HUMAN LIVER CYTOCHROMES P450

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We propose to continue our studies on human P450 enzymes involved in the metabolism of xenobiotics, with major emphasis placed on those proteins comprising the CYP2C gene subfamily, namely P4502C8, P4502C9, P4502C18, and P4502C19. The CYP2C enzymes are of particular relevance in that one or more of these proteins may underlie several polymorphisms of oxidative drug metabolism in man. Thus, our plan includes the specific CYP2C enzymes involved in the hydroxylation of S-mephenytoin (S-MEPH) and tolbutamide (TOL), two therapeutic agents that exhibit marked interindividual differences with regards to their hepatic metabolism. The involvement of 2C8, 2C9, 2C18, and 2C19 will first be determined in systems reconstituted with these enzymes purified from human liver. Subsequent studies will employ intact liver microsomes with inhibitory antibodies to the appropriate CYP2C enzyme(s) in order to corroborate the results obtained with the purified reconstituted proteins. Another of our aims is to identify human CYP2C proteins that exhibit aberrant functional properties, i.e., those responsible for the S-MEPH and TOL metabolic polymorphisms. For this purpose, we will first assess the capacity of individual human liver samples to oxidize S-MEPH and TOL. Antibody-based quantitation of CYP2C enzymes previously characterized as the major S-MEPH and TOL hydroxylases will allow identification of subjects who possess CYP2C proteins with normal immunoreactivity yet poor metabolic activity. After their purification, these CYP2C enzymes will be subjected to detailed physical characterization, including amino acid sequence analysis of protease-derived peptides, to ascertain the specific changes in protein primary structure that give rise to alterations in function. In terms of P450 enzyme expression, our primary goal is to first resolve whether any of the liver CYP2C proteins are inducible by xenobiotics (e.g., phenobarbital and rifampicin). For these studies, we will employ human hepatocytes, an established in vitro system allowing for manipulation of the cellular mileau but precluding the ethical considerations inherent to in vivo human experimentation. Since hepatocytes will be derived from livers provided mainly by procurement agencies, these investigations can also reveal the suitability of such material for studying P450 function and regulation, a question of importance of both academic and industrial scientists. Our overall goals are to define a role for the CYP2C gene subfamily enzymes in hepatic drug metabolism and, ultimately, to describe a biochemical basis for the interindividual variations noted in their function.