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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Accelerated Urinary Excretion of Methylmercury following Administration of Its Antidote N-Acetylcysteine Requires Mrp2/Abcc2, the Apical Multidrug Resistance-Associated Protein

Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York (M.S.M., D.A.A., T.W.C., N.B.); and Department of Chemistry, Texas Southern University, Houston, Texas (T.A.S.-W.)

Received March 14, 2007; accepted April 10, 2007.

	Abstract

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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 [¹⁴C]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 (TR-) Wistar rats. The TR- rats excreted only 30% 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 TR- and control Wistar rats. Transport of MeHg-NAC was lower in vesicles prepared from TR- rats, whereas transport of MeHg-cysteine was similar in control and TR- 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.

Once absorbed, MeHg has a half-life of around 45 to 70 days in humans (Clarkson, 2002). In the case of exposure to high levels of MeHg, the only way to counteract its deleterious effects is to accelerate its excretion. Past and current therapies for removing MeHg from the body include hemodialysis, exchange transfusion, thiol resin, and chelation therapy, with the latter seen as the least invasive and most commonly used method (Clarkson et al., 1973, 1981; Al-Abbasi et al., 1978; Elhassani, 1982; Lund et al., 1984). The most widely used chelating agents are dimercaptosuccinic acid and dimercaptopropanesulfonate (DMPS). Both are water-soluble, anionic, sulfhydryl-containing compounds to which MeHg readily bind. However, another agent, the amino acid derivative N-acetyl-L-cysteine (NAC), has more recently been shown to be effective in increasing urinary MeHg excretion in mice (Ballatori et al., 1998a). Mice administered NAC in their drinking water after methylmercury exposure excreted 47 to 54% of the MeHg dose in 48 h compared with 4 to 10% in control animals.

Although NAC produces a profound acceleration of urinary MeHg excretion, the mechanism for this effect is unknown. NAC itself is excreted in urine at high concentrations (Borgström et al., 1986; Aposhian et al., 1995; Ballatori et al., 1998a), suggesting that the MeHg-NAC complex may also be excreted into urine. The MeHg-NAC complex appears to be an excellent substrate for the renal organic anion transporter-1, Oat1/Slc22a6, with an apparent K_m value of 31 µM (Koh et al., 2002). Because Oat1/Slc22a6 is localized to the basolateral membrane (Tojo et al., 1999; Sekine et al., 2000), it is most likely a major route of entry for the MeHg-NAC complex into renal tubule cells. The present study tested the hypothesis that MeHg is transported from the cells into renal tubular fluid as the anionic MeHg-NAC complex, using the apical multidrug resistance-associated protein-2 (Mrp2/Abcc2) protein. Mrp2 belongs to subfamily C of the ATP binding cassette (ABC) superfamily of transporters (gene symbol Abcc). It is an organic anion transport pump with a broad range of substrates. In hepatocytes, Mrp2 functions to transport glutathione, glucuronide, and sulfate conjugates, as well as glutathione itself across the canalicular membrane into bile (Ballatori, 1994, 2002; Borst et al., 2000; Kruh and Belinsky, 2003). Mrp2 has been localized to the renal brush-border membrane (Schaub et al., 1997) and may have a similar excretory function in the kidney. The present results demonstrate that NAC is highly effective in accelerating urinary MeHg excretion in rats, that Mrp2 plays a major role in urinary MeHg excretion after NAC administration, and that the transported molecule is most likely the MeHg-NAC complex.

	Materials and Methods

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Materials. Male and female wild-type Wistar rats (200-250 g) were purchased from Charles River Laboratories (Kingston, NY). The animals were housed in our on-site animal care facility with access to food and water ad libitum until the time of experimentation. Transport-deficient (TR-) Wistar rats were taken from our breeding colony, which was established with animals kindly provided by Dr. Mary Vore at the University of Kentucky (Lexington, KY). Experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. [¹⁴C]MeHg (20.2 mCi/mmol) was obtained from American Radiolabeled (St. Louis, MO). Other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, MO) or J.T. Baker (Philipsburg, NJ).

