Introduction

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Methylmercury (MeHg) is a common environmental pollutant and a potent neurotoxicant (Clarkson, 2002). Once ingested, MeHg rapidly distributes to all tissues within the body, including its target organ, the brain (Clarkson, 1972). The only way to prevent or ameliorate toxicity once MeHg has been ingested is to accelerate its elimination from the body. Strategies for removing methylmercury include hemodialysis, exchange transfusion, and chelation therapy, with the latter being the least invasive and most common therapeutic intervention (Al-Abbasi et al., 1978; Clarkson et al., 1981; Elhassani, 1982; Lund et al., 1984). One agent that has been used successfully for chelation therapy in humans is the sulfhydryl-containing anionic compound dimercaptopropanesulfonate (DMPS). More recent studies in experimental animals indicate that a relatively simple, nontoxic amino acid derivative, N-acetylcysteine (NAC), may be even more effective in enhancing MeHg elimination in poisoned animals (Ballatori et al., 1998a,b). Mice that received NAC in the drinking water starting at 48 h after MeHg administration excreted from 47 to 54% of the MeHg dose in urine over the subsequent 48 h, compared with 4 to 10% in control animals (Ballatori et al., 1998b).

Although NAC and DMPS both produce a profound acceleration of urinary MeHg excretion, the mechanism for this effect is unknown. NAC and DMPS are themselves excreted in urine at high concentrations (Borgstrom et al., 1986; Aposhian et al., 1995; Ballatori et al., 1998b). The present study tested the hypothesis that the anionic MeHg-NAC and MeHg-DMPS complexes are cleared from peritubular blood by kidney organic anion transporters, in particular by Oat1 and Oat3 (Lopez-Nieto et al., 1997; Sekine et al., 1997; Sweet et al., 1997; Wolff et al., 1997; Cha et al., 2001). To date, Oat1 and Oat3 are the only organic anion transporters that have been localized to the basolateral membrane of proximal tubules (Tojo et al., 1999; Sekine et al., 2000). Oat1 is localized to the S2 region of the renal proximal tubule (Tojo et al., 1999) and functions to take up a range of relatively small hydrophilic organic anions in exchange for intracellular -ketoglutarate (Sweet and Pritchard, 1999). Uwai et al. (1998) demonstrated that hydrophilic dicarboxylates with a five-carbon backbone or longer, but not those with only three or four carbons, are able to inhibit Oat1-mediated p-aminohippurate (PAH) transport. In contrast, the preferred substrates for Oat3 are larger and more hydrophobic compounds such as estrone sulfate, and this transporter does not seem to function as an -ketoglutarate exchanger (Cha et al., 2001).

The present results demonstrate that MeHg-NAC and MeHg-DMPS are high-affinity substrates for Oat1 but are comparatively poor substrates for Oat3, indicating that Oat1 may provide a route of MeHg entry into the renal tubular cells in animals treated with these therapeutic agents.

	Materials and Methods

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Reagents and Animals. [³H]PAH (4.08 Ci/mmol) and [³H]estrone sulfate (40 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA), and [¹⁴C]MeHg (20.2 mCi/mmol) from American Radiolabeled (St. Louis, MO). -Glu-cys was a gift from the Kohjin Co. (Tokyo, Japan). Other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO) or J.T. Baker (Philipsburg, NJ). Mature Xenopus laevis frogs were purchased from Nasco (Fort Atkinson, WI). Animals were maintained under a constant light cycle at a room temperature of 18°C.

Synthesis of Capped cRNA. The cDNAs for rat Oat1 and Oat3 were prepared as described previously (Sweet et al., 1997; Li et al., 1998). Capped cRNA was transcribed in vitro with T7 RNA polymerase (Ambion, Austin, TX), the cRNA was precipitated with lithium chloride, and resuspended in RNase-free water for oocyte injection. When electrophoresed on an RNA gel, these cRNAs gave single bands corresponding to the predicted sizes for Oat1 and Oat3 messages.

