Our overall objectives are to characterize how the functional diversity and regulation of the multiple forms of cytochrome P450 affect the capacity of the organism and particularly the fetus, newborn nd mother to respond to the insult of foreign chemicals (xenobiotics). A determination of how physiologic processes influence the metabolism of xenobiotics and conversely, how xenobiotics can alter the metabolism of hormones and other endogenous substances may provide a key to understanding how the capacity to metabolize xenobiotics is regulated during pregnancy and the perinatal period. This understanding can facilitate the selection of drugs for which adequate detoxicative pathways are available and the identification of factors that determine the susceptibility of the immature to the detrimental effects of foreign chemicals. The focus of this proposal is the Cyp4A family of cytochrome P450 monooxygenases which includes a prostagiandin w-hydroxylase that is greatly elevated in the rabbit lung during pregnancy. The Cyp4A gene that is differentially expressed during pregnancy in the rabbit lung is being cloned and characterized. Gene tmnsfer experiments will be employed to identify cis-acting control elements of this gene. In addition, footprinting assays will be used to determine whether the progesterone receptor binds to this control element of the gene. The Cyp4A family is diverse as indicated by the identification and characterization of several Cyp4A cDNAs in our laboratory. The corresponding enzymes have been expressed in eukaryotic cells by transient transfection of the cDNAs in appropriate expression vectors. The capacities of these enzymes to catalyze the w-hydroxylation of fatty acids including arachidonic acid, prostaglandins as well as drugs such as sodium valproate will be 11 investigated. Human cDNAs will also be cloned in order to define the diversity of Cyp4A genes in humans by sequence analysis and expression of the enzyme from the cloned CDNA. Some Cyp4A P450s are induced by hypolipemic drugs such as clofibrate or the plasticizer diethylhexylphthalate. Cell lines will be identified that respond to clofibrate with the induction of Cyp4A enzymes as judged by hybridization of mRNAs with CDNA probes and immunoblot analysis of microsomal proteins. Cyp4A genes encoding the inducible enzymes will be cloned using the cDNAs as probes. Regulatory regions of the clofibrate-responsive gene will be identified by gene transfer experiments as described earlier for the Cyp4A gene induced by progesterone. DNA-protein binding assays will be used to identify the protein factors that interact with the clofibrate-responsive cis-acting control elements identified in the previous experiments. The identification and characterization of the cellular components that mediate the induction of P450 genes in response to foreign chemicals is a central question regarding our capacity to regulate the metabolism of toxic chemicals.
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