Christy C. Bridges

TRANSPORT OF INORGANIC MERCURY AND METHYLMERCURY IN TARGET TISSUES AND ORGANS

Owing to the prevalence of mercury in the environment, the risk of human exposure to this toxic metal continues to increase. Following exposure to mercury, this metal accumulates in numerous organs, including brain, intestine, kidneys, liver, and placenta. Although a number of mechanisms for the transport of mercuric ions into target organs were proposed in recent years, these mechanisms have not been characterized completely. This review summarizes the current literature related to the transport of inorganic and organic forms of mercury in various tissues and organs. This review identifies known mechanisms of mercury transport and provides information on additional mechanisms that may potentially play a role in the transport of mercuric ions into target cells.

Mercuric Conjugates of Cysteine Are Transported by the Amino Acid Transporter System b0,+: Implications of Molecular Mimicry

Humans and other mammals continue to be exposed to various forms of mercury in the environment. The kidneys, specifically the epithelial cells lining the proximal tubules, are the primary targets where mercuric ions accumulate and exert their toxic effects. Although the actual mechanisms involved in the transport of mercuric ions along the proximal tubule have not been defined, current evidence implicates mercuric conjugates of cysteine, primarily 2-amino-3-(2-amino-2-carboxyethylsulfanylmercuricsulfanyl)propionic acid (Cys-S-Hg-S-Cys), as the most likely transportable species of inorganic mercury (Hg(2+)). Because Cys-S-Hg-S-Cys and the amino acid cystine (Cys-S-S-Cys) are structurally similar, it was hypothesized that Cys-S-Hg-S-Cys might act as a molecular mimic of cystine at one or more of the amino acid transporters involved in the luminal absorption of this amino acid. One such candidate is the Na(+)-independent heterodimeric transporter system b(0,+). Therefore, the transport of Cys-S-Hg-S-Cys and cystine was studied in MDCK II cells that were or were not stably transfected with b(0,+)AT-rBAT. Transport of Cys-S-Hg-S-Cys and cystine across the luminal plasma membrane was similar in the transfected cells, indicating that Cys-S-Hg-S-Cys can behave as a molecular mimic of cystine at the site of system b(0,+). Moreover, only the b(0,+)AT-rBAT transfectants became selectively intoxicated during exposure to Cys-S-Hg-S-Cys. These findings indicate that system b(0,+) likely contributes to the nephropathy induced by Hg(2+) in vivo. These data represent the first direct molecular evidence for the participation of a specific transporter in the luminal uptake of a large divalent metal cation in proximal tubular cells.

Homocysteine, system b0,+ and the renal epithelial transport and toxicity of inorganic mercury.

Proximal tubular epithelial cells are major sites of homocysteine (Hcy) metabolism and are the primary sites for the accumulation and intoxication of inorganic mercury (Hg(2+)). Previous in vivo data from our laboratory have demonstrated that mercuric conjugates of Hcy are transported into these cells by unknown mechanisms. Recently, we established that the mercuric conjugate of cysteine [2-amino-3-(2-amino-2-carboxy-ethylsulfanylmercuricsulfanyl)propionic acid; Cys-S-Hg-S-Cys], is transported by the luminal, amino acid transporter, system b(0,+). As Cys-S-Hg-S-Cys and the mercuric conjugate of Hcy (2-amino-4-(3-amino-3-carboxy-propylsulfanylmercuricsulfanyl)butyric acid; Hcy-S-Hg-S-Hcy) are similar structurally, we hypothesized that Hcy-S-Hg-S-Hcy is a substrate for system b(0,+). To test this hypothesis, we analyzed the saturation kinetics, time dependence, temperature dependence, and substrate specificity of Hcy-S-Hg-S-Hcy transport in Madin-Darby canine kidney (MDCK) cells stably transfected with system b(0,+). MDCK cells are good models in which to study this transport because they do not express system b(0,+). Uptake of Hg(2+) was twofold greater in the transfectants than in wild-type cells. Moreover, the transfectants were more susceptible to the toxic effects of Hcy-S-Hg-S-Hcy than wild-type cells. Accordingly, our data indicate that Hcy-S-Hg-S-Hcy is transported by system b(0,+) and that this transporter likely plays a role in the nephropathy induced after exposure to Hg(2+). These data are the first to implicate a specific, luminal membrane transporter in the uptake and toxicity of mercuric conjugates of Hcy in any epithelial cell.

