Mechanism of Glutathione Conjugate Dependent Toxicity

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DESCRIPTION (provided by applicant): A significant portion of the US population is exposed to toxic, halogenated alkenes (e.g., trichloroethylene, tetrachloroethylene and tetrafluoroethylene) in the workplace and environment. These compounds are metabolized in part to halogenated cysteine S-conjugates, which are thought to be the major toxicants. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine (TFEC), the cysteine S-conjugate of tetrafluoroethylene, is chosen here as a representative toxic, halogenated cysteine S-conjugate. Toxic, halogenated cysteine S-conjugates are converted to pyruvate, ammonia and a reactive (thioacylating) fragment by cysteine S-conjugate ?-lyases. In vivo, the kidney and, to some extent, liver and brain, are susceptible. Previously, we showed that (i) a high-Mr cysteine S-conjugate ?-lyase in rat kidney co-purifies with mitochondrial HSP70 and protein disulfide isomerase, and contains mitochondrial aspartate aminotransferase (mitAspAT) (ii) several aminotransferases [mitochondrial branched-chain aminotransferase (BCATm), cytosolic branched-chain aminotransferase, alanine-glyoxylate aminotransferase II, mitAspAT] possess cysteine S-conjugate ?-lyase activity, but are themselves inactivated during turnover (syncatalytic inactivation), and (iii) exposure of PC12 cells and astrocytes in culture to TFEC causes selective loss of key mitochondrial enzymes of energy metabolism, including mitAspAT and ?-ketoglutarate dehydrogenase complex (KGDHC). Others have shown that KGDHC and branched-chain keto acid dehydrogenase complex (BCKAD) are targets of TFEC in rat kidney cells in vivo, and that halogenated cysteine S-conjugates are metabolic poisons of isolated kidney and liver mitochondria. The PI and coworkers have suggested that KGDHC and BCKAD are sensitive to inactivation due to toxicant channeling involving mitAspAT and BCATm, respectively. The overall goal of the present proposal is to determine the mechanism by which mitochondrial metabolism is poisoned by TFEC/TFEC thioacylating fragment. Accordingly, our aims are to determine: a) the effects of TFEC on respiration, Ca 2+ homeostasis, membrane potential and swelling in isolated rat liver, brain and kidney mitochondria, and correlate such pathological changes with loss of key mitochondrial enzyme activities, b) the mechanism whereby mitAspAT and BCATm are syncatalytically inactivated by TFEC, and c) the mechanism of toxicant (TFEC thioacylating fragment) transfer (channeling) from mitAspAT to KGDHC and from BCATm to BCKAD. The findings should elucidate the link between exposure to certain halogenated xenobiotics and impaired energy metabolism, and may suggest a means to minimize the toxic effects in heavily exposed individuals.