The broad, long-term objectives of this research are to understand the properties of glutathione transferase Z1- 1 (GSTZ1-1) and its role in the elimination of haloacetic acids, particularly dichloroacetic acid (DCA) across the human lifespan. This is important because while DCA has therapeutic benefits in treating certain solid tumors, lactic acidosis and pulmonary arterial hypertension, it exhibits marked individual variability in pharmacokinetics, and can cause side effects at high doses, particularly in adults. The first step in DCA metabolism is dechlorination to glyoxylate, catalyzed by GSTZ1-1. Also known as maleylacetoacetate isomerase, and playing an important role in catabolism of tyrosine, GSTZ1-1 is expressed mainly in the liver and is the only enzyme known to catalyze the dehalogenation of haloacetic acids, including DCA. Although it is well recognized that DCA inhibits its own metabolism and the metabolism of maleylacetoacetate and its decarboxylation product, maleylacetone, almost certainly because DCA inactivates GSTZ1-1, the reasons for the marked individual variability in pharmacokinetics of DCA after repeated doses are only beginning to be understood. Most adults clear repeated doses of DCA more slowly than most children. Persons with different GSTZ1-1 haplotypes appear to differ in their response to repeated doses of DCA. Recent in vitro work showed that chloride concentration affected the rate of inactivation of GSTZ1-1 by DCA in a haplotype-dependent manner. In addition to its well-known presence in liver cytosol, GSTZ1-1 has recently been found to exist in the mitochondrial matrix. This is the same location as pyruvate dehydrogenase kinase, DCA's pharmacodynamics site of action. Most of the downstream metabolism of the DCA metabolite, glyoxylate, occurs in the mitochondria suggesting the importance of this site in the overall disposition of DCA. This application seeks to examine age-related changes in expression and activity of GSTZ1-1 across the human lifespan and ascertain the properties of the newly-discovered mitochondrial enzyme.
Three specific aims are proposed. The first specific aim will investigate age-related changes in hepatic GSTZ1-1 expression and activity in mitochondria and cytosol. The second specific aim will examine the properties of the mitochondrial enzyme, with respect to the relative loss of mitochondrial versus cytosolic GSTZ1-1 following in vivo exposure to DCA, and the ability of the mitochondrial GSTZ1-1/MAAI to convert the physiologically important substrate of GSTZ1-1, maleylacetone, to fumarylacetone. The third specific aim will use in vitro studies to investigate sensitivity of mitochondrial GSTZ1-1 to inactivation by DCA and the role of chloride and haplotype as determinants of the rate and mechanism of inactivation of cytosolic, mitochondrial and expressed GSTZ1-1 by DCA.
Glutathione transferase Z1 (GSTZ1-1), the only enzyme known to convert the metabolic regulator and putative anticancer drug, dichloroacetate, to glyoxylate, has recently been identified in a new part of the liver cell, the mitochondrial matrix, which is the site of dichloroacetate's therapeutic interaction with its therapeutic target, pyruvat dehydrogenase kinase. The side effects of dichloroacetate vary between individuals of different ages and genetic makeup and are likely to be related to the properties of GSTZ1-1. This application seeks to investigate age-related and genetic changes in cytosolic and mitochondrial GSTZ1-1 and study the properties and stability of mitochondrial GSTZ1-1 in order to better understand its critical role in biotransformation of dichloroacetate.
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