Transport and Toxicity of Methylmercury-Cysteine in Cultured BeWo Cells.

Mercury is a heavy metal toxicant that is prevalent throughout the environment. Organic forms of mercury, such as methylmercury (MeHg), can cross the placenta and can lead to lasting detrimental effects in the fetus. The toxicological effects of MeHg on the placenta itself have not been clearly defined. Therefore, the purpose of the current study was to assess the transport of MeHg into placental syncytiotrophoblasts and to characterize the mechanisms by which MeHg exerts its toxic effects. Cultured placental syncytiotrophoblasts (BeWo) were used for these studies. The transport of radioactive MeHg was measured to identify potential mechanisms involved in the uptake of this compound. The toxicological effects of MeHg on BeWo cells were determined by assessing visible pathological change, autophagy, mitochondrial viability, and oxidative stress. The findings of this study suggest that MeHg compounds are transported into BeWo cells primarily by sodium-independent amino acid carriers and organic anion transporters. The MeHg altered mitochondrial function and viability, decreased mitophagy and autophagy, and increased oxidative stress. Exposure to higher concentrations of MeHg inhibited the ability of cells to protect against MeHg-induced injury. The findings show that MeHg is directly toxic to syncytiotrophoblasts and may lead to disruptions in the fetal/maternal transfer of nutrients and wastes.

Reaction of Cyanide with Hg0-Contaminated Gold Mining Tailings Produces Soluble Mercuric Cyanide Complexes.

Elemental mercury (Hg0) contamination in artisanal and small-scale gold mining (ASGM) communities is widespread, and Hg0-contaminated tailings are often reprocessed with cyanide (-CN) to extract residual gold remaining after amalgamation. Hg0 reacts with -CN under aerobic conditions to produce Hg(CN)42- and other Hg(CN)nn-2 complexes. The production of solvated Hg(CN)nn-2 complexes increases upon agitation in the presence of synthetic and authentic Hg0-contaminated tailings that aid in dispersing the Hg0, increasing its reactive surface area. Adult rats were exposed to various concentrations of Hg(CN)2, and accumulation in organs and tissues was quantified using direct mercury analysis. The primary site of Hg(CN)2 accumulation was the kidney, although accumulation was also detected in the liver, spleen, and blood. Little accumulation was observed in the brain, suggesting that Hg(CN)2 complexes do not cross the blood-brain barrier. Renal tissue was particularly sensitive to the effects of Hg(CN)2, with pathological changes observed at low concentrations. Hg(CN)2 complexes are handled by mammalian systems in a manner similar to other inorganic species of Hg, yet appear to be more toxic to organ systems. The findings from this study are the first to show that Hg(CN)2 complexes are highly stable complexes that can lead to cellular injury and death in mammalian organ systems.

Disposition of methylmercury over time in a 75% nephrectomized rat model.

Chronic kidney disease (CKD) is a highly relevant clinical condition that is characterized by the permanent loss of functional nephrons. Individuals with CKD may exhibit impaired renal clearance, which may alter corporal handling of metabolites and xenobiotics. Methylmercury (MeHg) is an important environmental toxicant to which humans are exposed to on a regular basis. Given the prevalence of CKD and ubiquitous presence of MeHg in the environment, it is important to understand how mercuric ions are handled in patients with CKD. Therefore, the purpose of the current study was to characterize the disposition of MeHg over time in a rat model of CKD (i.e., 75% nephrectomized (NPX) rats). Control and NPX rats were exposed intravenously (iv) to a non-nephrotoxic dose of MeHg (5 mg/kg) once daily for1, 2, or 3 d and the amount of MeHg in organs, blood, urine, and feces determined. The accumulation of MeHg in kidneys and blood of controls was significantly greater than that of NPX animals. In contrast, MeHg levels in brain and liver of controls were not markedly different from corresponding NPX rats. In all organs examined, accumulation of MeHg increased over the course of exposure, suggesting that urinary and fecal elimination are not sufficient to fully eliminate all mercuric ions. The current findings are important in that the disposition of mercuric ions in rats with normal renal function versus renal insufficiency following exposure to MeHg for a prolonged period differ and need to be taken into account with respect to therapeutic management.

