Stable expression of human cytochrome P450 3A4 in V79 cells and its application for metabolic profiling of ergot derivatives

European Journal of Pharmacology: Environmental Toxicology and Pharmacology Pub Date : 1995-10-06 DOI:10.1016/0926-6917(95)00016-X

Reimund Rauschenbach, Hille Gieschen, Manfred Husemann, Birgit Salomon, Michael Hildebrand

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引用次数: 21

Abstract

Expression of human cytochrome P450 (CYP) in heterologous cells is a means of specifically studying the role of these enzymes in drug metabolism. The complete cDNA encoding CYP3A4 (PCN1) was inserted into an expression vector containing the strong myeloproliferative sarcoma virus promoter in combination with the enhancer of the cytomegalovirus and stably expressed in V79 Chinese hamster cells. The presence of genomically integrated CYP3A4 cDNA cell clones was confirmed by polymerase chain reaction analysis. Transcription was detected by reverse transcribed polymerase chain reaction analysis. Functional expression could be demonstrated by conversion of testosterone to the specific 6β-hydroxylated product. In recombinant V79 cells expressing CYP3A4 about 6% of the substrate was converted to 6β-hydroxytestosterone. The metabolism of two dopaminergic ergot derivatives was investigated in live recombinant V79 cells. Both lisuride and terguride were monodeethylated.

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人细胞色素P450 3A4在V79细胞中的稳定表达及其在麦角衍生物代谢谱中的应用

人细胞色素P450 (CYP)在异源细胞中的表达是专门研究这些酶在药物代谢中的作用的一种手段。将编码CYP3A4 (PCN1)的完整cDNA插入含有强骨髓增殖性肉瘤病毒启动子和巨细胞病毒增强子的表达载体中，在V79中国仓鼠细胞中稳定表达。聚合酶链反应证实存在基因组整合的CYP3A4 cDNA细胞克隆。逆转录聚合酶链式反应检测转录。功能性表达可以通过将睾酮转化为特定的6β-羟基化产物来证明。在表达CYP3A4的重组V79细胞中，约6%的底物转化为6β-羟睾酮。在活的重组V79细胞中研究了两种多巴胺能麦角衍生物的代谢。lisuride和terguide均被单去乙基化。

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European Journal of Pharmacology: Environmental Toxicology and Pharmacology

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