Mechanism of Glutathione Conjugate Dependent Toxicity

描述（由申请人提供）：很大一部分美国人口在工作场所和环境中暴露于有毒的卤化烯烃（例如，三氯乙烯，四氯乙烯和四氟乙烯）。这些化合物部分被代谢为卤代半胱氨酸s缀合物，这被认为是主要的毒物。S-（1,1,2,2-四氟乙基）- l -半胱氨酸（TFEC）是四氟乙烯的半胱氨酸S-缀合物，这里选择它作为具有代表性的有毒卤化半胱氨酸S-缀合物。有毒的卤化半胱氨酸s -缀合物通过半胱氨酸s -缀合物裂解酶转化为丙酮酸、氨和活性（硫酰化）片段。在体内，肾脏，在某种程度上，肝脏和大脑都是易感的。先前，我们证明了(i)高mr半胱氨酸s -共轭物？与线粒体HSP70和蛋白二硫异构酶共纯化，含有线粒体天冬氨酸转氨酶（mitAspAT）。（ii）几种转氨酶[线粒体支链转氨酶（BCATm），细胞质支链转氨酶，丙氨酸-乙醛酸转氨酶ii， mitAspAT]具有半胱氨酸s偶联物？-裂解酶活性，但自身在周转期失活（合催化失活），并且（iii） PC12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选择性丧失，包括mitAspAT和？-酮戊二酸脱氢酶复合物。其他研究表明KGDHC和支链酮酸脱氢酶复合物（BCKAD）是活体大鼠肾细胞TFEC的靶点，而卤代半胱氨酸s偶联物是离体肾脏和肝脏线粒体的代谢性毒物。PI和同事们认为KGDHC和BCKAD分别对mitAspAT和BCATm的毒性通道失活敏感。本提案的总体目标是确定线粒体代谢被TFEC/TFEC硫酰化片段毒害的机制。因此，我们的目标是确定：a) TFEC对离体大鼠肝、脑和肾线粒体的呼吸、ca2 +稳态、膜电位和肿胀的影响，并将这些病理变化与线粒体关键酶活性的丧失联系起来；b) TFEC协同灭活mitAspAT和BCATm的机制；c)有毒物质（TFEC硫酰化片段）从mitAspAT转移（通道）到KGDHC，从BCATm转移到BCKAD的机制。研究结果应阐明暴露于某些卤化异种生物与能量代谢受损之间的联系，并可能提出一种方法，以尽量减少对大量暴露的个体的毒性作用。

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.