Multidrug Resistance Proteins and the Renal Elimination of Inorganic Mercury Mediated by 2,3-Dimercaptopropane-1-Sulfonic Acid and Meso-2,3-dimercaptosuccinic Acid

Current therapies for inorganic mercury (Hg2+) intoxication include administration of a metal chelator, either 2,3-dimercaptopropane-1-sulfonic acid (DMPS) or meso-2,3-dimercaptosuccinic acid (DMSA). After exposure to either chelator, Hg2+ is rapidly eliminated from the kidneys and excreted in the urine, presumably as an S-conjugate of DMPS or DMSA. The multidrug resistance protein 2 (Mrp2) has been implicated in this process. We hypothesize that Mrp2 mediates the secretion of DMPS- or DMSA-S-conjugates of Hg2+ from proximal tubular cells. To test this hypothesis, the disposition of Hg2+ was examined in control and Mrp2-deficient TR- rats. Rats were injected i.v. with 0.5 μmol/kg HgCl2 containing 203Hg2+. Twenty-four and 28 h later, rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h after HgCl2 exposure. The renal and hepatic burden of Hg2+ in the saline-injected TR- rats was greater than that of controls. In contrast, the amount of Hg2+ excreted in urine and feces of TR- rats was less than that of controls. DMPS, but not DMSA, significantly reduced the renal and hepatic content of Hg2+ in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of Hg2+, which was greater in the controls, was also observed following DMPS treatment. Experiments utilizing inside-out membrane vesicles expressing MRP2 support these observations by demonstrating that DMPS- and DMSA-S-conjugates of Hg2+ are transportable substrates of MRP2. Collectively, these data support a role for Mrp2 in the DMPS- and DMSA-mediated elimination of Hg2+ from the kidney.

MRP2 and the handling of mercuric ions in rats exposed acutely to inorganic and organic species of mercury.

Mercuric ions accumulate preferentially in renal tubular epithelial cells and bond with intracellular thiols. Certain metal-complexing agents have been shown to promote extraction of mercuric ions via the multidrug resistance-associated protein 2 (MRP2). Following exposure to a non-toxic dose of inorganic mercury (Hg²+), in the absence of complexing agents, tubular cells are capable of exporting a small fraction of intracellular Hg²+ through one or more undetermined mechanisms. We hypothesize that MRP2 plays a role in this export. To test this hypothesis, Wistar (control) and TR(-) rats were injected intravenously with a non-nephrotoxic dose of HgCl₂ (0.5 μmol/kg) or CH₃HgCl (5 mg/kg), containing [²⁰³Hg], in the presence or absence of cysteine (Cys; 1.25 μmol/kg or 12.5mg/kg, respectively). Animals were sacrificed 24 h after exposure to mercury and the content of [²⁰³Hg] in blood, kidneys, liver, urine and feces was determined. In addition, uptake of Cys-S-conjugates of Hg²+ and methylmercury (CH₃Hg+) was measured in inside-out membrane vesicles prepared from either control Sf9 cells or Sf9 cells transfected with human MRP2. The amount of mercury in the total renal mass and liver was significantly greater in TR⁻ rats than in controls. In contrast, the amount of mercury in urine and feces was significantly lower in TR⁻ rats than in controls. Data from membrane vesicles indicate that Cys-S-conjugates of Hg²+ and CH₃Hg+ are transportable substrates of MRP2. Collectively, these data indicate that MRP2 plays a role in the physiological handling and elimination of mercuric ions from the kidney.

