Mechanism of Glutathione Conjugate Dependent Toxicity

描述（由申请人提供）：相当一部分美国人口暴露于有毒的卤代烯烃（例如，三氯乙烯，四氯乙烯和三氯乙烯）的工作场所和环境。这些化合物部分代谢为卤代半胱氨酸S-共轭物，这被认为是主要的毒物。在此选择S-（1，1，2，2-四氟乙基）-L-半胱氨酸（TFEC），其为C1的半胱氨酸S-缀合物，作为代表性毒性卤化半胱氨酸S-缀合物。有毒的卤代半胱氨酸S-共轭物被半胱氨酸S-共轭物转化为丙酮酸盐、氨和反应性（硫酰化）片段。裂合酶在体内，肾脏以及在某种程度上肝脏和大脑是易感的。以前，我们表明，（i）高Mr半胱氨酸S-共轭？大鼠肾脏中的裂合酶与线粒体HSP 70和蛋白质二硫键异构酶共纯化，并含有线粒体天冬氨酸氨基转移酶（mitAspAT）（ii）几种氨基转移酶[线粒体支链氨基转移酶（BCATm），胞质支链氨基转移酶，丙氨酸-乙醛酸氨基转移酶II，mitAspAT]具有半胱氨酸S-缀合物？裂解酶活性，但本身在周转过程中失活（协同催化失活），和（iii）培养物中的PC 12细胞和星形胶质细胞暴露于TFEC会导致能量代谢的关键线粒体酶（包括mitAspAT和？酮戊二酸脱氢酶复合物（KGDHC）。其他研究表明，KGDHC和支链酮酸脱氢酶复合物（BCKAD）是体内大鼠肾细胞中TFEC的靶点，卤代半胱氨酸S-缀合物是分离的肾脏和肝脏线粒体的代谢毒物。PI及其同事认为KGDHC和BCKAD对分别涉及mitAspAT和BCATm的毒物通道导致的失活敏感。本提案的总体目标是确定线粒体代谢被TFEC/TFEC硫代酰化片段毒害的机制。因此，我们的目标是确定：a）TFEC对分离的大鼠肝、脑和肾线粒体中的呼吸、Ca 2+稳态、膜电位和肿胀的影响，并将这些病理变化与关键线粒体酶活性的丧失相关联，B）TFEC使mitAspAT和BCATm合成催化失活的机制，以及c）毒物（TFEC硫酰化片段）从mitAspAT转移（通道）到KGDHC和从BCATm转移（通道）到BCKAD的机制。研究结果应阐明暴露于某些卤化异生物质和受损的能量代谢之间的联系，并可能建议一种方法，以尽量减少在严重暴露的个人的毒性作用。

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.