Urine Collection from Anesthetized Rats. Rats were anesthetized with sodium pentobarbital (55-60 mg/kg body weight i.p.), and additional anesthetic was administered as required throughout the experiment. Body temperature was monitored and maintained at 37°C with a heat lamp controlled by a Tele-Thermometer and a rectal probe (Yellow Springs Instrument, Yellow Springs, OH). The right jugular vein was exposed, and its distal end was ligated. The vein was cannulated with a 60-cm length of PE-50 tubing filled with infusion solution (140 mM glucose) and sutured in place. The glucose solution was infused at a rate of 4.1 ml/h using a model 341B syringe pump (Sage Instruments, Boston, MA). The trachea was isolated, and a 2.7-cm length of PE-205 tubing was inserted and tied in place. A small incision was made in the abdomen to expose the urinary bladder. The bladder was cannulated with a 20-cm length of PE-50 tubing, which was flared at the end. Urine was collected in tared 12- x 75-mm test tubes in 30-min intervals for 4.5 to 5.5 h. After 1 h, a bolus of [¹⁴C]MeHg (2 ml/kg of 0.5 µmol MeHg/kg with 5 µCi ¹⁴C/kg) was injected into the jugular cannula over approximately 2 min. After the 2nd h of urine collection, either a low or high dose of NAC (0.42 or 0.84 mmol/kg, in 140 mM glucose; adjusted to pH 7) was infused over 30 min. In the two dose experiments, NAC was administered again at 4 h. At the end of the experiment, 2.0 to 3.0 ml of blood was collected from the dorsal aorta in a 5-ml syringe containing 50 µl of heparin (1000 U/ml). The liver, one kidney, and the brain were then removed and weighed.

Urine Collection from Rats in Metabolic Cages. Female wild-type and TR- Wistar rats were placed individually in stainless steel metabolic cages (Lab Products Inc., Rochelle Park, NJ) and were allowed to acclimate to the cages for 3 days. All rats were injected i.p. with [¹⁴C]MeHg (2 ml/kg of 0.5 µmol of MeHg/kg with 5 µCi/kg ¹⁴C). After 48 h, some of the wild-type and TR- Wistar rats were given NAC in their drinking water (10 mg/ml), whereas others served as control. Urine and feces were collected every 24 h, and [¹⁴C]MeHg content was analyzed. At the end of the experiments, tissues were removed and weighed as described above.

Determination of [¹⁴C]MeHg Content. A small cross-section of each tissue was taken for analysis of [¹⁴C]MeHg content. Tissue, fecal, and blood samples (approximately 0.2-0.4 g of tissue, 1.0 ml of blood) were placed in preweighed 20-ml glass vials. Solvable (Packard Instrument Company, Meriden, CT) was added to each vial (1.0 ml/0.1 g tissue or 2.0 ml/1.0 ml blood), which was then heated to 60°C for 2 to 3 h. The vials were allowed to cool to room temperature, and 0.1 ml of 0.1 M EDTA/1.0 ml of Solvable was added to each vial of blood. H₂O₂ (30%) was then added in 0.1-ml aliquots (0.3 ml/1.0 ml Solvable for blood or 0.1 ml/1.0 ml Solvable for tissues), with swirling between additions. After standing at room temperature for 15 to 30 min, the vials were capped tightly and heated to 60°C for 1 h and allowed to cool back to room temperature before counting. For urine, 5.0 ml of Opti-Fluor (Packard Instrument Company) was added to 50 to 100 µl of urine. For the other tissues, 5.0 ml of Opti-Fluor was added to 200 µl of the solubilized solution. After the addition of scintillation fluid, the samples were allowed to sit overnight at room temperature before counting. Samples were counted in a Beckman LS 6500 scintillation counter (Beckman Coulter, Fullerton, CA). Total blood volume was estimated as 6% of body weight.

