The overall objective of this laboratory is to identify those factors that underlie human susceptibility to methylmercury (MeHg) poisoning. MeHg is a highly toxic environmental pollutant: clinical and experimental studies demonstrate that exposure to MeHg results primarily in neurologic damage characterized by ataxia, sensory disturbances and changes in the mental state. The only way to prevent or ameliorate toxicity once MeHg has been ingested is to accelerate its removal from the body. Approximately 90% of the total excretion in humans or animals exposed to MeHg occurs via the gastrointestinal tract. Gastrointestinal excretion is in turn determined primarily by biliary secretion. Our recent studies provide the first direct demonstration of the mechanism by which MeHg crosses the blood-brain barrier to reach its target tissue, and of the mechanism by which MeHg is transported across the liver cell canalicular membrane into bile. These observations not only provide a new framework for understanding transport mechanisms for toxic metals, but also provide a mechanistic basis for the rational design of therapeutic strategies in MeHg intoxication. The overall objective of the proposed studies is to characterize these MeHg transport systems at the cellular and molecular level. Mechanisms of MeHg transport across the luminal and abluminal membranes of brain capillary endothelial cells, the cells that constitute the blood-brain barrier, will be characterized by: A) Testing the role of plasma GSH and of luminal membrane-associated gamma-glutamyltransferase in MeHg uptake into isolated brain capillary endothehal cells. B) Examining the mechanism of MeHg efflux from brain capillary endothelial cells, and C) Examining the molecular mechanism of MeHg-L-cysteine transport on the System L amino acid carrier. We propose to characterize the expression of the brain capillary transporter in Xenopus laevis oocytes, and use the direct expression cloning strategy to identify the cDNA for the transporter. If successful, cloning will yield important structural information on this amino acid carrier, and will allow production of molecular probes with which to define its role in MeHg disposition and toxicity. Hepatocanalicular transport of MeHg will be examined by: A) Testing whether MeHg-SG is transported across the canalicular membrane into bile by the recently cloned high Km canalicular GSH carrier (RcGshT), and/or by the low Km GSH/glutathione S-conjugate carrier recently identified in our laboratory, and B) Testing whether MeHg is reabsorbed from bile across the canalicular membrane as the cysteine complex on System L carriers.
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