MRP2 involvement in renal proximal tubular elimination of methylmercury mediated by DMPS or DMSA

2, 3-Dimercaptopropane-1-sulfonic acid (DMPS) and meso-2, 3-Dimercaptosuccinic acid (DMSA) are dithiols used to treat humans exposed to methylmercury (CH(3)Hg(+)). After treatment, significant amounts of mercury are eliminated rapidly from the kidneys and are excreted in urine. In the present study, we extended our previous studies by testing the hypothesis that MRP2 mediates the secretion of DMPS or DMSA S-conjugates of CH(3)Hg(+). To test this hypothesis, the disposition of mercury was assessed in control and Mrp2-deficient (TR(-)) rats exposed intravenously to a 5.0-mg/kg dose of CH(3)HgCl. Twenty-four and 28 h after exposure, groups of four control and four TR(-) rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h later. Renal and hepatic contents of mercury were greater in saline-injected TR(-) rats than in controls. In contrast, the amounts of mercury excreted in urine and feces by TR(-) rats were less than those by controls. DMPS and DMSA significantly reduced the renal and hepatic content of mercury in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of mercury (which was greater in the controls) was also observed. Our findings in inside-out membrane vesicles prepared from hMRP2-transfected Sf9 cells show that uptake of DMPS and DMSA S-conjugates of CH(3)Hg(+) was greater in the vesicles containing hMRP2 than in control vesicles. Overall, these dispositional findings indicate that MRP2 does play a role in DMPS- and DMSA-mediated elimination of mercury from the kidney.

MRP2 and the DMPS- and DMSA-Mediated Elimination of Mercury in TR− and Control Rats Exposed to Thiol S-Conjugates of Inorganic Mercury

Cysteine (Cys) and homocysteine (Hcy)-S-conjugates of inorganic mercury (Hg2+) are transportable species of Hg2+ that are taken up readily by proximal tubular cells. The metal chelators, 2,3-dimercaptopropane-1-sulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA), have been used successfully to extract Hg2+ from these cells, presumably via the multidrug resistance protein (Mrp2). In the current study, we tested the hypothesis that Mrp2 is involved in the DMPS- and DMSA-mediated extraction of Hg2+ following administration of Hg2+ as an S-conjugate of Cys or Hcy. To test this hypothesis, control and TR(-) (Mrp2-deficient) rats were injected with 0.5 micromol/kg HgCl2 (containing 203Hg2+) conjugated to 1.25 micromol/kg Cys or Hcy. After 24 and 28 h, rats were treated with saline or 100 mg/kg DMPS or DMSA. Tissues were harvested 48 h after Hg2+ exposure. The renal and hepatic burden of Hg2+ was greater in saline-injected TR- rats than in corresponding controls. Accordingly, the content of Hg2+ in the urine and feces was less in TR- rats than in controls. Following treatment with DMPS or DMSA, the renal content of Hg2+ in both groups of rats was reduced significantly and the urinary excretion of Hg2+ was increased. In liver, the effect of each chelator appeared to be dependent upon the form in which Hg2+ was administered. In vitro experiments provide direct evidence indicating that DMPS and DMSA-S-conjugates of Hg2+ are substrates for Mrp2. Overall, these data support our hypothesis that Mrp2 is involved in the DMPS and DMSA-mediated extraction of the body burden of Hg2+.

Relationships between the Renal Handling of DMPS and DMSA and the Renal Handling of Mercury

Within the body of this review, we provide updates on the mechanisms involved in the renal handling mercury (Hg) and the vicinal dithiol complexing/chelating agents, 2,3-bis(sulfanyl)propane-1-sulfonate (known formerly as 2,3-dimercaptopropane-1-sulfonate, DMPS) and meso-2,3-bis(sulfanyl)succinate (known formerly as meso-2,3-dimercaptosuccinate, DMSA), with a focus on the therapeutic effects of these dithiols following exposure to different chemical forms of Hg. We begin by reviewing briefly some of the chemical properties of Hg, with an emphasis on the high bonding affinity between mercuric ions and reduced sulfur atoms, principally those contained in protein and nonprotein thiols. A discussion is provided on the current body of knowledge pertaining to the handling of various mercuric species within the kidneys, focusing on the primary cellular targets that take up and are affected adversely by these species of Hg, namely, proximal tubular epithelial cells. Subsequently, we provide a brief update on the current knowledge on the handling of DMPS and DMSA in the kidneys. In particular, parallels are drawn between the mechanisms participating in the uptake of various thiol S-conjugates of Hg in proximal tubular cells and mechanisms by which DMPS and DMSA gain entry into these target epithelial cells. Finally, we discuss factors that permit DMPS and DMSA to bind intracellular mercuric ions and mechanisms transporting DMPS and DMSA S-conjugates of Hg out of proximal tubular epithelial cells into the luminal compartment of the nephron, and promoting urinary excretion.