Preparation of Liver Plasma Membrane Vesicles. The methods for isolating plasma membranes were modified from Blitzer and Donovan (1984) and Smith and Ploch (1991). In brief, tissue was homogenized in sterile, ice-cold 5 mM HEPES/Tris buffer, pH 7.5, with a Dounce homogenizer and loose-fitting pestle. Membranes were pelleted by centrifugation (10,000g, 20 min), and the mixed plasma membranes were resuspended and homogenized with a tight-fitting (type B) pestle. The homogenate was then spun through a two-step sucrose gradient [16 and 35% (w/v) sucrose density] in an SA-600 rotor (30,000g, 40 min). The discrete band formed at the 16/35% interface was collected and washed twice by diluting to 40 ml with transport buffer (250 mM sucrose, 10 mM HEPES/Tris, pH 7.5, 20 mM KCl, and 0.20 mM CaCl₂) and collected at 15,000g for 10 min). The final pellet was resuspended in 0.5 to 1 ml of transport buffer and was passed through a 25-gauge needle (10x) and stored in aliquots at -80°C. Protein was determined according to Lowry et al. (1951), using bovine serum albumin as a standard.

Transport Measurements. Frozen membrane suspensions were quickly thawed by immersion in a 37°C water bath, diluted to the desired protein concentration, and passed repeatedly (10x) through a 25-gauge needle. To prevent the membrane potential from influencing the transport of substrates, the experiments were voltage-clamped using the potassium ionophore valinomycin (10 µg/mg protein) in the presence of 20 mM KCl in the incubation and vesicle suspension media. In all experiments, an excess of NAC was used to ensure that MeHg was present as the NAC complex. Specific details regarding the NAC/MeHg ratio are given in the legends. Uptake into membrane vesicles was measured by a rapid Millipore filtration technique (Millipore Corporation, Billerica, MA; Meier et al., 1984; Ballatori et al., 1986). Membrane suspensions (5-16 µg of protein in 20 µl) were preincubated at 37°C for 10 min. Uptake studies were initiated by the addition of 80 µl of transport buffer, also prewarmed, containing the respective radiolabeled substrates. Transport was terminated by the addition of 0.9 ml of ice-cold stop solution. Unless otherwise indicated, the stop solution consisted of 250 mM sucrose, 10 mM HEPES/Tris, pH 7.5, 20 mM KCI, and 500 µM DMPS. DMPS, a water-soluble compound containing two sulfhydryl groups, was used to remove MeHg nonspecifically bound to the outside of the vesicles. Membrane vesicle-associated ligand was separated from free ligand by rapid filtration through a 0.45-µm filter (Millipore, HAWP). Vesicles were then washed with an additional 4 ml of ice-cold stop solution. Filters containing radioisotopes were placed in 5 ml of Opti-Fluor (PerkinElmer Life and Analytical Sciences, Boston, MA) and counted in a Beckman LS 6500 scintillation counter.

Statistical Analysis. Differences between NAC doses were analyzed by analysis of variance, followed by Student's t test. Statistical significance was assigned at p < 0.05.

	Results

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NAC Markedly Stimulates Urinary MeHg Excretion in Normal Wistar Rats but Is Largely Ineffective in Mrp2/Abcc2-Deficient Rats. Previous studies demonstrated that NAC is efficient at stimulating urinary excretion of MeHg in mice (Ballatori, 1998a,b), but other animal species were not examined. To test whether NAC is also effective in rats, wild-type male and female Wistar rats were injected i.v. with a nontoxic dose of [¹⁴C]MeHg (0.5 µmol/kg) and subsequently received NAC (0.42 or 0.84 mmol/kg i.v.). Urine was collected at 0.5-h intervals throughout the experiment and analyzed for [¹⁴C]MeHg. Control rats excreted trace amounts of [¹⁴C]MeHg in urine, whereas NAC was able to stimulate [¹⁴C]MeHg excretion in a dose-dependent fashion (Fig. 1, A and C). Males were able to excrete a slightly larger amount with a single equivalent dose of NAC (Fig. 1; Table 1). This gender difference became significant at the higher dose of NAC (Table 1). Urine flow did not appear to be significantly effected by NAC (Fig. 1, B and D). When a second dose of NAC was administered at 4 h, an equivalent amount of [¹⁴C]MeHg was excreted as after the first NAC dose (Fig. 2). In these two dose experiments, males and females were able to excrete comparable amounts of MeHg (5.9 ± 1.2 and 15.1 ± 3.2 for males versus 3.5 ± 1.2 and 14.8 ± 2.2 for females at low and high doses of NAC, respectively).

