Barwick J L, Quattrochi L C, Mills A S, Potenza C, Tukey R H, Guzelian P S
University of Colorado Health Sciences Center, Denver 80262, USA.
Mol Pharmacol. 1996 Jul;50(1):10-6.
Interindividual variation in the spontaneous and in the glucocorticoid-or rifampicin-inducible expression of the CYP3A cytochromes P450, the dominant froms of this supergene family that catalyze the oxidation of numerous drugs and environmental chemicals in human liver, remains largely unexplained, due in part to the lack of a validated animal model. We analyzed the 5'-flanking sequences of CYP3A genes from the rat (CYP3A23, CYP3A2), rabbit (CYP3A6), and human (CYP3A4, CYP3A5, CYP3A7) and found variable regions separated by three areas (consensus I, II, and III) of sequence homology immediately upstream of their respective promoters. We used trans-species gene transfer in cellulo as a new approach for determining the basis for qualitative differences among species in liver expression of different forms of CYP3A. When we transfected into cultured rat hepatocytes vectors containing 5'-flanking DNA from CYP3A23, CYP3A4, or CYP3A6 genes, we found that CAT activity was induced on treatment with dexamethasone or pregnenolone-16 alpha-carbonitrile only if consensus II sequences were included. Rifampicin treatment had no effect. When the same constructions containing consensus II were transfected into rabbit hepatocytes, increased activity was observed on treatment of the cells with dexamethasone or with rifampicin but not with pregnenolone-16 alpha-carbonitrile. These results suggest that the host cellular environment rather than the structure of the gene dictates the pattern of CYP3A inducibility. The application of this new model system will provide a unique technique for identifying mechanisms of induction and advancing the development of appropriate toxicological models for human safety assessment.
细胞色素P450 3A（CYP3A）是该超基因家族的主要形式，可催化人肝脏中多种药物和环境化学物质的氧化。CYP3A在自发状态下以及在糖皮质激素或利福平诱导下的个体间差异在很大程度上仍未得到解释，部分原因是缺乏经过验证的动物模型。我们分析了大鼠（CYP3A23、CYP3A2）、兔子（CYP3A6）和人类（CYP3A4、CYP3A5、CYP3A7）CYP3A基因的5'侧翼序列，发现在其各自启动子上游紧邻的区域有三个序列同源性区域（共有序列I、II和III）将可变区域分隔开。我们采用细胞内跨物种基因转移作为一种新方法，来确定不同形式的CYP3A在肝脏表达中物种间定性差异的基础。当我们将含有CYP3A23、CYP3A4或CYP3A6基因5'侧翼DNA的载体转染到培养的大鼠肝细胞中时，我们发现只有包含共有序列II时，地塞米松或孕烯醇酮-16α-腈处理才能诱导氯霉素乙酰转移酶（CAT）活性。利福平处理没有效果。当将含有共有序列II的相同构建体转染到兔子肝细胞中时，用 地塞米松或利福平处理细胞时观察到活性增加，但用孕烯醇酮-16α-腈处理则没有。这些结果表明，宿主细胞环境而非基因结构决定了CYP3A的诱导模式。这种新模型系统的应用将为确定诱导机制以及推进用于人类安全评估的合适毒理学模型的开发提供一种独特技术。