Exposure to mixtures of mercury, cadmium, lead, and arsenic alters the disposition of single metals in tissues of Wistar rats.

Humans throughout the world are exposed regularly to mixtures of environmental toxicants. Four of the most common heavy metal toxicants in the environment are mercury (Hg), cadmium (Cd), lead (Pb), and arsenic (As). Numerous studies have assessed the effects and disposition of individual metals in organ systems; however, humans are usually exposed to mixtures of toxicants or metals rather than to a single toxicant. Therefore, the purpose of the current study was to test the hypothesis that exposure to a mixture of toxic heavy metals alters the disposition of single metals in target organs. Wistar rats (Rattus norvegicus) were exposed to Hg, Cd, Pb, or As as a single metal or as a mixture of metals. Rats were injected intravenously for three days, following which kidneys, liver, brain, and blood were harvested. Samples were analyzed for content of Hg, Cd, Pb, and As via inductively coupled plasma mass spectrometry. In general, exposure to a mixture of metals reduced accumulation of single metals in target organs. Interestingly, exposure to mixtures of metals with Pb and/or As increased the concentration of these metals specifically in the liver. The findings from this study indicate that exposure to mixtures of toxic heavy metals may alter significantly the distribution and accumulation of these metals in target organs and tissues.

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.

Structural characterization of 1,3-propanedithiols that feature carboxylic acids: Homologues of mercury chelating agents.

The molecular structures of a series of 1,3-propanedithiols that contain carboxylic acid groups, namely rac- and meso-2,4-dimercaptoglutaric acid (H4DMGA) and 2-carboxy-1,3-propanedithiol (H3DMCP), have been determined by X-ray diffraction. Each compound exhibits two centrosymmetric intermolecular hydrogen bonding interactions between pairs of carboxylic acid groups, which result in a dimeric structure for H3DMCP and a polymeric tape-like structure for rac- and meso-H4DMGA. Significantly, the hydrogen bonding motifs observed for rac- and meso-H4DMGA are very different to those observed for the 1,2-dithiol, rac-2,3-dimercaptosuccinic acid (rac-H4DMSA), in which the two oxygen atoms of each carboxylic acid group hydrogen bond to two different carboxylic acid groups, thereby resulting in a hydrogen bonded sheet-like structure rather than a tape. Density functional theory calculations indicate that 1,3-dithiolate coordination to mercury results in larger S-Hg-S bond angles than does 1,2-dithiolate coordination, but these angles are far from linear. As such, κ2-S2 coordination of these dithiolate ligands is expected to be associated with mercury coordination numbers of greater than two. In vivo studies demonstrate that both rac-H 4 DMGA and H3DMCP reduce the renal burden of mercury in rats, although the compounds are not as effective as either 2,3-dimercaptopropane-1-sulfonic acid (H3DMPS) or meso-H4DMSA.

Proximal tubular transport of Metallothionein-Mercury complexes and protection against nephrotoxicity.

Mercury (Hg) is an important environmental toxicant to which humans are exposed on a regular basis. Mercuric ions within biological systems do not exist as free ions. Rather, they are bound to free sulfhydryl groups (thiols) on biological molecules. Metallothionein (MT) is a cysteine-rich, metal-binding protein that has been shown to bind to heavy metals and reduce their toxic effects in target cells and organs. Little is known about the effect of MT on the handing and disposition of Hg. Therefore, the current study was designed to test the hypothesis that overexpression of MT alters the corporal disposition of Hg and reduces its nephrotoxicity. Furthermore, the current study examined the transport of Hg-MT complexes in isolated proximal tubules. Rats were treated with saline or Zn followed by injection with a non-nephrotoxic (0.5 µmol kg-1), moderately nephrotoxic (1.5 µmol kg-1), or significantly nephrotoxic (2.25 µmol kg-1) dose of HgCl2 (containing radioactive Hg). Pretreatment with Zn increased mRNA expression of MT and enhanced accumulation of Hg in the renal cortex of male and female rats. In addition, injection with Zn also protected animals from Hg-induced nephrotoxicity. Studies using isolated proximal tubules from rabbit kidney demonstrated that Hg-MT is taken up rapidly at the apical and basolateral membranes. The current findings suggest that at least part of this uptake occurs through an endocytic process. This study is the first to examine the uptake of Hg-MT complexes in isolated proximal tubules. Overall, the findings of this study suggest that supplementation with Zn may be a viable strategy for reducing the risk of Hg intoxication in at-risk populations.