View larger version (27K): [in this window] [in a new window]   Fig. 1. NAC stimulates urinary MeHg excretion in wild-type Wistar rats but is less effective in Mrp2-defficient TR- rats. [14C]MeHg excretion and urine flow were measured in wild-type male (A and B) and female (C and D) Wistar rats and TR- male (E and F) and female (G and H) Wistar rats. [14C]MeHg (0.5 µmol/kg) was injected i.v. at 1 h, and NAC (0.42 or 0.84 mmol/kg) was administered from 2 to 2.5 h. Urine was collected in 0.5-h intervals in tared test tubes. Data are presented as percentage of the total dose, ±S.E. (n = 4-7 for male rats and n = 4-8 for female rats).

View larger version (23K): [in this window] [in a new window]   Fig. 2. A second dose of NAC is equally as effective at stimulating urinary MeHg excretion. [14C]MeHg excretion in wild-type male (A) and female (B) rats was measured after NAC doses of 0.42 or 0.84 mmol/kg. [14C]MeHg (0.5 µmol/kg) was injected i.v. at 1 h and NAC administered from 2 to 2.5 and from 4 to 4.5 h. Urine was collected in 0.5-h intervals. Data are presented as percentage of the total dose, ±S.E. (n = 5-7 for male rats and n = 3-4 for female rats).

TR- Wistar rats were used to investigate the role of Mrp2 in the urinary excretion of MeHg after NAC administration. TR- rats have a mutation in their Mrp2 gene that introduces a premature stop codon, thus creating a nonfunctional truncated protein (Paulusma et al., 1996). In the absence of NAC, the TR- rats also excreted trace amounts of MeHg into urine (Fig. 1, E-H). However, in contrast with wild-type Wistar rats, NAC was much less effective at stimulating urinary MeHg excretion in the Mrp2-deficient animals. Cumulative MeHg dose excreted in urine over this time interval was only 11 to 34% of that observed in wild-type rats (Table 1).

To confirm the effects noted in this short-term study in anesthetized rats, a longer term study was performed in which the rats received an injection of [¹⁴C]MeHg (0.5 µmol/kg i.p.), and after 48 h, some animals were given NAC in their drinking water (10 mg/ml) for the next 10 days. No significant differences were observed in the amount of water consumed among the different groups of rats. In the absence of NAC in the drinking water, urinary MeHg excretion was minimal in both wild-type and TR- rats. However, NAC administration in drinking water resulted in a dramatic increase in urinary MeHg excretion in wild-type rats but had no affect on MeHg excretion in TR- rats (Fig. 3). Fecal excretion of MeHg was unaffected by NAC in both the wild-type and TR- rats (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Cumulative urinary MeHg excretion during NAC administration in drinking water. The rats received i.p. injection of [14C]MeHg (0.5 µmol/kg) on day 0, and after 48 h, some animals were given NAC in their drinking water (10 mg/ml) for the next 10 days. Each data point represents cumulative average of the net MeHg content of 24-h urinary output, ±S.E. (n = 4).

View larger version (16K): [in this window] [in a new window]   Fig. 4. Residual MeHg content of selected tissues after 10 days following NAC administration in drinking water. The kidney, liver, brain, spleen, and a sample of whole blood were removed from wild-type (WT) and TR- female rats following the final urine collection, and samples were analyzed for radioactivity. Data are presented as percentage of the total dose, ±S.E. (n = 4). *, significantly different from wild-type animals, p < 0.05.

ATP-Dependent Transport of the MeHg-NAC Complex in Liver Plasma Membrane Vesicles. Liver plasma membrane vesicles isolated from wild-type and TR- rats were employed to further demonstrate the involvement of Mrp2 in the transport of the MeHg-NAC complex. ATP-dependent transport of MeHg-NAC was significantly higher in liver membrane vesicles prepared from wild-type rats versus TR- rats, whereas there was no difference in MeHg transport when given as the L-cysteine complex (Fig. 5).

View larger version (13K): [in this window] [in a new window]   Fig. 5. ATP-dependent transport of MeHg complexed with NAC and cysteine. Membrane vesicles (8 µg) prepared from livers of wild-type (WT) (black bars) and TR- (white bars) rats were incubated for 5 min in transport buffer containing an ATP-regenerating system and 5 µM [14C]MeHg-cysteine or 5 µM[14C]MeHg-NAC in the presence of 5 mM ATP or 10 mM NaCl at 37°C. NAC or cysteine concentration was 50 µM, and DMPS in stop solution was 500 µM. Data are presented as ATP-dependent uptake after the subtraction of uptake in the presence of NaCl, ±S.E. (n = 3-5 independent experiments). *, significantly different from wild-type vesicles, p < 0.05.

