The broad objective of the proposed research is to investigate the metabolism and toxicity of halogenated hydrocarbons. This competitive renewal application will be focused on the glutathione transferase zeta (GSTZ 1-1)-catalyzed metabolism of a-haloacids, particularly dichloroacetate (DCA). There are three Specific Aims: 1. Investigate the GSTZ 1-1 -catalyzed biotransformation of a-haloacids, including the mechanism of the DCA-induced inactivation of polymorphic variants of hGSTZ 1-1, the covalent modification of rat liver proteins by DCA, the in vivo inactivation of rat liver GSTZ1-l by DCA, the inhibition or inactivation of (3STZ1-1 by the tyrosine metabolites maleylacetone (MA) and maleylacetoacetate (MAA), the regulation of rat liver GSTZ1 expression, the organ and tissue distribution of GSTZ 1-1 by immunohistochemistry, and development of selective, fluorescent substrates for assaying (3STZ 1-1 activity. 2. Investigate the structure and function of GSTZ 1-1, including the active-site residues involved in the binding of glutathione and in catalysis with a-haloacids, MAA, and MA, investigation of residues involved in the inactivation of GSTZ 1-1 by DCA, and the relevance of structural changes in allelic variants that differ in their susceptibility to inactivation by DCA. 3. Investigate the capacity of GSTs other than GSTZ 1-1 to catalyze the isomerization of MAA and MA. The proposed studies are health-related for several reasons. First, DCA is present in finished drinking water supplies consumed by an estimated 170 million individuals in the U.S. DCA is also carcinogenic in both rats and mice. Hence, there is a need for more information about the fate and biological effects of DCA and other dihaloacetates to understand better the risk associated with human exposure to a-haloacids. Second, DCA is used in the clinical management of congenital lactic acidosis and has been proposed as a therapeutic agent for a range of disease states, which supports the need for a better understanding of the fate of DCA. Finally, the finding that GSTZ1- 1 also catalyzes the isomerization to MAA to fumarylacetoacetate (FAA) indicates that DCA-induced inactivation of GSTZ 1-1 may decrease the formation of FAA. If, as has been proposed, FAA plays a role in liver tumor formation in hereditary tyrosinemia type 1 (HT- 1), inactivation of GSTZ1-1 by DCA would be expected to prevent the formation of FAA. Hence, DCA may also find use in the management of HT-1. It is, therefore, important to explore the mechanism by which DCA and other dihaloacetates inactivate GSTZ1-1.
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