New insights into the metabolism of organomercury compounds: mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine γ-lyase.

Anthropogenic practices and recycling in the environment through natural processes result in release of potentially harmful levels of mercury into the biosphere. Mercury, especially organic forms, accumulates in the food chain. Mercury reacts readily with sulfur-containing compounds and often exists as a thiol S-conjugate, such as the l-cysteine (Cys)-S-conjugate of methylmercury (CH(3)Hg-S-Cys) or inorganic mercury (Cys-S-Hg-S-Cys). These S-conjugates are structurally similar to l-methionine and l-cystine/l-cystathionine, respectively. Bovine and rat glutamine transaminase K (GTK) catalyze transamination of sulfur-containing amino acids. Recombinant human GTK (rhGTK) has a relatively open catalytic active site, and we report here that this enzyme, like the rat and bovine enzymes, can also utilize sulfur-containing l-amino acids, including l-methionine, l-cystine, and l-cystathionine as substrates. The current study extends this list to include mercuric S-conjugates, and shows that CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are substrates and reversible inhibitors of rhGTK. The homocysteine S-conjugates, Hcy-S-Hg-S-Hcy and CH(3)Hg-S-Hcy, are also inhibitors. Finally, we show that HgCl(2), CH(3)Hg-S-Cys and Cys-S-Hg-S-Cys are potent irreversible inhibitors of rat cystathionine γ-lyase. The present study broadens our knowledge of the biochemistry of mercury compounds by showing that Cys S-conjugates of mercury interact with enzymes that catalyze transformations of biologically important sulfur-containing amino acids.

Hepatic processing of mercuric ions facilitates delivery to renal proximal tubules.

Mercury (Hg) is a toxic heavy metal to which humans are exposed on a regular basis. Hg has a high affinity for thiol-containing biomolecules with the majority of Hg in blood being bound to albumin. The current study tested the hypothesis that circulating Hg-albumin complexes are taken up into hepatocytes and processed to form Hg-glutathione (GSH) conjugates (GSH-Hg-GSH). Subsequently, GSH-Hg-GSH conjugates are exported from hepatocytes into blood via multidrug resistance transporters (MRP) 3 and 5. To test this hypothesis, the portal vein and hepatic artery in Wistar rats were ligated to prevent delivery of Hg to the liver. Ligated and control rats were injected with HgCl2 or GSH-Hg-GSH (containing radioactive Hg) and the disposition of Hg was assessed in various organs. Renal accumulation of Hg was reduced significantly in ligated rats exposed to HgCl2. In contrast, when rats were exposed to GSH-Hg-GSH, the renal accumulation of Hg was similar in control and ligated rats. Experiments using HepG2 cells indicate that Hg-albumin conjugates are taken up by hepatocytes and additional experiments using inside-out membrane vesicles showed that MRP3 and MRP5 mediate the export of GSH-Hg-GSH from hepatocytes. These data are the first to show that Hg-albumin complexes are processed within hepatocytes to form GSH-Hg-GSH, which is, in part, exported back into blood via MRP3 and MRP5 for eventual excretion in urine.

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 [²°³Hg], 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 [²°³Hg] 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.

Prophylactic supplementation with selenium alters disposition of mercury in aged rats.

