MOLECULAR TOXICOLOGY OF PLACENTAL CARBOXYLESTRASE

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APPLICANT'S ABSTRACT: Carboxylesterases are known to play an important role in xenobiotic metabolism and detoxication of pesticides The long-term objective of the proposed studies is to determine the molecular basis for the existence of multiple forms of human carboxylesterases, and to determine the structure, function and regulation of these enzymes. The central hypothesis of the proposed project is that the multiple forms of carboxylesterases expressed in human placenta are distinct gene products that have different substrate specificities, and are independently regulated. The specific aims of the project are (1) to isolate from a human placental library full-length cDNAs encoding two carboxylesterases (HP-1 and HP-2), and (2) to characterize by biochemical, immunochemical and molecular techniques the structure, function and regulation of these enzymes. As part of enzymatic and immunochemical studies, recombinant enzymes and antibodies against the recombinant proteins will be produced. To test the hypothesis that HP-1 and HP-2 have different substrate specificities, the recombinant enzymes will be examined for their ability to hydrolyze selected esters and amides, and their ability to catalyze the transesterification of cocaine and the synthesis of fatty acid ethyl esters. Immunoprecipitation from placental samples will be conducted to compare enzymatic characteristics between the natural and recombinant enzymes. To test the hypothesis that HP-1 and HP-2 are independently regulated, the MRNA and protein levels will be determined with samples from individual placenta and cultured placental slices treated with xenobiotics. Samples for these studies will cover a broad range of genetic (i.e., ethnic) and environmental (i.e., smoking) factors. Some progress has been made toward the goals of the project. The enzymatic activity of placental microsomes to hydrolyze several esters has been determined. Two partial cDNAs (348 bp each) that apparently encode two distinct carboxylesterases have been isolated from human placentas. In addition to hydrolyzing numerous xenobiotics such as drugs and pesticides, carboxylesterases are known to catalyze a transesterification reaction and fatty acid ethyl ester synthesis. Ethylcocaine, a more potent metabolite of cocaine in mediating lethality and cardiac toxicity than the parent compound, is formed through the transesterification reaction. The accumulation of ethyl esters due to alcohol abuse during pregnancy has been shown to contribute significantly to the development of fetal alcohol syndrome. Therefore, the experiments outlined in this project will contribute significantly to our basic understanding of carboxylesterases as a family of enzymes involved in the detoxication and bioactivation of xenobiotics.