Role Of Altered Membrane Function In Xenobiotic Toxicity

Environmental Health Sciences

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Abstract

Renal secretory transport of organic anions (OA) and organic cations (OC) controls the excretion of most foreign chemicals and/or their metabolites. Our current focus is on the mechanisms, driving forces, and molecular biology of the renal transport proteins which mediate drug and xenobiotic elimination, specifically the organic anion (OAT) and organic cation (OCT) transport systems. Two OATs appear to be of primary importance in renal xenobiotic elimination, OAT1 and OAT3. In collaboration with Sweet and Nigam (UC San Diego), we have obtained, and functionally characterized, an OAT3 knockout (KO) mouse. Kidneys of the KO mice lost the ability to transport OAT3 substrates, e.g., estrone sulfate (ES) and taurocholate. Liver expresses little OAT3 in wild type (WT) and showed no deficit. Choroid plexus transport of all OA substrates was markedly depressed, indicating that OAT3 is the major transporter present at the blood/CSF barrier. Substrates shared by OAT1 and OAT3 showed a partial deficit in renal transport, with no evidence of compensatory increase of OAT1 in the kidney of the KO. In parallel studies, we examined the mechanism of OAT3 transport. Contrary to the previously accepted notion, OAT3 was shown to be a dicarboxylate/OA exchanger. Flux studies and electro-physiological measurements are under way to determine the stoichiometry of the exchange process. Our molecular work has focused on site specific mutation of conserved residues involved with PKC and PKA regulation of transport activity and of charged residues believed to be involved in substrate and counterion binding to the carrier. Several of these have proven to be critical for function and the basis for the functional changes are under investigation. Finally, we have also shown that 1) several mercapturic acids (i.e., N-acetylcysteine S-conjugates) are endogenous substrates for both rat and human OAT1, 2) renal accumulation of mercury-sulfhydryl conjugates are OAT1 substrates and mediate the marked uptake and toxicity of mercury (Hg) toward the renal proximal tubule, and 3) methyl-Hg antidotes, e.g., dimercaptopropane sulfonate (DMPS), form conjugates that are transported by OAT1, but not OAT3. The data demonstrate that the binding of mercury and methyl-mercury to sulfhydryl groups, e.g., with glutathione or its metabolites, is protective relative to free mercury. However, several mercury complexes (e.g., mercury-cysteine and mercury-N-acetyl-cysteine) are much more toxic to OAT1 expressing cells than to controls lacking this transporter, suggesting that transport increases their entry and toxicity. Consistent with this hypothesis, renal toxicity of the Hg-complexes may be blocked in vitro and in vivo by probenecid and p-aminohippurate, competitive inhibitors of OAT1.