	Discussion

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MeHg has a half-life for elimination in humans of 45 to 70 days, which allows this toxicant to accumulate. Under normal conditions, the main route of MeHg elimination is via excretion through bile and the intestine into feces, whereas very little is detectable in the urine. In cases in which there is a high body burden, the only way to minimize toxicity is by increasing its elimination rate. Current therapy for MeHg poisoning is via the administration of a chelating agent and its subsequent elimination from the body. The currently used chelating agents dimercaptosuccinic acid and DMPS are anionic, dithiol-containing compounds that can complex with free metals, including essential metals, and are excreted. Previous work has demonstrated that NAC is also able to increase the urinary excretion of MeHg, at least in mice (Ballatori et al., 1998a). NAC is used clinically in the treatment of acetaminophen overdose and possesses a number of desirable properties as a potential MeHg antidote; it is nontoxic, can be administered orally, it selectively forms a stable complex with MeHg via its thiol group, NAC itself is readily excreted, and NAC does not alter the distribution of essential trace metals.

The present data confirm the ability of NAC to mobilize MeHg and provide insight into the potential mechanism. In previous studies, mice that received NAC in their drinking water were able to excrete 47 to 54% of an acute MeHg dose in 48 h compared with around 10% in controls (Ballatori et al., 1998a). The increased excretion was due to an increase in urinary excretion. Although NAC might be expected to increase biliary MeHg excretion via Mrp2, this potential stimulatory effect of NAC is counterbalanced by a decline in blood MeHg levels and thus decreased hepatic uptake (Ballatori et al., 1998a), which would limit the amount of MeHg excreted into bile. The present experiments in rats demonstrate that NAC is also able to increase the urinary excretion rate, with up to 15% of the MeHg dose being excreted in just 5 h versus a trace amount in control animals. The rapid increase seen in these experiments is most likely due to the use of an i.v. infusion of NAC, rather than administering NAC in their drinking water. This achieves a high blood concentration of NAC rapidly, as well as standardizes the dose for each rat. However, the effect is transient because the accelerated urinary excretion of MeHg returns to basal level shortly after withdrawal of the NAC. NAC was able to increase the urinary excretion rate of methylmercury in a dose-dependent manner in both male and female rats. A second, identical dose of NAC was able to elicit a similar response when administered 2 h after the first dose, and there were only small differences between males and females in their response to NAC. The small gender differences observed may be partially explained by the expression of transporters involved in the transport of MeHg-NAC. The kidney expression of the basolateral membrane-localized Oat1 is greater in male than in female rats (Buist et al., 2002). However, in mice, no gender expression differences for Mrp2 have been observed in the kidney (Maher et al., 2005).

Previous work in our laboratory has shown that the MeHg-NAC complex is a substrate for the Oat1 transporter (Koh et al., 2002), and this finding has recently been confirmed (Zalups and Ahmed, 2005). Oat1 is localized to the basolateral membrane in renal tubular epithelial cells, where it can take up organic anions, including MeHg-NAC, in exchange for intracellular -ketoglutarate (Sweet et al., 1997; Uwai et al., 1998). Once the complex is in the cell, the MeHg can redistribute to other intracellular thiols, especially glutathione. Under our experimental conditions, however, it is likely that a portion will stay as a complex with NAC, due to the high NAC concentration. Glutathione complexes have been shown to be substrates for Mrp2, the apically localized organic anion and conjugate pump (Ballatori, 2002), and this may also be true of the MeHg-NAC complex. This hypothesis was tested using TR- Wistar rats. TR- rats have a natural point mutation in their Mrp2 gene causing the formation of an early stop codon and a nonfunctional, truncated protein. NAC administration to these rats had minimal effects at the lower dose tested and only a very modest effect at the higher dose. Again, the male rats were somewhat more responsive than female rats. The observation that there is still a modest increase in MeHg excretion in the TR- rats indicates that although Mrp2 is the major contributor to MeHg-NAC excretion, it is not the only mechanism involved. Simple glomerular filtration probably accounts for much of this increase; however, other transporters may also be involved. Mrp4, another member of the MRP/CFTR/ABCC family, has also been localized to the apical membrane of renal tubular cells, and a compensatory increase in the expression of Mrp4 in the liver and kidney of TR- rats has been reported (Chen et al., 2005). Mrp4 has some overlapping substrate specificity with Mrp2 and may also be able to transport the MeHg-NAC complex. However, such a possible compensatory role of Mrp4 was not evident in the study in which animals were placed on drinking water containing NAC for 10 days (Fig. 3). There was no significant difference in urinary MeHg excretion in wild-type rats without NAC or in TR- rats with or without NAC in drinking water.

