HUMAN P450 ENZYMES AND THEIR TOXICOLOGICAL IMPACT

CYP 2 E1将多种底物代谢为肝毒素， 对人类有重要的毒性 毒性的严重程度 在生物转化过程中由这种P450产生的是由其 表达的程度。 因此，了解CYPE 2 E1参与的机制 调节提供了确定细胞损伤的基础， 有毒物质 我们以前的研究主要集中在人CYP 2 E1上， 帮助确定了管理其监管和潜力的几个方面 在与酒精滥用相关的疾病状态中的作用。 我们希望 继续研究人类CYP 2 E1调节机制 并将这些研究扩展到包括CYP 4A 11。 这两种P450酶 参与细胞脂肪酸浓度的调节， 代谢花生四烯酸和脂肪酸，如月桂酸。 此外，几种异生素诱导剂和生理状态引起 两种P450的表达增强，表明这些酶是共同的。 类似的机制。 一个可能在基因中很重要的因素 表达并且因此对两种酶共同的是氧化应激。 这 条件可以通过P450的催化活性来调用;特别是 与CYP 2 E1相关的活性。 在此期间，P450 “泄漏”电子到氧气中产生活性氧中间体 （ROI）。 考虑到这一点，目前的提案将确定 ROIs参与CYP 2 E1和CYP 4A 11的表达， 人肝细胞的培养物。 初步研究表明， 氧化剂处理导致两者的微粒体浓度更高 P450 此外，这两种酶的mRNA水平升高。具体 目标一包含设计用于确定氧化剂， 在人肝细胞中产生更高的CYP 2 E1和CYP 4A 11表达。 在具体目标二中，提出了实验来鉴定P450- 外源性物质介导的代谢构成ROI的主要来源 如果产生足够的水平引起CYP 2 E1的诱导， CYP4A11。 具体目标三中描述的实验将确定 导致CYP 2 E1和CYP 4A 11 mRNA水平升高的分子事件。 总之，我们的目标将决定是否投资回报率，牵连在 几种病理性疾病和P450介导的 底物的氧化，将提高CYP 4A 11和CYP 2 E1浓度 在人类肝细胞中的作用，以及控制其增强的机制。

CYP2E1 metabolizes a variety of substrates to hepatotoxins making it toxicologically significant to humans. The severity of toxicity produced by this P450 during biotransformation is determined by its extent of expression. Thus understanding mechanisms involved in CYPE2E1 regulation provides a basis for determining cellular damage produced by toxicants. Our previous studies have focused on human CYP2E1 and have helped define several aspects governing its regulation and its potential role in disease states associated with alcohol abuse. We wish to continue our studies ascertaining mechanisms of human CYP2E1 regulation and extend these studies to include CYP4A11. Both of these P450 enzymes are involved in the regulation of cellular fatty acid concentrations by metabolizing arachidonic acid and fatty acids, such as laurate. Moreover, several xenobiotic inducers and physiological states cause enhanced expression of both P450s, suggesting these enzymes are co- regulated by similar mechanisms. A factor that may be important in gene expression and hence common to both enzymes is oxidative stress. This condition may be invoked by the catalytic activity of P450s; especially those activities associated with CYP2E1. During catabolism, this P450 "leaks" electrons to oxygen producing reactive oxygen intermediates (ROIs). With this in mind, the current proposal will determine the participation of ROIs in the expression of CYP2E1 and CYP4A11 in primary cultures of human hepatocytes. Preliminary studies demonstrate that oxidant treatment results in higher microsomal concentrations of both P450s. Furthermore, mRNA levels of both enzymes are elevated. Specific aim one contains experiments designed to determine whether the oxidants, produce greater expression of CYP2E1 and CYP4A11 in human hepatocytes. In specific aim two, experiments are proposed to identify whether P450- mediated metabolism of xenobiotics constitutes the major source of ROIs and if sufficient levels are generated to cause induction of CYP2E1 and CYP4A11. Experiments described in specific aim three will determine the molecular events leading to enhanced levels of CYP2E1 and CYP4A11 mRNA. Taken together, our aims will determine whether ROIs, implicated in several pathological disorders and generated during P450-mediated oxidation of substrates, will enhance CYP4A11 and CYP2E1 concentrations in human hepatocytes, and mechanisms governing their enhancement.