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 research focus is the biochemistry of these transport proteins; their development, expression, and control; and their impact on the toxicity of xenobiotics. We have previously characterized the mechanisms and energetics of both processes and cloned several of these transport proteins (rOAT1, hOAT1, rOCT2, and hOCT2). These transporters have all been stably transfected into MDCK cells, a polarized renal cell line. This model was used to characterize its function and cellular localization in a mammalian system. In particular, we have used the hOAT1 MDCK line to demonstrate that the stoichiometry of this human xenobiotic transporter is one organic anion exchanged for one dicarboxylate, yielding net entry of positive charge during transport. Thus, hOAT1 is able tap both the inside negative membrane potential and the in>out dicarboxylate gradient, explaining why this transporter is so very effective. We have also generated cDNA constructs in which green fluorescent protein (GFP) is linked in frame with both OAT1 and OCT2. When transfected into oocytes, cell lines, or isolated tubules, these constructs produce functional, fluorescent proteins which can be followed optically in the living cell. These studies have confirmed the basolateral targeting of both rOAT1 and rOCT2. The latter result was particularly important, for it resolved a controversy regarding both the subcellular localization and function role of rOCT2. Recent 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. We have also shown that a number of mercapturic acids (i.e., N-acetylcysteine S-conjugates) are endogenous substrates for both rat and human OAT1. In addition, using the hOAT1 transfected cell line we have assessed the putative role of organic anion transport in the marked toxicity of mercury toward the renal proximal tubule. The data demonstrate that the binding of 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 also toxic, but only to the hOAT1 expressing cells. The toxicity of the Hg-complexes is blocked by probenecid, a potent inhibitor of OAT function.