Mechanism of Glutathione Conjugate Dependent Toxicity

谷胱甘肽缀合物依赖性毒性机制

• 批准号: 6805315
• 负责人: Arthur Joseph Cooper
• 金额: $ 35.55万
• 依托单位: WINIFRED MASTERSON BURKE MED RES INST
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6805315
• 关键词: SDS polyacrylamide gel electrophoresis; alkenes; bioenergetics; brain; chemical conjugate; cytotoxicity; environmental exposure; environmental toxicology; enzyme activity; fluorocarbon polymers; glutathione; halogenation; kidney; laboratory rat; liquid chromatography mass spectrometry; liver; membrane potentials; mitochondria; toxin metabolism; transaminases

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.

描述（由申请人提供）：很大一部分美国人在工作场所和环境中接触有毒卤代烯烃（例如三氯乙烯、四氯乙烯和四氟乙烯）。这些化合物部分代谢为卤化半胱氨酸 S-结合物，被认为是主要毒物。 S-(1,1,2,2-四氟乙基)-L-半胱氨酸 (TFEC)，四氟乙烯的半胱氨酸 S-缀合物，在这里被选为具有代表性的有毒卤代半胱氨酸 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 α-裂合酶转化为丙酮酸、氨和反应性（硫代酰化）片段。在体内，肾脏以及某种程度上的肝脏和大脑都容易受到影响。之前，我们发现（i）大鼠肾脏中的高Mr半胱氨酸S-缀合α-裂合酶与线粒体HSP70和蛋白质二硫键异构酶共纯化，并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)、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-缀合 α-裂合酶活性，但它们本身在周转过程中失活（联催化失活），并且 (iii) 将培养的 PC12 细胞和星形胶质细胞暴露于 TFEC 会导致选择性丢失密钥 能量代谢的线粒体酶，包括 mitAspAT 和 β-酮戊二酸脱氢酶复合物 (KGDHC)。其他人表明，KGDHC 和支链酮酸脱氢酶复合物 (BCKAD) 是体内大鼠肾细胞中 TFEC 的靶标，卤化半胱氨酸 S-缀合物是离体肾脏和肝脏线粒体的代谢毒物。 PI 和同事提出，KGDHC 和 BCKAD 对失活敏感，因为毒物通道分别涉及 mitAspAT 和 BCATm。本提案的总体目标是确定 TFEC/TFEC 硫代酰化片段毒害线粒体代谢的机制。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、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联系起来，b) TFEC 联催化灭活 mitAspAT 和 BCATm 的机制，以及 c) 有毒物质（TFEC 硫代酰化片段）从 mitAspAT 转移（通道）至 KGDHC，以及从 BCATm 转移（通道）至 BCKAD。研究结果应阐明接触某些卤化异生物质与能量代谢受损之间的联系，并可能提出一种方法来最大限度地减少严重接触个体的毒性作用。