Toxicological Significance of Alkylbenzene Metabolism

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DESCRIPTION (provided by applicant): The long-term objective of this proposal is to better understand how the proteins of the P450 monooxygenase system are organized in the endoplasmic reticulum, the role of P450-P450 interactions on the function of these enzymes, and how these interactions may make individuals susceptible to alkylbenzene-induced toxicity by alterations in P450-mediated hydrocarbon metabolism. Alkylbenzenes are produced in extensive quantities throughout the world, with simple aromatic hydrocarbons being major components of gasoline and used in 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. 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. These conditions raise basic questions as to how the enzymes of this microsomal electron transport chain are organized. During the previous grant period we demonstrated that P450s interact through the formation of heteromeric P450 complexes. We have identified complexes between CYP2B4-CYP1A2 as well as CYP1A2-CYP2E1. These interactions were shown to have a profound effect on xenobiotic metabolism, largely due to an alteration in the manner in which NADPH-cytochrome P450 reductase transfers electrons to P450s in the heteromeric complex. Interestingly, we did not observe an effect on P450 function from CYP2B4-CYP2E1 complexes. The proposed studies are designed to extend our investigation and address questions related to the organization of reductase and P450, their interactions within the endoplasmic reticulum, and how these interactions affect xenobiotic metabolism, including the metabolism of alkylbenzenes. We plan to continue our characterization of these interactions, examining (1) the functional consequences of P450-P450 interactions, (2) the structural basis for these interactions by identifying the region(s) responsible for P450-P450 complex formation, and (3) the organizational consequences to P450-P450 complex formation (i.e. how do such interactions affect their regional distribution in the endoplasmic reticulum). 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.