Lucy Joshee

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 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+.

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+.

Aging and the disposition and toxicity of mercury in rats

Progressive loss of functioning nephrons, secondary to age-related glomerular disease, can impair the ability of the kidneys to effectively clear metabolic wastes and toxicants from blood. Additionally, as renal mass is diminished, cellular hypertrophy occurs in functional nephrons that remain. We hypothesize that these nephrons are exposed to greater levels of nephrotoxicants, such as inorganic mercury (Hg(2+)), and thus are at an increased risk of becoming intoxicated by these compounds. The purpose of the present study was to characterize the effects of aging on the disposition and renal toxicity of Hg(2+) in young adult and aged Wistar rats. Paired groups of animals were injected (i.v.) with either a 0.5μmol·kg(-1) non-nephrotoxic or a 2.5μmol·kg(-1) nephrotoxic dose of mercuric chloride (HgCl2). Plasma creatinine and renal biomarkers of proximal tubular injury were greater in both groups of aged rats than in the corresponding groups of young adult rats. Histologically, evidence of glomerular sclerosis, tubular atrophy, interstitial inflammation and fibrosis were significant features of kidneys from aged animals. In addition, proximal tubular necrosis, especially along the straight segments in the inner cortex and outer stripe of the outer medulla was a prominent feature in the renal sections from both aged and young rats treated with the nephrotoxic dose of HgCl2. Our findings indicate 1) that overall renal function is significantly impaired in aged rats, resulting in chronic renal insufficiency and 2) the disposition of HgCl2 in aging rats is significantly altered compared to that of young rats.

Novel Hg2+-Induced Nephropathy In Rats And Mice Lacking Mrp2: Evidence Of Axial Heterogeneity In The Handling Of Hg2+ Along The Proximal Tubule

The role of the multi-resistance protein 2 (Mrp2) in the nephropathy induced by inorganic mercuric mercury (Hg(2+)) was studied in rats (TR(-)) and mice (Mrp2(-/-)), which lack functional Mrp2, and control animals. Animals were exposed to nephrotoxic doses of HgCl2. Forty-eight or 24 hours after exposure, tissues were harvested and analyzed for Hg content and markers of injury. Histological analyses revealed that the proximal tubular segments affected pathologically by Hg(2+) were significantly different between Mrp2-deficient animals and controls. In the absence of Mrp2, cellular injury localized almost exclusively in proximal tubular segments in the subcapsular (S1) to midcortical regions (early S2) of the kidney. In control animals, cellular death occurred mainly in the proximal tubular segments in the inner cortex (late S2) and outer stripe of the outer medulla (S3). These differences in renal pathology indicate that axial heterogeneity exists along the proximal tubule with respect to how mercuric ions are handled. Total renal and hepatic accumulation of mercury was also greater in animals lacking Mrp2 than in controls, indicating that Mrp2 normally plays a significant role in eliminating mercuric ions from within proximal tubular cells and hepatocytes. Analyses of plasma creatinine, BUN, and renal expression of Kim-1 and Ngal tend to support the severity of the nephropathies detected histologically. Collectively, our findings indicate that a fraction of mercuric ions is normally secreted by Mrp2 in early portions of proximal tubules into the lumen and then is absorbed downstream in straight portions, where mercuric species typically induce toxic effects.

Glutathione status and the renal elimination of inorganic mercury in the mrp2(-/-) mouse.

Multidrug resistance-associated proteins (MRP) 2 and 4 are localized in proximal tubular epithelial cells and participate in the renal elimination of xenobiotics. MRP2 has also been implicated in the renal and hepatic elimination of mercury. The current study tested the hypothesis that MRP2 and MRP4 are involved in renal and hepatic handling of inorganic mercury (Hg2+). We examined the disposition of Hg2+ in Mrp2−/− mice and assessed the transport of mercuric conjugates in inside-out membrane vesicles containing human MRP4. Since MRP2 has been shown to utilize glutathione (GSH) for transport of select substrates, we examined renal concentrations of GSH and cysteine and the expression of glutamate cysteine ligase (GCL) in Mrp2−/− and FVB mice. The effect of Hg2+ exposure on renal GSH levels was also assessed in these mice. Our data suggest that MRP2, but not MRP4, is involved in proximal tubular export of Hg2+. In addition, GSH levels are greater in Mrp2−/− mice and exposure to Hg2+ reduced renal levels of GSH. Expression of GCL was also altered in Mrp2−/− mice under normal conditions and following exposure to HgCl2. This study provides important novel data regarding the transport of Hg2+ and the effect of Hg2+ exposure on GSH levels.

