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.

Protective role of melatonin in myocardial oxidative damage induced by mercury in murine model.

This study was designed to investigate the electrophysiological, hemodynamic and biochemical parameters of mercuric chloride and methylmercury exposure on cardiovascular functions and its modulation by melatonin in vivo. Wistar albino rats were divided into six group containing 10 animals each. Mercuric chloride (3.75 µM/L) in drinking water and methylmercury (0.5 mg/kg/day) through gavage, given for 1 month, induced a statistically significant increase (p < 0.001) in left ventricular end diastolic pressure, blood and cardiac tissue mercury content and myocardial lipid peroxides compared to control. Significant attenuation (p < 0.05) of baroreflex sensitivity and depletion of myocardial endogenous antioxidants (p < 0.001) viz. Reduced glutathione (GSH) and superoxide dismutase (SOD) were also found in the mercury-exposed groups as compared to control group. Mercury exposure followed by subacute treatment with melatonin (4 µg/mL/day) in drinking water for 1 month significantly lowered (p < 0.01) left ventricular end diastolic pressure and lipid peroxide levels and increased baroreceptor sensitivity (p < 0.001) and also levels of GSH and SOD (p < 0.001) as compared to mercury-exposed rats. The results of our study provide clear evidence that elevated oxidative stress and altered baroreflex mechanisms caused by mercury intoxication may be the contributing factors responsible for impairment of cardiovascular functions and melatonin may exhibit cardioprotective property against subacute heavy metal intoxication and enhance the antioxidant defense against mercury-induced oxidative myocardial injury in rats.

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.

Seventy-five percent nephrectomy and the disposition of inorganic mercury in 2,3-dimercaptopropanesulfonic acid-treated rats lacking functional multidrug-resistance protein 2.

In the present study, we evaluated the disposition of inorganic mercury (Hg(2+)) in sham-operated and 75% nephrectomized (NPX) Wistar and transport-deficient (TR(-)) rats treated with saline or the chelating agent meso-2,3-dimercaptosuccinic acid (DMSA). Based on previous studies, DMSA and TR(-) rats were used as tools to examine the potential role of multidrug-resistance protein 2 (MRP2) in the disposition of Hg(2+) during renal insufficiency. All animals were treated with a low dose (0.5 mumol/kg i.v.) of mercuric chloride (HgCl(2)). At 24 and 28 h after exposure to HgCl(2), matched groups of Wistar and TR(-) rats received normal saline or DMSA (intraperitoneally). Forty-eight hours after exposure to HgCl(2), the disposition of Hg(2+) was examined. A particularly notable effect of 75% nephrectomy in both strains of rats was enhanced renal accumulation of Hg(2+), specifically in the outer stripe of the outer medulla. In addition, hepatic accumulation, fecal excretion, and blood levels of Hg(2+) were enhanced in rats after 75% nephrectomy, especially in the TR(-) rats. Treatment with DMSA increased both the renal tubular elimination and urinary excretion of Hg(2+) in all rats. DMSA did not, however, affect hepatic content of Hg(2+), even in the 75% NPX TR(-) rats. We also show with real-time polymerase chain reaction that after 75% nephrectomy and compensatory renal growth, expression of MRP2 (only in Wistar rats) and organic anion transporter 1 is enhanced in the remaining functional proximal tubules. We conclude that MRP2 plays a significant role in the renal and corporal disposition of Hg(2+) after a 75% reduction of renal mass.

Cystine alters the renal and hepatic disposition of inorganic mercury and plasma thiol status.

