Methylmercury (MeHg) readily crosses cell membrane barriers to reach its target tissue, the brain. Although it is generally assumed that this rapid transport is due to simple diffusion, recent studies have demonstrated that MeHg is transported as a hydrophilic complex, and possibly as an L-cysteine complex on the ubiquitous L-type large neutral amino acid transporters (LATs). To test this hypothesis, studies were carried out in Xenopus laevis oocytes expressing two of the major L-type carriers in humans, LAT1-4F2 heavy chain (4F2hc) and LAT2-4F2hc. Oocytes expressing LAT1-4F2hc or LAT2-4F2hc demonstrated enhanced uptake of [(14)C]MeHg when administered as the L-cysteine or D,L-homocysteine complexes, but not when administered as the D-cysteine, N -acetyl-L-cysteine, penicillamine or GSH complexes. Kinetic analysis of transport indicated that the apparent affinities ( K (m)) of MeHg-L-cysteine uptake by LAT1 and LAT2 (98+/-8 and 64+/-8 microM respectively) were comparable with those for methionine (99+/-9 and 161+/-11 microM), whereas the V (max) values were higher for MeHg-L-cysteine, indicating that it may be a better substrate than the endogenous amino acid. Uptake and efflux of [(3)H]methionine and [(14)C]MeHg-L-cysteine were trans -stimulated by leucine and phenylalanine, but not by glutamate, indicating that MeHg-L-cysteine is both a cis - and trans -substrate. In addition, [(3)H]methionine efflux was trans -stimulated by leucine and phenylalanine even in the presence of an inwardly directed methionine gradient, demonstrating concentrative transport by both LAT1 and LAT2. The present results describe a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers. These transport proteins may therefore participate in metal ion homoeostasis and toxicity.

N-Acetylcysteine (NAC) and dimercaptopropanesulfonate (DMPS) are sulfhydryl-containing compounds that produce a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned animals, but the molecular mechanism for this effect is unknown. NAC and DMPS are themselves excreted in urine in high concentrations. The present study tested the hypothesis that the complexes formed between MeHg and these anionic chelating agents are transported from blood into proximal tubule cells by the basolateral membrane organic anion transporters (Oat) 1 and Oat3. Xenopus laevis oocytes expressing rat Oat1 showed increased uptake of [ 14 C]MeHg when complexed with either NAC or DMPS but not when complexed with L-cysteine, glutathione, dimercaptosuccinate, penicillamine, or ␥-glutamylcysteine. In contrast, none of these MeHg complexes were transported by Oat3-expressing oocytes. The apparent K m values for Oat1-mediated transport were 31 Ϯ 2 M for MeHg-NAC and 9 Ϯ 2 M for MeHg-DMPS, indicating that these are relatively high-affinity substrates. Oat1-mediated uptake of [ 14 C]MeHg-NAC and [ 14 C]MeHg-DMPS was inhibited by prototypical substrates for Oat1, including p-aminohippurate (PAH), and was trans-stimulated when oocytes were preloaded with 2 mM glutarate but not glutamate. Conversely, efflux of [ 3 H]PAH from Oat1-expressing oocytes was trans-stimulated by glutarate, PAH, NAC, DMPS, MeHg-NAC, MeHg-DMPS, and a mercapturic acid, indicating that these are transported solutes. [3 H]PAH uptake was competitively inhibited by NAC (K i of 2.0 Ϯ 0.3 mM) and DMPS (K i of 0.10 Ϯ 0.02 mM), providing further evidence that these chelating agents are substrates for Oat1. These results indicate that the MeHg antidotes NAC and DMPS and their mercaptide complexes are transported by Oat1 but are comparatively poor substrates for Oat3. This is the first molecular identification of a transport mechanism by which these antidotes may enhance urinary excretion of toxic metals.

N-Acetylcysteine (NAC) is a sulfhydryl-containing compound that produces a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned mice, but the molecular mechanism for this effect is poorly defined. MeHg readily binds to NAC to form the MeHg-NAC complex, and recent studies indicate that this complex is an excellent substrate for the basolateral organic anion transporter (Oat)-1, Oat1/Slc22a6, thus potentially explaining the uptake from blood into the renal tubular cells. The present study tested the hypothesis that intracellular MeHg is subsequently transported across the apical membrane of the cells into the tubular fluid as a MeHg-NAC complex using the multidrug resistance-associated protein-2 (Mrp2/Abcc2). NAC markedly stimulated urinary [14 C]MeHg excretion in wild-type Wistar rats, and a second dose of NAC was as effective as the first dose in stimulating MeHg excretion. In contrast with the normal Wistar rats, NAC was much less effective at stimulating urinary MeHg excretion in the Mrp2-deficient (TRϪ) Wistar rats. The TRϪ rats excreted only ϳ30% of the MeHg excreted by the wild-type animals. To directly test whether MeHg-NAC is a substrate for Mrp2, studies were carried out in plasma membrane vesicles isolated from livers of TRϪ and control Wistar rats. Transport of MeHg-NAC was lower in vesicles prepared from TRϪ rats, whereas transport of MeHg-cysteine was similar in control and TRϪ rats. These results indicate that Mrp2 is involved in urinary MeHg excretion after NAC administration and suggest that the transported molecule is most likely the MeHg-NAC complex.