From a toxicological standpoint, P4502E1 (2E1), the principal form of P450 inducible by ethanol consumption, has proven unique among these hemeproteins in that it metabolizes a variety of xenobiotics to hepatotoxic and/or carcinogenic products. Thus, our finding of a 2E1 enzyme in man, with catalytic and inductive properties similar to the rodent orthologs, has begun to shed light on the probable etiology of certain disease states fostered by alcohol abuse. In order to further understand the role of 2E1 in alcohol-promoted pathology, we wish to continue our studies on the functional and regulatory attributes of this human liver enzyme. Our plan includes assessing whether 2E1 participates in the bioactivation of cocaine and certain nitrosamines (N-nitroso-2,6- dimethylmorpholine and N-nitrosopyrrolidine), agents whose cytotoxicity is enhanced in the alcoholic. 2E1 involvement in these reactions of toxicological impact will be determined using purified enzyme systems as well as intact hepatic microsomes together with anti-2E1 immunoglobulins. Another of our aims is to identify """"""""variant' human 2E1 proteins that exhibit aberrant functional properties. For this purpose, we will first assess the capacity of individual human liver samples to oxidize the ketone body acetone to acetol, a 2E1-specific process that may embody the enzyme's """"""""physiological"""""""" function. Enzyme immunoquantitation will then allow us to identify those individuals who possess 2E1 proteins with ample immunoreactivity yet poor metabolic activity. After their purification, these catalytically-deficient 2E1 proteins will be subjected to detailed physical characterization, including amino acid sequence analysis of protease-derived peptides, to ascertain whether specific changes in protein primary structure are responsible for alterations in function. In terms of the regulation of 2E1 expression, our aim is to resolve the molecular mechanism via which hepatic enzyme levels are enhanced by alcohol intake. Liver specimens from active drinkers as well as abstainers will be probed with 2E1 cDNAS an antibodies in order to address the following issues: a) whether exposure of hepatocytes to ethanol results in coordinate induction of 2E1 protein and encoding mRNA or whether enzyme levels increase independently of alterations in 2E1 transcripts and b) whether 2E1 enzyme induction, which can be considered an adaptive response to alcohol consumption, is inherent to all or only to certain individuals. Finally, we will examine the human CYP2E1 gene itself, searching for restriction fragment length polymorphisms (RFLPs) that may underlie the differences among individuals in 2E1 enzyme function or its expression. Our overall goal is to describe the caused by excessive alcohol intake and, ultimately;y, to the apparent interindividual variations thereof.
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