AS-GSH CONJUGATION LIMITS AS AVAILABILITY & TOXICITY

这项工作的总体目标是了解更多关于排毒 和砷（As）的清除，并检查As蛋白的作用， 结合物在毒性中起作用。有待检验的主要假设是， 体内As的清除依赖于两个独立的途径，一个主要的途径 包括As-GSH结合物的形成和转运， 依赖于As离子的运输;此外，未能降低 由于B GSH结合允许形成金属-蛋白硫醇， 对细胞有毒。该项目利用了最新的进展， 光谱和分子遗传学来解决这些问题。虽然 普遍认为，谷胱甘肽共轭物的砷是必不可少的， 解毒，没有令人信服的证据表明它们的存在， vivo.研究人员已经合成了砷三谷胱甘肽（ATG）， 甲基砷二谷氨酸（MADG），并确定了他们的尿液中， γ-谷氨酰转肽酶（GGT）缺陷型小鼠和野生型胆汁 通过使用液相色谱/质谱（LCMS）和LC/电感耦合 MS（LC/ICP-MS）。使用多药耐药相关的其他初步数据 蛋白2（MRP 2）缺乏的大鼠已经表明， 依赖于这个基因。他们还使用了γ-谷氨酰半胱氨酸合成酶 （GGCS）缺陷胚胎发育细胞缺乏合成能力， GSH，并证明这些细胞是As敏感的。他们会把这些 研究和开发方法，以研究其他砷-巯基结合物在体内。他们 将检验As-GSH结合物的形成及其 As-半胱氨酸衍生物在数量上是As中最重要的途径 间隙他们将使用缺乏MDR蛋白和MRP的啮齿动物来测试 假设这些蛋白质在As-GSH排泄中起作用。他们有 确定酵母缺乏蛋白质的泛素/蛋白酶体途径， 降解对As敏感。他们将使用这些，GGCS缺陷， GSH过量产生的哺乳动物细胞和泛素/蛋白酶体缺陷的小鼠 功能，以检验未能形成As-GSH结合物允许 形成对细胞有毒的As-蛋白巯基缀合物。

The overall goal of this work is to learn more about the detoxification and clearance of arsenic (As) and to examine the role that As-protein conjugates play in toxicity. The major hypothesis to be tested is that clearance of As in vivo is dependent on two separate pathways, a major one involving formation and transport of As-GSH conjugates and a minor one dependent on transport of As ions; further, failure to reduce the reactivity of As b GSH conjugation allows the formation of metal-protein thiols that are toxic to the cell. The project takes advantage of recent advances in mass spectrometry and molecular genetics to address these issues. Although it is widely postulated that GSH conjugates of As are essential for its detoxification, there is no convincing demonstration of their existence in vivo. The investigators have synthesized arsenic triglutathione (ATG) and methyl arsenic diglutathione (MADG) and identified them in the urine of gamma-glutamyl transpeptidase (GGT)-deficient mice and the bile of wild type mice by use of liquid chromatography/mass spec (LCMS) and LC/inductive coupled MS (LC/ICP-MS). Other preliminary data using multidrug resistance-associated protein 2 (MRP2)-deficient rats have shown that As excretion into bile is dependent on this gene. They have also used gamma-glutamyl cysteine synthetase (GGCS)-deficient embryos to develop cells that lack the ability to synthesize GSH and demonstrated that these cells are As-sensitive. They will extend these studies and develop methods to study other As-thiol conjugates in vivo. They will test the hypothesis that formation of As-GSH conjugates and their As-cysteine derivatives are quantitatively the most important pathway in As clearance. They will use rodents deficient in MDR proteins and MRPs to test the hypothesis that these proteins function in As-GSH excretion. They have determined that yeast deficient in the ubiquitin/proteasome pathway for protein degradation is sensitive to As. They will use these, GGCS-deficient and GSH-overproducing mammalian cells, and mice deficient in ubiquitin/proteasome function to test the hypothesis that failure to form As-GSH conjugates allows formation of As-protein thiol conjugates that are toxic to the cell.