HUMAN P450 ENZYMES AND THEIR TOXICOLOGICAL IMPACT

CYP2E1 将多种底物代谢为肝毒素，使其 对人类具有重要的毒理学意义。 毒性的严重程度 P450 在生物转化过程中产生的量由其决定 表达的程度。 从而了解 CYPE2E1 涉及的机制 调节为确定细胞损伤提供了基础 有毒物质。 我们之前的研究主要集中在人类 CYP2E1 上，并已 帮助定义了管理其监管及其潜力的几个方面 在与酒精滥用相关的疾病状态中的作用。 我们希望 继续我们的研究以确定人类 CYP2E1 调节机制 并将这些研究扩展到包括 CYP4A11。 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，例如月桂酸。 此外，一些外源性诱导剂和生理状态会导致 两种 P450 的表达增强，表明这些酶是共同的 受类似机制监管。 基因中可能重要的一个因素 表达，因此这两种酶的共同点是氧化应激。 这个 该条件可能是由 P450 的催化活性引起的；尤其是 那些与 CYP2E1 相关的活动。 在分解代谢过程中，P450 将电子“泄漏”给氧气，产生活性氧中间体 （投资回报率）。 考虑到这一点，当前的提案将确定 ROI参与原代细胞中CYP2E1和CYP4A11的表达 人肝细胞培养物。 初步研究表明 氧化剂处理导致两者的微粒体浓度更高 P450s。 此外，两种酶的 mRNA 水平均升高。具体 目标一包含旨在确定氧化剂是否， 在人肝细胞中产生更高的 CYP2E1 和 CYP4A11 表达。 在具体目标二中，建议进行实验以确定 P450- 外源物质介导的代谢构成了 ROI 的主要来源 如果产生足够的水平来诱导 CYP2E1 和 CYP4A11。 具体目标三中描述的实验将确定 导致 CYP2E1 和 CYP4A11 mRNA 水平升高的分子事件。 总而言之，我们的目标将决定投资回报率是否涉及 P450 介导过程中产生的几种病理性疾病 底物氧化，将提高 CYP4A11 和 CYP2E1 浓度 在人类肝细胞中，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.