In the present study, we determined whether cystine can inhibit, under certain conditions, the renal tubular uptake of inorganic mercury in vivo. We co-injected (i.v.) cystine with a non-toxic dose of mercuric chloride to rats and then studied the disposition of inorganic mercury during the next 24 h. We also determined if pretreatment with cystine influences the disposition of administered inorganic mercury. Moreover, plasma thiol status was examined after the intravenous administration of cystine with or without mercuric chloride. During the initial hour after co-injection, the renal tubular uptake of mercuric ions was diminished significantly relative to that in control rats. The inhibitory effects of cystine were evident in both the renal cortex and outer stripe of the outer medulla. In contrast, the renal accumulation of mercury increased significantly between the 1st and 12th hour after co-treatment. Urinary excretion and fecal excretion of mercury were greatly elevated in the rats co-treated with cystine and mercuric chloride. Thus, when cystine and mercury are administered simultaneously, cystine can serve as an inhibitor of the renal tubular uptake of mercury during the initial hour after co-treatment. In rats pretreated with cystine, the renal uptake of inorganic mercury was enhanced significantly relative to that in rats not pretreated with cystine. This enhanced accumulation of inorganic mercury correlated with the increased circulating concentrations of the reduced cysteine and glutathione. Additionally, the present findings indicate that thiol status is an important determinant of renal and hepatic disposition, and urinary and fecal excretion, of inorganic mercury.

Induction of anti-metallothionein antibody and mercury treatment decreases bone mineral density in mice.

Mercuric chloride (HgCl2) is an industrial agent with toxic effects on the immune system, kidney, lung, and nervous tissue, but little is known about its effect on bone. Metallothionein (MT) is a cysteine-rich metal-binding protein that exerts cytoprotective effects against heavy metal toxins. It has been reported that the susceptibility of renal and pulmonary toxicity of mercury was markedly enhanced in MT-null mice compared to control mice. However, there is no report about the effects of anti-metallothionein (anti-MT) Ab induction on mercury toxicity. We investigated the effect of anti-MT Ab induction on mercury-induced bone injury. BALB/c mice were injected with MT (10 microg/mouse ic) five times to induce anti-MT Ab and then treated with HgCl2 (1 mg/kg sc) three times per week for 3 weeks. MT immunization plus HgCl2 treatment dramatically decreased bone mineral density (BMD), and the humoral bone formation indices, alkaline phosphatase (ALP) activity and osteocalcin. MT immunization or HgCl2 treatment alone did not affect either BMD or serum ALP activity and osteocalcin levels. MT immunization impeded HgCl2-induced increase of MT expression in the liver and led to an increase of mercury in serum and the liver but a decrease in the kidney. Furthermore, serum titers of IgE and IgG1 were significantly elevated in the MT-immunized plus HgCl2 treatment group compared with those in the HgCl2 treatment group. Similar results were also observed in splenic secretions of IL-4 and IL-10 based on anti-CD3 Ab stimulation. Taken together, our results indicate that anti-MT Ab induction causes mercury-induced bone injury in BALB/c mice and also enhances mercury-related immune disorders.

Structural basis of the antagonism between inorganic mercury and selenium in mammals.

Mercuric chloride toxicity in mammals can be overcome by co-administration of sodium selenite. We report a study of the mutual detoxification product in rabbit plasma, and of a Hg-Se-S-containing species synthesized by addition of equimolar mercuric chloride and sodium selenite to aqueous, buffered glutathione. Chromatographic purification of this Hg-Se-S species and subsequent structural analysis by Se and Hg extended X-ray absorption fine structure (EXAFS) spectroscopy revealed the presence of four-coordinate Se and Hg entities separated by 2.61 A. Hg and Se near-edge X-ray absorption spectroscopy of erythrocytes, plasma, and bile of rabbits that had been injected with solutions of sodium selenite and mercuric chloride showed that Hg and Se in plasma samples exhibited X-ray absorption spectra that were essentially identical to those of the synthetic Hg-Se-S species. Thus, the molecular detoxification product of sodium selenite and mercuric chloride in rabbits exhibits similarities to the synthetic Hg-Se-S species. The underlying molecular mechanism for the formation of the Hg-Se-S species is discussed.

Basolateral uptake of mercuric conjugates of N-acetylcysteine and cysteine in the kidney involves the organic anion transport system.