Mercury (Hg) is a prevalent environmental toxicant to which older individuals are particularly susceptible. Selenium (Se) has been used as an antidote following exposure to Hg. However, little is known about the effect of prophylactic supplementation with Se on the handling of Hg. The current study was designed to test the hypothesis that oral pre-treatment with Se alters the corporal disposition of Hg and reduces the risk of Hg-induced toxicity. Young and aged rats were gavaged for 10 days with sodium selenite or saline. On day 11, rats were injected intravenously with 0.5 µmol HgCl2·kg-1·2 mL-1 normal saline. After 24 h, rats were euthanized and organs and tissues were harvested for determination of Hg content. Accumulation of Hg in the kidney was reduced significantly by pre-treatment with Se in both young and aged rats. In the renal cortex, the magnitude of the reduction was greater in aged rats than in young rats but in the outer stripe of the outer medulla, the magnitude of the reduction was similar between groups of rats. Urinary excretion of Hg was also reduced in rats pre-treated with Se. In contrast, the hepatic and hematologic burden of Hg increased in rats pre-treated with Se. Fecal excretion of Hg was decreased significantly by pre-treatment with Se in young rats but not in aged rats. These data suggest that prophylactic supplementation with Se alters the corporal disposition of Hg in a way that may reduce Hg-induced toxicity in target organs.

Sex differences in renal handling of inorganic mercury in aged rats.

The sex of an individual/animal has been shown to play an important role in many biological processes. Furthermore, sex may also be a factor in the way environmental toxicants, such as heavy metals, are handled by organisms. However, the effect of sex on the handling and disposition of heavy metals, such as mercury (Hg), has not been shown. Aging has also been shown to be a factor in the accumulation of heavy metals in that older individuals tend to have higher burdens of these metals. Therefore, the purpose of the current study was to evaluate the effect of sex on the accumulation of mercury in aged animals. Aged male and female rats were injected intravenously with 0.5 µmol or 2.0 µmol·kg-1 HgCl2 (containing radioactive Hg) and organs were harvested after 24 h. In general, the renal accumulation of Hg was significantly greater in males than in females. Similarly, urinary excretion of Hg was greater in males than in females. There were no significant differences between males and females in the burden of Hg in other organs. Sex differences in the renal accumulation of Hg may be related to differences in the expression of membrane transporters involved in the uptake of mercuric species into tubular epithelial cells. The results of the current study illustrate the need to evaluate both sexes when assessing the renal effects of environmental toxicants.

Chronic kidney disease in pregnant mothers affects maternal and fetal disposition of mercury.

Chronic kidney disease (CKD) affects over 15 % of the adults in the United States. Pregnant women with CKD present an additional challenge in that they are at increased risk for adverse events such as preterm birth. Exposure to environmental toxicants, such as methylmercury, may exacerbate maternal disease and increase the risk of adverse fetal outcomes. We hypothesized that fetuses of mothers with CKD are more susceptible to accumulation of methylmercury than fetuses of healthy mothers. The current data show that when mothers are in a state of renal insufficiency, uptake of mercury in fetal kidneys is enhanced significantly. Accumulation of Hg in fetal kidneys may be related to the flow of amniotic fluid, maternal handling of Hg, and/or underdeveloped mechanisms for cellular export and urinary excretion. The results of this study indicate that renal insufficiency in mothers leads to significant alterations in the way toxicants such as mercury are handled by maternal and fetal organs.

Co-administration of Selenium with Inorganic Mercury Alters the Disposition of Mercuric Ions in Rats.

Mercury (Hg) is a common environmental toxicant to which humans are exposed regularly through occupational and dietary means. Although selenium supplementation has been reported to prevent the toxic effects of Hg in animals, the mechanisms for this prevention are not well understood. The purpose of the current study was to determine the effects of selenium on the disposition and toxicity of Hg. Wistar rats were injected intravenously with a non-nephrotoxic dose (0.5 µmol kg-1) or a nephrotoxic dose (2.5 µmol kg-1) of HgCl2 (containing radioactive Hg) with or without co-administration of sodium selenite (Na2SeO3). Twenty-four hours after exposure, rats were euthanized, and organs were harvested. Co-administration of SeO32- with HgCl2 reduced the renal burden of Hg and the urinary excretion of Hg while increasing the amount of Hg in blood and spleen. We propose that Hg reacts with reduced selenite in the blood to form large Hg-Se complexes that are unable to be filtered at the glomerulus. Consequently, these complexes remain in the blood and are able to accumulate in blood-rich organs. These complexes, which may have fewer toxic effects than other species of Hg, may be eliminated slowly over the course of weeks to months.

