N-Acetylcysteine & Accelerated Urinary Excretion of Methylmercury

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

S. Madejczyk,

A. Aremu,

Tracey A. -Willis,

W. son and

Nazzareno Ballatori

+ Author Affiliations

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.)

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@...

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Abstract

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.

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The main human exposure to the common environmental toxicant methylmercury (MeHg) is from eating fish in which the compound has bioaccumulated. Once ingested, MeHg is absorbed in the intestine and readily distributes to all tissues of the body, including its target organ, the brain, and the developing fetus. The fetal brain is much more susceptible to the toxic effects of methylmercury than is the mature brain (son, 1972, 2002).

Once absorbed, MeHg has a half-life of around 45 to 70 days in humans (son, 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 (son 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 Km 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.

http://jpet.aspetjournals.org/content/322/1/378.full