X. laevis Oocyte Preparation and Microinjection. Isolation of X. laevis oocytes was performed as described by Goldin (1992) and previously employed in our laboratory (Ballatori et al., 1996; Li et al., 1998). Frogs were anesthetized by immersion for 15 min in ice-cold water containing 0.3% tricaine (Sigma-Aldrich). Oocytes were removed from the ovary and washed with OR-2 solution (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 5 mM HEPES-Tris, pH 7.5) and incubated at room temperature with gentle shaking for 90 min in OR-2 solution supplemented with 2 mg/ml of collagenase (type IA; Sigma-Aldrich). Oocytes were transferred to fresh collagenase solution after the first 45 min of incubation. Collagenase was removed by extensive washing in OR-2 solution at room temperature. Stage V and VI defolliculated oocytes were selected and incubated at 18°C in modified Barth's solution [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO_3, 0.82 mM MgSO_4, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, and 20 mM HEPES-Tris, pH 7.5], supplemented with gentamicin (0.5 mg/ml). After 2 h of incubation, oocytes were injected with 50 nl of Oat1 or Oat3 cRNA (10-20 ng/oocyte), or sterile water for controls. Injected oocytes were cultured at 18°C with a daily change of modified Barth's medium containing gentamicin. Healthy oocytes with a clean, brown animal half and a distinct equator line were selected for experiments.

Transport Measurements in Oocytes. Uptake studies were performed 3 days after injection of cRNA. Six oocytes were incubated at 25°C for 1 h in 100 µl of modified Barth's solution in the presence of [³H]PAH or of various [¹⁴C]MeHg-mercaptide complexes and other substrates as described in the figure legends. Uptake was stopped by adding 2.5 ml of ice-cold modified Barth's solution, and oocytes were washed three times each with 2.5 ml of ice-cold modified Barth's solution. Two oocytes were placed in a polypropylene scintillation vial and were dissolved with 0.2 ml of 10% SDS. Radioisotope was counted in a Beckman model 6500 scintillation counter (Beckman Coulter, Fullerton, CA) after addition of 5 ml of Opti-Fluor (Packard Instruments, Downers Grove, IL).

Statistical Analyses. Data were analyzed by analysis of variance (analysis of variance), and the two-tailed Student's t test was used for comparisons with a control. P values < 0.05 were considered statistically significant.

	Results

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MeHg-NAC and MeHg-DMPS Are Substrates for Oat1 but Are Poor Substrates for Oat3. Rat Oat1 or Oat3 cRNA was injected into X. laevis oocytes, and functional expression was assessed 3 days later by measuring uptake of [³H]PAH or [³H]estrone sulfate (Figs. 1A and 2A, respectively). PAH uptake was enhanced in Oat1-expressing oocytes (Fig. 1A), and estrone sulfate uptake was enhanced in Oat3-expressing oocytes (Fig. 2A). Although not shown in Fig. 2, PAH is also a substrate for Oat3 but it is transported with a low catalytic efficiency compared with estrone sulfate (Sweet et al., 2002). Likewise, estrone sulfate is a relatively poor substrate for Oat1 (Sweet et al., 2002).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Uptake of [3H]PAH or of [14C]MeHg mercaptide complexes in X. laevis oocytes expressing rat Oat1. Oocytes were injected with 20 ng of Oat1 cRNA, cultured for 3 days, and then incubated for 1 h with 20 µM [3H]PAH (A) or 20 µM [14C]MeHg (B) in the presence of 100 µM amino acids and thiols. DMSA, dimercaptosuccinic acid. Values are means ± S.E., n = 3 independent experiments, each performed in triplicate. *, P < 0.05; significantly different from control.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Uptake of [3H]estrone sulfate or of [14C]MeHg mercaptide complexes in Oat3- expressing oocytes. Oocytes were injected with 20 ng of Oat3 cRNA, cultured for 3 days, and then incubated for 1 h with 50 nM [3H]estrone sulfate (A) or 20 µM [14C]MeHg (B) in the presence of 100 µM amino acids, organic anions, and thiols. Values are means ± S.E., n = 3 independent experiments, each performed in triplicate.

Kinetics of MeHg-NAC and MeHg-DMPS Uptake by Oat1. Kinetic analysis of [¹⁴C]MeHg-NAC uptake further suggested that it is a high-affinity substrate for Oat1, with an apparent K_m value of 31 ± 2 µM (Fig. 3A). An even higher affinity for the MeHg-DMPS complex is suggested by an apparent K_m of 9 ± 2 µM (Fig. 3B). These K_m values are comparable with those for PAH uptake on Oat1, which range from 5 to 70 µM (Sweet and Pritchard, 1999). The V_max values for MeHg-NAC and MeHg-DMPS were similar (22 ± 4 and 20 ± 3 pmol/oocyte/h, respectively; Fig. 3).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Concentration dependence of [14C]MeHg-NAC and [14C]MeHg-DMPS uptake rates in Oat1-expressing oocytes. Oocytes were incubated in MeHg-mercaptide concentrations of 5, 10, 25, 50, 100, and 200 µM with a thiol to MeHg ratio of 3:1. Insets illustrate Eadie-Hofstee plots of substrate uptake measured at 1 h. Data points represent values for Oat1-expressing oocytes (20 ng cRNA) minus water-injected oocytes. Values are means ± S.E., n = 3 separate experiments, each performed in triplicate.

