Glutathione and cysteine conjugates of a variety of haloalkenes are nephrotoxic and mutagenic through a mechanism involving metabolism by the enzyme, renal cysteine conjugate beta-lyase (beta-lyase), located in the cells of the proximal tubule. This mechanism, which represents one of the most well characterized models of renal bioactivation, is thought to involve the formation of a reactive intermediate capable of covalently modifying cellular macromolecules. It is our hypothesis, based on studies examining the toxicity of S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBC), that a series of events, beginning with mitochondrial dysfunction, followed by deregulation of cytosolic Ca 2+, and the subsequent activation of catabolic enzymes, contribute to the alteration of """"""""critical"""""""" macromolecules and ultimately lead to cell death. However, the relationship between these processes and their role in cell injury has not been clearly established. Furthermore, it appears that the chemical reactivity of the products of betalyase-metabolism is determined by the structure of the parent haloalkene, suggesting that multiple mechanisms of cysteine conjugate toxicity may exist. In the proposed studies we will use freshly isolated renal epithelial cells to study the mechanism(s) responsible for cysteine conjugate toxicity. The two major goals of the proposed studies are: First to investigate the role of specific pathways or processes (1, mitochondrial injury; 2, covalent binding; 3, lipid peroxidation; 4, cytosolic Ca2+ deregulation; and 5, stimulation of phospholipase and/or endonuclease activity) in the proximal tubular cell injury and death associated with beta-lyase-mediated, cysteine conjugate toxicity; and Second to use a structure/activity approach to identify similarities and differences in the process of cell injury induced by four different toxic cysteine conjugates (PCBC, S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC)). In addition to furthering our understanding of cysteine conjugate toxicity, these studies will also provide valuable information on the reaction of proximal tubular epithelial cells to injury. With the information gained from these investigations, future studies will be designed to explore the mechanism(s) of proximal tubular cell injury at the molecular level by identifying the critical macromolecular targets of toxic cysteine conjugates.
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