Potential mechanisms of cellular injury following exposure to a physiologically relevant species of inorganic mercury.

Mercury is a toxic metal that is found ubiquitously in the environment. Humans are exposed to different forms of mercury via ingestion, inhalation, and/or dermal absorption. Following exposure, mercuric ions may gain access to target cells and subsequently lead to cellular intoxication. The mechanisms by which mercury accumulation leads to cellular injury and death are not understood fully. Therefore, purpose of this study was to identify the specific intracellular mechanisms that are altered by exposure to inorganic mercury (Hg2+). Normal rat kidney (NRK) cells were exposed to a physiologically relevant form of Hg2+, as a conjugate of cysteine (10 µM or 50 µM). Alterations in oxidative stress were estimated by measuring lipid peroxidation and mitochondrial oxidative stress. Alterations in actin and tubulin were measured using specific fluorescent dyes. Calcium levels were measured using Fluo-3 AM Calcium Indicator while autophagy was identified with Premo™ Autophagy Sensor LC3B-GFP. The current findings show that exposure to Hg2+ leads to enhanced oxidative stress, alterations in cytoskeletal structure, increases in intracellular calcium, and enhanced autophagy. We have established a more complete understanding of intoxication and cellular injury induced by a relevant form of Hg2+ in proximal tubule cells.

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 (Hg(2+)) 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, Hg(2+) 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 Hg(2+) from proximal tubular cells. To test this hypothesis, the disposition of Hg(2+) was examined in control and Mrp2-deficient TR(-) rats. Rats were injected i.v. with 0.5 mumol/kg HgCl(2) containing (203)Hg(2+). Twenty-four and 28 h later, rats were injected with saline, DMPS, or DMSA. Tissues were harvested 48 h after HgCl(2) exposure. The renal and hepatic burden of Hg(2+) in the saline-injected TR(-) rats was greater than that of controls. In contrast, the amount of Hg(2+) 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 Hg(2+) in both groups of rats, with the greatest reduction in controls. A significant increase in urinary and fecal excretion of Hg(2+), 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 Hg(2+) are transportable substrates of MRP2. Collectively, these data support a role for Mrp2 in the DMPS- and DMSA-mediated elimination of Hg(2+) from the kidney.

MRP2 and the Transport Kinetics of Cysteine Conjugates of Inorganic Mercury.

Human exposure to mercuric species occurs regularly throughout the world. Mercuric ions may accumulate in target cells and subsequently lead to cellular intoxication and death. Therefore, it is important to have a thorough understanding of how transportable species of mercury are handled by specific membrane transporters. The purpose of the current study was to characterize the transport kinetics of cysteine (Cys)-S-conjugates of inorganic mercury (Cys-S-Hg-S-Cys) at the site of the multidrug resistance-associated transporter 2 (MRP2). In order to estimate the maximum velocity (V max) and Michaelis constant (K m) for the uptake of Cys-S-Hg-S-Cys mediated by MRP2, in vitro studies were carried out using radioactive Cys-S-Hg-S-Cys (5 µM) and inside-out membrane vesicles made from Sf9 cells transfected with MRP2. The V max was estimated to be 74.3 ± 10.1 nmol mg protein-1 30 s-1 while the K m was calculated to be 63.4 ± 27.3 µM. In addition, in vivo studies were utilized to measure the disposition of inorganic mercury (administered dose 0.5 µmol kg-1 in 2 mL normal saline) over time in Wistar and TR¯ (Mrp2-deficient) rats. These studies measured the disposition of mercuric ions in the kidney, liver, and blood. In general, the data suggest that the initial uptake of mercuric conjugates into select target cells is rapid followed by a period of slower uptake and accumulation. Overall, the data indicate that MRP2 transports Cys-S-Hg-S-Cys in a manner that is similar to that of other MRP2 substrates.

