Mechanism of Glutathione Conjugate Dependent Toxicity

描述(申请人提供)：相当一部分美国人在工作场所和环境中接触到有毒的卤代烯(如三氯乙烯、四氯乙烯和四氟乙烯)。这些化合物被部分代谢成卤代半胱氨酸S结合物，这被认为是主要的毒物。S-(1，1，2，2-四氟乙基)-L-半胱氨酸是四氟乙烯的半胱氨酸S偶联物，是典型的有毒卤代半胱氨酸S偶联物。有毒的卤化半胱氨酸S偶联物通过半胱氨酸S偶联物裂解酶转化为丙酮酸、氨和活性(硫代酰化)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，我们已经证明：(1)大鼠肾脏中的一个高MR半胱氨酸S结合裂解酶与线粒体HSP70和蛋白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[线粒体支链转氨酶(BCATm)、胞浆支链转氨酶(BCATm)、丙氨酸-乙氧基酸氨基转移酶II(MITASPAT)]具有半胱氨酸S结合裂解酶活性，但自身在周转(同步催化失活)过程中失活；(Iii)PC12细胞和星形胶质细胞暴露在TFEC培养中导致能量代谢的关键线粒体酶丢失，包括线粒体支链氨基转移酶(BCAT)和选择性丙二酸氨基转移酶复合体(KGDHC)。另有研究表明，KGDHC和支链酮酸脱氢酶复合体(BCKAD)是TFEC在体内作用的靶点，卤代半胱氨酸S结合物是分离的肾脏和肝脏线粒体的代谢毒物。PI和他的同事们认为KGDHC和BCKAD分别对涉及mitAspAT和BCATm的毒物通道失活敏感。本提案的总体目标是确定TFEC/TFEC硫代酰化片段毒化线粒体代谢的机制。因此，我们的目标是确定TFEC对大鼠肝、脑和肾线粒体呼吸、钙稳态、膜电位和肿胀的影响及其与线粒体关键酶活性丧失的关系，b)TFEC共同催化灭活mitAspAT和BCATm的机制，以及c)毒物(TFEC硫代酰化片段)从mitAspat转移(通道)到KGDHC和从BCATm转移到BCKAD的机制。这些发现应该会阐明接触某些卤代异生物质与能量代谢受损之间的联系，并可能提出一种将重度接触者的毒性影响降至最低的方法。

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.