AS-GSH CONJUGATION LIMITS AS AVAILABILITY & TOXICITY

这项工作的总体目标是了解更多关于戒毒的知识 和砷(As)的清除，并检测As-蛋白质的作用 结合物在毒性中起作用。需要检验的主要假设是 体内AS的清除依赖于两条不同的途径，一条主要途径 涉及AS-GSH偶联物和一个次要偶联物的形成和运输 依赖于砷离子的运输；此外，未能降低砷的反应性 作为b-谷胱甘肽的结合允许金属-蛋白质硫醇的形成 对细胞有毒性。该项目利用了质量方面的最新进展 光谱学和分子遗传学来解决这些问题。尽管它是 普遍认为AS的GSH偶联物是其必需的 解毒，没有令人信服的证据表明它们的存在 活着。研究人员合成了三硫代砷(ATG)和 甲基砷二还原型谷胱甘肽(MADG)的检测和鉴定 γ-谷氨酰转肽酶基因缺陷小鼠和野生型胆汁 小鼠LC/MS和LC/感应联用的研究 MS(LC/ICP-MS)。使用与多药耐药相关的其他初步数据 蛋白2(MRP2)缺陷的大鼠已表明，AS排泄到胆汁中是 依赖于这种基因。他们还使用了γ-谷氨酰半胱氨酸合成酶 (GGCs)缺陷胚胎发育缺乏合成能力的细胞 GSH，证明这些细胞是AS敏感的。他们将延长这些 研究和开发在体内研究其他硫醇结合物的方法。他们 将检验AS-GSH偶联物的形成和它们的 As-半胱氨酸衍生物在数量上是As中最重要的途径 通行证。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的排泄中起作用。他们有 确定酵母缺乏蛋白质的泛素/蛋白酶体途径 降解对砷很敏感。他们将使用这些GGC不足的和 GSH过多的哺乳动物细胞和泛素/蛋白酶体缺陷的小鼠 函数来检验这样的假设，即未能形成AS-GSH结合物会导致 形成对细胞有毒性的AS蛋白硫醇结合物。

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.