Glutathione plays a central role in cellular defense against reactive electrophiles and oxidants, two of the most common producers of cellular injury in both chemical toxicity and disease. Our overall objective is to identify mechanisms for disposition of glutathione S conjugates and mercapturic acids (S-substituted N-acetyl-L-cysteines) via a novel intrahepatic pathway. This pathway was recently described from our laboratory, and is thought to play an important role in hepatic detoxification of electrophiles and toxic metals such as mercury, mercapturic acid biosynthesis, bile formation, and glutathione homeostasis.
The specific aims are: (1) Define the role of the intrahepatic gamma- glutamyl cycle in mercapturic acid formation. In contrast to current models of mercapturic acid biosynthesis, our recent studies demonstrated that this detoxification pathway can occur entirely within the liver. Moreover, in contrast to current thinking, a representative mercapturic acid (S.2,4-dinitrophenyl-N-acetylcysteine, or DNP-NAC) was excreted by isolated rat liver almost exclusively into bile, and not into the sinusoidal circulation. To best this model and to identify the mechanisms involved, we plan to: A.- Examine sinusoidal uptake and disposition of structurally distinct cysteine S-conjugates and mercapturic acids, both in vivo and in the perfused liver (with ethyl iodide and 1-chloro-2,4-dinitrobenzene as model xenobiotics). B. Using isolated canalicular and basolateral membrane vesicles, define the driving forces and substrate specificities for hepatic transport of mercapturic acids, and C. Test the role of sinusoidal gamma-glutamyl transferase iii hepatic clearance of GSH and glutathione S-conjugates, and in mercapturic acid biosynthesis. (2) Express the novel canalicular ATP-independent, high affinity glutathione S-conjugate carrier in Xenopus laevis oocytes using poly(A)+ RNA isolated from rat liver, and characterize the properties of the transporter in the oocyte system. With oocytes as a functional assay for transport, use expression cloning strategy to identify the cDNA for the transporter. Cloning will not only yield important structural information on this glutathione S-conjugate transporter, but will allow production of molecular probes with which to define its role in xenobiotic metabolism in liver and the whole organism. It is anticipated that these studies will ultimately provide information critical for the prevention and treatment of toxic liver cell injury and its extrahepatic complications induced by reactive electrophilic compounds.
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