Toxicological Significance of Alkylbenzene Metabolism

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DESCRIPTION (provided by applicant): The long-term objective of this proposal is to supply information that will aid in identifying conditions under which individuals may be susceptible to alkylbenzene-induced toxicity. Alkylbenzenes are produced in extensive quantities throughout the world. Simple aromatic hydrocarbons (e.g. benzene, toluene, and ethylbenzene) are major components of unleaded gasoline and are also used in the production of a wide variety of consumer products. The P450 system is responsible for both aliphatic and aromatic hydroxylation of the aromatic hydrocarbons, with several forms, CYP1A2, CYP2B4, and CYP2E1, being implicated in hydrocarbon metabolism. The toxicity from many of the hydrocarbons is known to be due to bioactivation of a small percentage of the parent compound to reactive intermediates. This process requires a functional interaction between P450 and the flavoprotein NADPH-cytochrome P450 reductase. However, total P450 levels exceed those of reductase by a ratio of 20:1. In addition, there are multiple forms of P450, each having their own reductase binding characteristics and substrate dependencies. This raises the question: "How does a single reductase supply electrons to all the P450s?" A second question is: "Can one P450 influence the function of a second P450?" The proposed studies are designed to address questions related to the organization of P450 and reductase, focusing on the metabolism of alkylbenzenes. During the prior grant period, we identified important interactions among CYP2B4, CYP1A2 and reductase that have a substantial effect on substrate metabolism. The results are consistent with the formation of a CYP1A2-CYP2B4 complex having unusual reductase binding characteristics. We now propose to characterize these interactions, and to identify the region(s) responsible for the interactions among these proteins. We also intend to examine P450-P450 interactions in the CYP2E1/CYP1A2/reductase, and CYP2E1/CYP2B4/reductase systems, and to focus on the ability of these interactions to alter not only metabolism of hydrocarbons and other substrates, but also generation of reactive oxygen. These studies will increase our understanding of how the P450 electron transport chain is organized, and will provide new important information on the role of the P450 system in the bioactivation of aromatic hydrocarbons and the generation of reactive oxygen - a process that can have a significant influence on chemical toxicity.