Substrate and Inhibitor Specificity of Oat1. The substrate and inhibitor specificity of Oat1was assessed by measuring uptake of [³H]PAH, [¹⁴C]MeHg-NAC, and [¹⁴C]MeHg-DMPS in the presence of the compounds shown in Fig. 4. Oat1-mediated transport of all three substrates was markedly reduced by PAH, probenecid, S-dinitrophenyl-NAC, and butyl-NAC but was unaffected by the neutral amino acids L-alanine and L-methionine. N-Acetyl-L-methionine and ethyl-NAC also reduced uptake of these three substrates. In addition, DMPS and NAC were cis-inhibitors of PAH uptake (Fig. 4), suggesting that they may be potential substrates.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Cis-inhibition of [3H]PAH, [14C]MeHg-NAC and [14C]MeHg-DMPS uptake into Oat1-expressing oocytes by organic anions and mercapturic acids. Oocytes were exposed to 20 µM [3H]PAH, 10 µM[14C] MeHg-NAC, and 10 µM [14C]MeHg-DMPS in the absence (control) or presence of various compounds for 1 h. All inhibitors were present at a concentration of 1 mM, except for S-dinitrophenyl-NAC which was at 0.1 mM. Uptake values in water-injected oocytes were subtracted from those of Oat1-expressing oocytes. Values are means ± S.E. of four separate experiments, each performed in triplicate. All values are significantly different from control, P < 0.05 by Student's t test, except those for L-methionine and L-alanine.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   NAC and DMPS competitively inhibit Oat1-mediated [3H]PAH uptake in oocytes. Uptake of increasing concentrations of [3H]PAH was measured in the absence (control) or in the presence of 2 mM NAC or 0.5 mM DMPS. Uptake values in water-injected oocytes were subtracted from those of Oat1-expressing oocytes. Data represent the mean ± S.E. of three separate experiments.

trans-Stimulation of MeHg-NAC and MeHg-DMPS Transport on Oat1. Oat1 has been shown to function as a dicarboxylate-coupled anion exchanger (Sweet and Pritchard, 1999). To confirm that Oat1 mediates uptake of MeHg-complexes by exchange with specific intracellular dicarboxylates, the effect of an imposed glutarate gradient on MeHg transport was assessed. Oat1-mediated uptake of [³H]PAH, [¹⁴C]MeHg-NAC, and [¹⁴C]MeHg-DMPS was measured in oocytes that were microinjected with glutarate or glutamate to obtain intracellular concentrations of approximately 2 mM, or water for control. Figure 6 shows that uptake of all three substrates by Oat-1 expressing oocytes was increased when intracellular glutarate was added, but not when glutamate was added. The observed dicarboxylate-induced trans-stimulation of substrate uptake indicates that Oat1 mediates uptake of MeHg-NAC and MeHg-DMPS by exchange with intracellular dicarboxylates.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   trans-Stimulation of 20 µM [3H]PAH, 20 µM [14C]MeHg-NAC, and 20 µM [14C]MeHg-DMPS uptake in Oat1-expressing oocytes by intracellular glutarate. Uptake was measured for 1 h in both control and Oat1-expressing oocytes that had been preloaded with 2 mM glutamate, 2 mM glutamate, or water for control. Values are means ± S.E., n = 3 separate experiments, each performed in triplicate. *, P < 0.05; significantly different from control.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   trans-Stimulation of [3H]PAH efflux in Oat1-expressing oocytes by extracellular substrates. Efflux was measured for 1 h in the presence of the compounds shown. All compounds were present at a concentration of 1 mM, except for NAC, which was tested at both 1 and 5 mM. Values are means ± S.E., n = 6 independent experiments, each performed in quadruplicate. *, P < 0.05; significantly different from control.

