HUMAN P450 ENZYMES AND THEIR TOXICOLOGICAL IMPACT

它能将多种底物代谢成肝毒素，从而使其 对人类具有重要的毒理意义。毒性的严重性 这种P450在生物转化过程中产生的物质是由其 表达的程度。从而了解CYPE2E1的作用机制 法规为确定由 毒药。我们之前的研究主要是针对人类的细胞色素P450 2 1，并且已经 帮助确定了管理其监管和其潜力的几个方面 在与酗酒有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细胞色素P450-2的调控机制 并将这些研究扩展到包括CYP4A11。这两种P450酶 参与了细胞脂肪酸浓度的调节，通过 代谢花生四烯酸和脂肪酸，如月桂酸。 此外，几种异源诱导剂和生理状态会导致 两个P450的表达增强，表明这些酶是协同作用的 受到类似机制的监管。一个可能在基因中很重要的因素 氧化应激是两种酶共同的表达方式。这 条件可以通过P450的催化活性来调用；特别是 那些与细胞色素P450-2相关的活动。在分解代谢过程中，这个P450 “泄漏”电子到氧气，产生活性氧中间体 (ROIS)。考虑到这一点，当前的提案将决定 感兴趣区参与细胞色素P450_2E_1和细胞色素P4A_(11)的表达 人肝细胞培养。初步研究表明， 氧化剂处理导致两者微粒体浓度升高 P450。此外，这两种酶的mRNA水平都升高了。特定的 目标一包含的实验旨在确定氧化剂， 在人肝细胞中产生更高水平的细胞色素P4E1P4A11的表达。 在具体目标二中，提出了实验以确定P450- 外源物质的中介代谢是ROI的主要来源 如果产生了足够的水平来诱导CYP2E1和 细胞色素P4A11。具体目标三中描述的实验将确定 导致细胞色素P450 2E1和细胞色素P4A11基因表达水平升高的分子事件。 总而言之，我们的目标将决定ROI，是否牵涉到 在P450介导的过程中产生的几种病理障碍 底物的氧化，会提高细胞色素P4A11和细胞色素P42E1的浓度 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.