Placental and fetal disposition of mercuric ions in rats exposed to methylmercury: role of Mrp2.

Methylmercury is a prevalent environmental toxicant that can have deleterious effects on a developing fetus. Previous studies indicate that the multidrug resistance-associated protein 2 (Mrp2) is involved in renal and hepatic export of mercuric ions. Therefore, we hypothesize that Mrp2 is also involved in export of mercuric ions from placental trophoblasts and fetal tissues. To test this hypothesis, we assessed the disposition of mercuric ions in pregnant Wistar and TR(-) (Mrp2-deficient) rats exposed to a single dose of methylmercury. The amount of mercury in renal tissues (cortex and outer stripe of outer medulla), liver, blood, amniotic fluid, uterus, placentas and fetuses was significantly greater in TR(-) rats than in Wistar rats. Urinary and fecal elimination of mercury was greater in Wistar dams than in TR(-) dams. Thus, our findings suggest that Mrp2 may be involved in the export of mercuric ions from maternal and fetal organs following exposure to methylmercury.

Disposition of inorganic mercury in pregnant rats and their offspring

Environmental toxicants such as methylmercury have been shown to negatively impact fetal health. Despite the prevalence of inorganic mercury (Hg(2+)) in the environment and the ability of methylmercury to biotransform into Hg(2+), little is known about the ability of Hg(2+) to cross the placenta into fetal tissues. Therefore, it is important to understand the handing and disposition of Hg(2+) in the reproductive system. The purpose of the current study was to assess the disposition and transport of Hg(2+) in placental and fetal tissues, and to test the hypothesis that acute renal injury in dams can alter the accumulation of Hg(2+) in fetal tissues. Pregnant Wistar rats were injected intravenously with 0.5 or 2.5 μmol kg(-1) HgCl2 for 6 or 48 h and the disposition of Hg(2+) was measured. Accumulation of Hg(2+) in the placenta was rapid and dose-dependent. Very little Hg(2+) was eliminated during the initial 48 h after exposure. When dams were exposed to the low dose of HgCl2, fetal accumulation of Hg(2+) increased between 6h and 48 h, while at the higher dose, accumulation was similar at each time point. Within fetal organs, the greatest concentration of Hg(2+) (nmol/g) was localized in the kidneys, followed by the liver and brain. A dose-dependent increase in the accumulation of Hg(2+) in fetal organs was observed, suggesting that continued maternal exposure may lead to increased fetal exposure. Taken together, these data indicate that Hg(2+) is capable of crossing the placenta and gaining access to fetal organs in a dose-dependent manner.

Toxicological significance of renal Bcrp: Another potential transporter in the elimination of mercuric ions from proximal tubular cells

Secretion of inorganic mercury (Hg(2+)) from proximal tubular cells into the tubular lumen has been shown to involve the multidrug resistance-associated protein 2 (Mrp2). Considering similarities in localization and substrate specificity between Mrp2 and the breast cancer resistance protein (Bcrp), we hypothesize that Bcrp may also play a role in the proximal tubular secretion of mercuric species. In order to test this hypothesis, the uptake of Hg(2+) was examined initially using inside-out membrane vesicles containing Bcrp. The results of these studies suggest that Bcrp may be capable of transporting certain conjugates of Hg(2+). To further characterize the role of Bcrp in the handling of mercuric ions and in the induction of Hg(2+)-induced nephropathy, Sprague-Dawley and Bcrp knockout (bcrp(-/-)) rats were exposed intravenously to a non-nephrotoxic (0.5 μmol · kg(-1)), a moderately nephrotoxic (1.5 μmol · kg(-1)) or a significantly nephrotoxic (2.0 μmol · kg(-1)) dose of HgCl2. In general, the accumulation of Hg(2+) was greater in organs of bcrp(-/-) rats than in Sprague-Dawley rats, suggesting that Bcrp may play a role in the export of Hg(2+) from target cells. Within the kidney, cellular injury and necrosis was more severe in bcrp(-/-) rats than in controls. The pattern of necrosis, which was localized in the inner cortex and the outer stripe of the outer medulla, was significantly different from that observed in Mrp2-deficient animals. These findings suggest that Bcrp may be involved in the cellular export of select mercuric species and that its role in this export may differ from that of Mrp2.