	Discussion

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DMPS and NAC are organic anions that produce a dramatic acceleration of MeHg excretion in urine and are themselves excreted in urine at high concentrations. To account for this effect, the present study tested whether DMPS, NAC, and their corresponding MeHg complexes are substrates for Oat1 and Oat3, which are localized to the basolateral membrane of rat proximal tubule cells (Kojima et al., 2002). Our results demonstrate that MeHg-NAC and MeHg-DMPS are high-affinity substrates for Oat1 but are comparatively poor substrates for Oat3, and indicate that NAC and DMPS are also substrates for Oat1. These results identify a molecular mechanism by which these therapeutic agents enhance whole-body elimination of this toxic metal.

The present results also provide insight into the substrate specificity for Oat1. Specifically, these findings support the conclusion that carbon chain length is an important determinant of Oat1-mediated transport selectivity (Uwai et al., 1998; Pombrio et al., 2001). Uwai et al. (1998) demonstrated that dicarboxylates with a 5-carbon backbone or longer, but not those with only three or four carbons, are able to inhibit Oat1-mediated PAH transport. The present results indicate that MeHg-NAC, MeHg-DMPS, NAC, and DMPS, all of which have a molecular backbone containing five or more atoms (Fig. 8), are substrates for Oat1. In contrast, MeHg complexes with dimercaptosuccinic acid, penicillamine, and acetylpenicillamine, which have a molecular backbone containing only four atoms (Fig. 8), do not seem to interact with Oat1.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Structural specificity of Oat1-mediated transport of organic anions. Substrates for Oat1 seem to have a molecular backbone containing at least five atoms, whereas compounds that have a four-atom backbone do not seem to be transported.

Although the present study did not directly measure Oat1-mediated transport of NAC and DMPS, several lines of evidence indicate that these chelating agents are themselves substrates for Oat1. As noted above, these molecules possess the key structural features, namely a net negative charge and a five-atom backbone (Fig. 8). Moreover, NAC and DMPS were able to cis-inhibit (Fig. 4) and trans-stimulate PAH transport (Fig. 6), and both were competitive inhibitors of PAH uptake (Fig. 5). It is interesting to note that the K_i values for inhibition of PAH transport by NAC and DMPS were 2 and 0.1 mM, respectively, suggesting that they have a low affinity for Oat1 compared with PAH (K_m, 5 to 70 µM; Sweet et al., 2002), MeHg-NAC (K_m, 31 ± 2 µM), and MeHg-DMPS (K_m, 9 ± 2 µM). Thus, the addition of MeHg to NAC or DMPS seems to enhance affinity for the transporter, an effect that may be attributed to the increased hydrophobic character and molecular size of the resulting MeHg complexes (Fig. 8). This higher apparent affinity of the mercaptide complexes suggests that they are transported more efficiently than the parent chelating agents, which in turn would promote more MeHg complexation and excretion.

Oat1 is known to function as an -ketoglutarate-coupled anion exchanger at the basolateral membrane of the proximal tubule. The -ketoglutarate gradient across the basolateral membrane is large and provides a powerful driving force for organic anion uptake via Oat1. Indeed, the magnitude of this driving force is sufficiently large to account for efficient organic anion secretion, often mediating substrate clearance in a single pass through the kidney. These observations suggest that Oat1 mediates the initial active step in the process of organic anion secretion across the proximal tubule. The present results demonstrate that Oat1 is an anion exchanger coupling uptake of MeHg-NAC and MeHg-DMPS to gradients of glutarate, PAH, and mercapturic acids (Figs. 6 and 7).

Upon their uptake into proximal tubule cells, urinary secretion of organic anions requires efflux across the brush border membrane. However, the transporter(s) mediating export across the brush border membrane are presently unknown. Candidate transporters include the multidrug resistance-associated protein-2 (Mrp2), which has been localized to the brush border membrane (Schaub et al., 1997). In hepatocytes, Mrp2 functions to transport glutathione, glutathione S-conjugates, glutathione mercaptides, and other organic anions across the canalicular membrane into bile (Ballatori, 1994, 2002). Alternatively, other members of the Oat or Oatp families of transporters, yet to be localized to the brush border membrane, may mediate efflux of MeHg complexes.

The molecular identification of a mechanism mediating MeHg transport across cell membranes advances our understanding of MeHg toxicity and possible therapy. Thus, organic anion transporters may have an important role in facilitating urinary MeHg excretion, and may contribute to individual susceptibility to MeHg toxicity. Additional studies are needed to test these possibilities.