AS-GSH CONJUGATION LIMITS AS AVAILABILITY & TOXICITY

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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.