HUMAN LIVER CYTOCHROMES P450

人肝细胞色素 P450

• 批准号: 2852378
• 负责人: JUDY L RAUCY
• 金额: $ 48.35万
• 依托单位: LA JOLLA INST FOR MOLECULAR MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2852378
• 关键词: HTC cell; RNase protection assay; cytochrome P450; dexamethasone; drug metabolism; enzyme activity; enzyme induction /repression; gene deletion mutation; gene induction /repression; genetic enhancer element; genetic regulation; genetic regulatory element; genetic transcription; glucocorticoids; human tissue; laboratory rabbit; liver metabolism; microsomes; nucleic acid sequence; pharmacogenetics; reporter genes; rifamycins; transfection; western blottings

The marked inter-individual differences in drug oxidations observed in humans is often-times associated with particular subfamilies of P450 enzymes. Among the hepatic P450s to exhibit wide variations in substrate oxidations are those in the CYP2C subfamily. Factors contributing to this phenomenon include induction from exposure to certain xenobiotics. Investigations in our laboratory during the previous funding period noted that the antibiotic, rifampicin (RIF) induces proteins and mRNAs corresponding to CYP2C8, CYP2C9, and CYP2C19 in isolated human hepatocytes. These studies gave rise to the hypothesis that xenobiotic response elements (XRE) exist in the 5'-flanking regions of the CYP2C genes. Studies proposed in specific aim 1 are designed to isolate, clone, and sequence the 5'-upstream region of the human CYP2C19 gene. We hypothesize that isolation of this region will provide information regarding xenobiotics that affect expression of this P450. Moreover, isolation of this regulatory region of the CYP2C19 gene is a prerequisite for the experiments planned in specific aim 2. Experiments described in specific aim 2 will test the hypothesis that RIF enhances expression of the human CYP2C P450 genes. Mechanisms governing RIF- mediated induction of CYP2C8, CYP2C9, and CYP2C19 will be explored with our primary focus on distinguishing DNA elements within each CYP2C gene that are responsive to the chemical agent, RIF. In addition, investigation regarding CYP2C11 in rat hepatocytes have indicated that dexamethasone (DEX) enhances expression of this CYP2C enzyme which occurs via the glucocorticoid receptor (GR). Furthermore, RIF has recently been shown to activate the GR. These studies have directed us to hypothesize that the CYP2C enzymes in humans may also be induced by DEX through the GR. Interestingly, both CYP2C8 and CYP2C9 genes possess several GREs. Thus, we will examine whether DEX enhances human CYP2C expression and if glucocorticoid regulation is via the same response element as that identified for RIF. Finally, the function of the response elements will be tested in situ in specific aim 3. For these studies, cultured human hepatocytes will be treated with RIF and/or DEX and CYP2C mRNA and protein levels measured. In addition, catalytic activities representative of each CYP2C enzyme will be assessed to determine whether enhanced expression of the CYP2C genes ultimately leads to an increase in drug metabolism. In this manner, the function of the CYP2C enhancer elements can be verified in intact liver cells. Taken together, studies planned in this renewal application will extend previous investigation s on the human CYP2C enzymes and further examine causes for their inter-individual variability. Understanding molecular events associated with altered gene expression due to xenobiotic exposure can lead to appropriate mechanisms for identifying individuals with reduced or enhanced capacity to metabolize certain drugs.

在人类中观察到的药物氧化的显着个体间差异通常与 P450 酶的特定亚家族有关。在底物氧化方面表现出广泛变化的肝脏 P450 是 CYP2C 亚家族中的那些。造成这种现象的因素包括接触某些外源物质的诱导。我们实验室在上一个资助期间的调查表明，抗生素利福平 (RIF) 在分离的人肝细胞中诱导产生与 CYP2C8、CYP2C9 和 CYP2C19 相对应的蛋白质和 mRNA。这些研究提出了这样的假设：外源反应元件 (XRE) 存在于 CYP2C 基因的 5' 侧翼区域。具体目标 1 中提出的研究旨在分离、克隆和测序人类 CYP2C19 基因的 5' 上游区域。我们假设该区域的分离将提供有关影响该 P450 表达的异生素的信息。此外，分离 CYP2C19 基因的调节区域是特定目标 2 中计划的实验的先决条件。特定目标 2 中描述的实验将检验 RIF 增强人类 CYP2C P450 基因表达的假设。我们将探索 RIF 介导的 CYP2C8、CYP2C9 和 CYP2C19 诱导的机制，我们的主要重点是区分每个 CYP2C 基因内对化学试剂 RIF 做出反应的 DNA 元件。此外，对大鼠肝细胞中 CYP2C11 的研究表明，地塞米松 (DEX) 可以通过糖皮质激素受体 (GR) 增强这种 CYP2C 酶的表达。此外，RIF 最近被证明可以激活 GR。这些研究使我们推测人类的 CYP2C 酶也可能是 DEX 通过 GR 诱导的。有趣的是，CYP2C8 和 CYP2C9 基因都具有多个 GRE。因此，我们将检查 DEX 是否增强人 CYP2C 表达，以及糖皮质激素调节是否通过与 RIF 相同的反应元件进行。最后，将在特定目标 3 中原位测试响应元件的功能。对于这些研究，将用 RIF 和/或 DEX 处理培养的人肝细胞，并测量 CYP2C mRNA 和蛋白质水平。此外，将评估代表每种 CYP2C 酶的催化活性，以确定 CYP2C 基因表达的增强是否最终导致药物代谢的增加。通过这种方式，可以在完整的肝细胞中验证 CYP2C 增强子元件的功能。总而言之，本次更新申请中计划的研究将扩展先前对人类 CYP2C 酶的研究，并进一步检查其个体间差异的原因。了解与异生素暴露导致的基因表达改变相关的分子事件可以产生适当的机制来识别某些药物代谢能力降低或增强的个体。