HUMAN LIVER CYTOCHROMES P450

在人类中观察到的药物氧化的显著个体间差异往往与P450酶的特定亚家族有关。在肝脏P450中，底物氧化表现出很大差异的是那些在CYP2C亚家族中的P450。造成这一现象的因素包括接触某些外来生物物质所致的诱因。我们实验室在上一次资助期间的研究发现，抗生素利福平(RIF)在分离的人肝细胞中诱导了与CYP2C8、CYP2C9和CYP2C19对应的蛋白质和mRNAs。这些研究提出了一种假设，即外源反应元件(XRE)存在于CYP2C基因的5‘侧翼区。在特定目的1中提出的研究旨在分离、克隆和测序人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P450表达的外源物质的信息。此外，分离CYP2C19基因的这个调控区是进行特定目的2中计划的实验的先决条件。特定目的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，主要集中在区分每个CYP2C基因中对化学试剂RIF有反应的DNA元件。此外，有关大鼠肝细胞内细胞色素P450 2 C11的研究表明，地塞米松(DEX)可增强这种通过糖皮质激素受体(GR)发生的细胞色素P450 2 C酶的表达。此外，RIF最近被证明可以激活GR。这些研究指导我们假设，人类中的CYP2C酶也可能是由地塞米松通过GR诱导的。有趣的是，CYP2C8和CYP2C9基因都有几个GRE。因此，我们将研究DEX是否促进人的CYP2C表达，以及糖皮质激素的调节是否通过与RIF相同的反应元件来实现。最后，将在特定的目标3中原位测试反应元件的功能。在这些研究中，将用RIF和/或DEX处理培养的人肝细胞，并测量其CYP2C的mRNA和蛋白水平。此外，还将评估代表每种CYP2C酶的催化活性，以确定CYP2C基因表达的增强最终是否会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。综上所述，此次续签申请中计划进行的研究将扩展S之前对人类CYP2C酶的调查，并进一步研究其个体间差异的原因。了解与外来生物暴露引起的基因表达变化相关的分子事件可以导致适当的机制来识别某些药物代谢能力降低或增强的个人。

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.