muM β-萘黄酮处理导致 CYP1A2 mRNA 增加（15 倍）和活性（7-乙氧基试卤灵 O-脱烷基化，27 倍）。 UGT1A mRNA 由 β-萘黄酮（2 倍）诱导，MDR1（P-糖蛋白）mRNA 由利福平（3 倍）诱导。这些使用一些原型诱导剂的初步数据表明，Fa2N-4 细胞可以成为原代人肝细胞的可靠替代品，并且当与 Invader 技术结合使用时，可以为评估药物代谢酶和转运蛋白的诱导提供可靠的测定。
Induction of drug-metabolizing enzymes and transporters can cause drug-drug interactions and loss of efficacy. In vitro induction studies traditionally use primary hepatocyte cultures and enzyme activity with selected marker compounds. We investigated the use of a novel human hepatocyte clone, the Fa2N-4 cell line, as an alternative reagent, which is readily available and provides a consistent, reproducible system. We used the Invader assay to monitor gene expression in these cells. This assay is a robust, yet simple, high-throughput system for quantification of mRNA transcripts. CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1 transcripts were quantified from total RNA extracts from Fa2N-4 cells treated with a panel of known inducers and compared with vehicle controls. In addition, we used enzyme activity assays to monitor the induction of CYP1A2, CYP2C9, and CYP3A4. The Fa2N-4 cells responded in a similar manner as primary human hepatocytes. Treatment with 10 muM rifampin resulted in increases in CYP3A4 mRNA (17-fold) and activity (6-beta-hydroxytestoterone formation, 9-fold); and in CYP2C9 mRNA (4-fold) and activity (4'-hydroxydiclofenac formation, 2-fold). Treatment with 50 muM beta-naphthoflavone resulted in increases in CYP1A2 mRNA (15-fold) and activity (7-ethoxyresorufin O-dealkylation, 27-fold). UGT1A mRNA was induced by beta-naphthoflavone (2-fold), and MDR1 (P-glycoprotein) mRNA was induced by rifampin (3-fold). These preliminary data using a few prototypical inducers show that Fa2N-4 cells can be a reliable surrogate for primary human hepatocytes, and, when used in conjunction with the Invader technology, could provide a reliable assay for assessment of induction of drug-metabolizing enzymes and transporters.