HUMAN P450 ENZYMES AND THEIR TOXICOLOGICAL IMPACT

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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.