Pregnancy Alters Renal and Blood Burden of Mercury in Females.

Methylmercury (CH3Hg+), a common environmental toxicant, has serious detrimental effects in numerous organ systems. We hypothesize that a significant physiological change, like pregnancy, can alter the disposition and accumulation of mercury. To test this hypothesis, pregnant and non-pregnant female Wistar rats were exposed orally to CH3Hg+. The amount of mercury in blood and total renal mass was significantly lower in pregnant rats than in non-pregnant rats. This finding may be due to expansion of plasma volume in pregnant rats and dilution of mercury, leading to lower levels of mercury in maternal blood and kidneys.

Mouse monocytes (RAW CELLS) and the handling of cysteine and homocysteine S-conjugates of inorganic mercury and methylmercury.

Although there is evidence indicating that mononuclear phagocytes can take up mercury by some forms of endocytosis, very little is known about the potential for the uptake of mercuric species by carrier-mediated processes. Thus, we hypothesized that monocytes also possess mechanisms allowing these cells to take up inorganic mercury (Hg2+) and/or methylmercury (CH3Hg+) as cysteine (Cys) and/or homocysteine (Hcy) S-conjugates by certain membrane transport proteins. The specific thiol S-conjugates were chosen for study because our laboratory and those of some other investigators have demonstrated that these species of mercury are indeed transportable substrates for several membrane transport proteins in certain types of epithelial cells. We chose to use RAW 264.7 cells for our experiments. These cells represent an adherent line of mouse monocytes. Kinetic analyses for the uptake of Cys-Hg-Cys, CH3Hg-Cys, Hcy-Hg-Hcy, and CH3Hg-Hcy revealed that uptake occurred by a saturable, concentration-dependent mechanism, displaying Michaelis-Menten properties. Interestingly, in the cells exposed to the Cys or Hcy S-conjugate of Hg2+, significantly more Hg2+ was taken up in the presence of 140 mM sodium chloride (NaCl) than in the presence of 140 mM N-methyl-D-glucamine (NMDG), indicating that Na-dependent processes play more of a role in the uptake of these species of Hg2+ than sodium-independent ones. With respect to the uptake of CH3Hg+, rates of uptake of the Cys and Hcy S-conjugates of CH3Hg+ were similar under both Na-dependent and Na-independent conditions, although the levels of uptake of these mercuric species far exceeded the levels of uptake of the corresponding S-conjugate of Hg2+. Uptake of Hg2+ and CH3Hg+, as the Cys or Hcy S-conjugates, was also time-dependent. We also showed that when the temperature in the bathing medium was reduced to 4 degrees C, uptake of the Cys S-conjugates Hg2+ or CH3Hg+ was for the most part reduced to negligible levels in the RAW cells; indicating that the preponderance of uptake at 37 degrees C was not due primarily to simple diffusion and/or non-specific binding. Overall, the present findings strongly suggest that the uptake of the Cys and Hcy S-conjugates of Hg2+ and/or CH3Hg+ occurs in monocytes by one or more mechanisms involving carrier proteins.

Oral exposure of pregnant rats to toxic doses of methylmercury alters fetal accumulation.

Methylmercury (CH3Hg+) is an environmental toxicant that may lead to significant pathologies in exposed individuals. The current study assessed the disposition and toxicological effects of 2.5 or 7.5mgkg-1 CH3Hg+, conjugated to cysteine (Cys; Cys-S-CH3Hg) and administered orally to pregnant and non-pregnant Wistar and TR- rats. Rats were euthanized on gestational day 20 and the content of mercury in each fetus, amniotic sac, and placenta was determined. The brain, liver, and kidneys were removed from each fetus for estimation of mercury content. From the dams, a sample of blood, kidneys, liver, and brain were removed at the time of euthanasia. The findings from this study indicate that pregnancy leads to significant changes in the handling of mercuric ions, particularly in the liver. Furthermore, there are significant differences in the handling of non-nephrotoxic and nephrotoxic doses of Cys-S-CH3Hg by maternal and fetal organs.