HUMAN LIVER CYTOCHROMES P450

人肝细胞色素 P450

• 批准号: 6519541
• 负责人: JUDY L RAUCY
• 金额: $ 5.26万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519541
• 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著个体间差异通常与P450酶的特定亚家族相关。在肝P450中，表现出底物氧化的广泛变化的是CYP 2C亚家族中的那些。造成这一现象的因素包括接触某些异生物质的诱导。在上一个资助期内，我们实验室的研究发现，抗生素利福平（RIF）诱导分离的人肝细胞中对应于CYP 2C 8、CYP 2C 9和CYP 2C 19的蛋白质和mRNA。这些研究提出了外源性反应元件（XRE）存在于CYP 2C基因5 '侧翼区域的假设。具体目标1中拟定的研究旨在分离、克隆和测序人CYP 2C 19基因的5 '上游区域。我们假设，该区域的隔离将提供有关外源性物质影响P450表达的信息。此外，分离CYP 2C 19基因的该调控区是特定目标2中计划的实验的先决条件。具体目的2中描述的实验将检验RIF增强人CYP 2C P450基因表达的假设。将探索RIF介导的CYP 2C 8、CYP 2C 9和CYP 2C 19诱导的机制，我们的主要重点是区分每个CYP 2C基因内对化学试剂RIF有反应的DNA元件。此外，关于大鼠肝细胞中CYP 2C 11的研究表明，地塞米松（DEX）可增强该CYP 2C酶的表达，该酶通过糖皮质激素受体（GR）发生。此外，RIF最近已被证明激活GR。这些研究指导我们假设，CYP 2C酶在人类中也可能被DEX通过GR诱导。有趣的是，CYP 2C 8和CYP 2C 9基因都具有几个GR。因此，我们将检查DEX是否增强人CYP 2C表达，以及糖皮质激素调节是否通过与RIF相同的反应元件。最后，将在具体目标3中对响应元件的功能进行现场测试。对于这些研究，将用RIF和/或DEX处理人肝细胞培养物，并测量CYP 2C mRNA和蛋白水平。此外，将评估每种CYP 2C酶的代表性催化活性，以确定CYP 2C基因表达增强是否最终导致药物代谢增加。以这种方式，可以在完整的肝细胞中验证CYP 2C增强子元件的功能。综上所述，本次更新申请中计划的研究将扩展先前对人CYP 2C酶的研究，并进一步检查其个体间变异性的原因。了解与外源性暴露引起的基因表达改变相关的分子事件，可以找到适当的机制来识别某些药物代谢能力降低或增强的个体。