Effect of DMPS and DMSA on the Placental and Fetal Disposition of Methylmercury

Methylmercury (CH3Hg+) is a serious environmental toxicant. Exposure to this metal during pregnancy can cause serious neurological and developmental defects in a developing fetus. Surprisingly, little is known about the mechanisms by which mercuric ions are transported across the placenta. Although it has been shown that 2,3-dimercaptopropane-1-sulfonate (DMPS) and 2,3-dimercaptosuccinic acid (DMSA) are capable of extracting mercuric ions from various organs and cells, there is no evidence that they are able to extract mercury from placental or fetal tissues following maternal exposure to CH3Hg+. Therefore, the purpose of the current study was to evaluate the ability of DMPS and DMSA to extract mercuric ions from placental and fetal tissues following maternal exposure to CH3Hg+. Pregnant Wistar rats were exposed to CH3HgCl, containing [203Hg], on day 11 or day 17 of pregnancy and treated 24 h later with saline, DMPS or DMSA. Maternal organs, fetuses, and placentas were harvested 48 h after exposure to CH3HgCl. The disposition of mercuric ions in maternal organs and tissues was similar to that reported previously by our laboratory. The disposition of mercuric ions in placentas and fetuses appeared to be dependent upon the gestational age of the fetus. The fetal and placental burden of mercury increased as fetal age increased and was reduced by DMPS and DMSA, with DMPS being more effective. The disposition of mercury was examined in liver, total renal mass, and brain of fetuses harvested on gestational day 19. On a per gram tissue basis, the greatest amount of mercury was detected in the total renal mass of the fetus, followed by brain and liver. DMPS and DMSA reduced the burden of mercury in liver and brain while only DMPS was effective in the total renal mass. The results of the current study are the first to show that DMPS and DMSA are capable of extracting mercuric ions, not only from maternal tissues, but also from placental and fetal tissues following maternal exposure to CH3Hg+.

System B0,+ and the Transport of Thiol-S-Conjugates of Methylmercury

Methylmercury (CH3Hg+) is a clinically relevant toxicant that is the most abundant form of mercury found in the environment. After exposure, it accumulates in the kidneys, liver, and central nervous system. The mechanisms by which this toxicant is taken up by target cells are only now beginning to be understood. Some experimental data support a hypothesis involving molecular mimicry, whereby thiol conjugates of methylmercury (especially a cysteine S-conjugate) mimic one or more amino acids and are transported into target cells by amino acid transporters. In the present study, we tested the hypothesis that Cys and homocysteine (Hcy) S-conjugates of methylmercury (CH3Hg-S-Cys and CH3Hg-S-Hcy, respectively) mimic one or more amino acids at the site of the Na+-dependent amino acid transporter, system B0,+. In the kidneys, system B0,+ is situated on the luminal plasma membrane of proximal tubular epithelial cells. To test our hypothesis, we measured uptake of CH3Hg-S-Cys and CH3Hg-S-Hcy in Xenopus laevis oocytes injected with water or capped RNA encoding mouse ATB0,+. Analyses of time course, substrate specificity, and saturation kinetics showed that the uptake of CH3Hg-S-Cys and CH3Hg-S-Hcy was 5- to 10-fold greater in oocytes expressing ATB0,+ than in corresponding water-injected controls. Moreover, the transport of CH3Hg-S-Cys and CH3Hg-S-Hcy was inhibited by substrates transported by system B0,+. Finally, our data indicate that CH3Hg-S-Cys and CH3Hg-S-Hcy may mimic of one or more amino acids (e.g., methionine) that are normally transported by system B0,+. To our knowledge, this is the first report implicating system B0,+ in the transport of any mercuric species.