One potential complicating factor in these studies is the demethylation of the [¹⁴C]MeHg. However, demethylation rates for MeHg are relatively slow (Norseth and Clarkson, 1970; Friberg and Mottet, 1989). In rats, demethylation rates are estimated to be about 3 to 5% per day (Norseth and Clarkson, 1970) and, thus, should not influence the present short-term studies. In the long-term study (Figs. 3 and 4), the conversion of MeHg to inorganic Hg might lead to an underestimate of the total amount of Hg that is excreted. However, NAC is relatively selective for MeHg and does not affect the elimination rate of inorganic mercury (Ballatori et al., 1998a). Therefore, the majority of the excreted [¹⁴C] over the 12-day experiment should reflect the amount of MeHg excreted.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Mechanism of NAC-stimulated renal excretion of MeHg. Oat1 is an -ketoglutarate-coupled anion exchanger at the basolateral membrane of proximal tubule cells. In this proposed model, Mrp2 is localized to the brush-border membrane and uses energy in the form of ATP to transport the MeHg-NAC complex from the renal tubular cells into the renal tubular lumen for excretion via the urine. There may be intracellular ligand exchange and interplay of transporters, including the formation of the glutathione complex (MeHg-SG) and transport of this complex by Mrp2 or formation of the L-cysteine complex (MeHg-Cys) and transport of this complex via amino acid carriers.

The proposed model that Mrp2 is the apical transporter of MeHg-NAC was further investigated by examining its role in the transport of the MeHg-NAC complex in membrane vesicles derived from livers of TR- and wild-type rats. The findings illustrated in Fig. 5 support the hypothesis that the MeHg-NAC complex is a substrate for Mrp2. Therefore, based on the previous finding that MeHg-NAC complex is a substrate for Oat1, which has been localized to the basolateral membrane of the renal tubular cells, it is highly probable that when Oat1 transports the MeHg-NAC complex into the renal tubular cells, Mrp2 is mainly responsible for its transport into the tubular lumen for excretion via the urine (Fig. 6). In addition, the MeHg-cysteine complex may be formed as the highly labile MeHg redistributes to other intracellular thiols. This complex has been shown to be transported via both system L amino acid exchanger (Simmons-Willis et al., 2002) and the B^0,+ carrier (Bridges and Zalups, 2006) and possibly by other amino acid carriers; thus, these carriers may play a role in the excretion and reuptake of MeHg in renal tubule cells. Although these data provide insight into the mechanism of MeHg-NAC transport, additional studies are needed to establish the NAC dose that may be therapeutically relevant for potential use as an antidote in poisoned individuals and to assess the use of NAC as a potential biomonitoring agent of MeHg exposure.

	Footnotes

 
This study was supported in part by National Institutes of Health Grants DK48823 and ES06484, by National Institute of Environmental Health Sciences Center Grant ES01247, and by Environmental Toxicology Training Grant ES07026.

M.S.M. and D.A.A. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.122812.

ABBREVIATIONS: MeHg, methylmercury; DMPS, dimercaptopropanesulfonate; NAC, N-acetyl-L-cysteine; Oat, organic anion transporter; Mrp2, multidrug resistance-associated protein-2; ABC, ATP binding cassette; TR-, transport-deficient.

Address correspondence to: Dr. Ned Ballatori, Department of Environmental Medicine, University of Rochester School of Medicine, 575 Elmwood Avenue, Box EHSC, Rochester, NY 14642. E-mail: ned_ballatori{at}urmc.rochester.edu

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