Renal uptake and disposition of administered inorganic mercury were studied in rats that had undergone an acute bilateral ureteral ligation shortly before being injected intravenously with a nontoxic 0.5 micromol/kg dose of inorganic mercury with or without 2 micromol/kg N-acetylcysteine or cysteine. Bilateral ureteral ligation was performed in an attempt to reduce glomerular filtration to negligible levels, which in turn permitted the study of the basolateral uptake of inorganic mercury. The disposition of mercury was studied in the kidneys, liver, and blood 1 h after treatment. In rats given only mercuric chloride, the renal burden of mercury was approximately 20% of the administered dose of mercury. This confirms previous observations implicating a basolateral mechanism in the renal uptake of inorganic mercury. Coadministration of inorganic mercury with either N-acetylcysteine or cysteine caused a significant increase in the renal uptake of mercury 1 h after treatment, particularly in the rats treated with inorganic mercury plus N-acetylcysteine. The enhanced uptake of mercury in the kidneys was due to increased uptake of mercury in the renal cortex and outer stripe of the outer medulla. Interestingly, the rate of uptake of mercury was so great in the rats treated with inorganic mercury plus N-acetylcysteine that the renal burden of mercury was virtually equivalent to that generally detected in normal animals administered the same dose of inorganic mercury as mercuric chloride. Pretreatment with para-aminohippuric acid (PAH) (which is a potent inhibitor of the organic anion transport system) caused significant reductions in the renal uptake and burden of inorganic mercury in all the rats administered inorganic mercury, regardless of whether the inorganic mercury was coadministered with N-acetylcysteine or cysteine. Overall, the findings from the present study provide additional evidence that there is basolateral uptake of inorganic mercury in the kidneys, and that the primary or sole mechanism is dependent on the activity of the organic anion transporter. The present findings also show that cysteine and N-acetylcysteine enhance the basolateral uptake of mercuric ions in the kidney when they are coadministered with inorganic mercury, presumably in the form of mercuric conjugates. Moreover, it appears that mercuric conjugates of N-acetylcysteine are taken up more avidly at the basolateral membrane than mercuric conjugates of cysteine. Furthermore, the basolateral uptake of mercuric conjugates of N-acetylcysteine or cysteine in the kidney is due primarily to a mechanism involving the organic anion transport system.

Toxicokinetics and disposition of inorganic mercury and cadmium in channel catfish after intravascular administration.

To better understand the distribution and elimination of inorganic mercury (Hg) and cadmium (Cd) in fishes, channel catfish (Ictalurus punctatus) were administered either 6.4 micrograms/kg 203Hg as HgCl2 or 4.0 micrograms/kg 109Cd as CdCl2 via a dorsal aortic cannula. Blood, plasma, and red blood cells (RBCs) were serially sampled up to 156 (Hg) or 335 (Cd) days. The fraction of the injected dose remaining in the animal (Xf) was also determined at selected times by whole animal counting. The blood concentration and Xf-time profiles were simultaneously fitted to a three-compartment toxicokinetic model. The plasma concentration-time profile was also separately fitted to the same three-compartment model for comparison of parameter estimates. Toxicokinetic analysis of the blood concentration and Xf-time profile provided the following values: steady-state volume of distribution = 13.8 +/- 2.8 ml/g (Hg), 41.4 +/- 0.3 ml/g (Cd); total body clearance = 0.021 +/- 0.0006 ml/day/g (Hg), 0.0031 +/- 0.0008 ml/day/g (Cd); biological half-life (t1/2, beta) = 722 +/- 309 days (Hg), 9627 +/- 2206 days (Cd). Estimates of the t1/2 beta were up to 94 times longer if determined by simultaneous fitting of the blood concentration and Xf-time profiles. A time-dependent accumulation of Hg and Cd by RBCs was observed with maximum RBC concentrations of Hg and Cd occurring at 7 and 12 days after injection. After injection, the tissues with the highest accumulation of Hg were the liver, trunk and head kidney, muscle, and skin, but the amount of Hg in the liver gradually increased over 156 days. Most of the Cd was accumulated by the liver and trunk kidney within 24 hr, with little change occurring after 335 days. This study demonstrates the usefulness of intravascular injection and simultaneous analysis of blood and whole body amount data in determining the elimination of metals from fishes.

Effect of different renal glutathione levels on renal mercury disposition and excretion in the rat.

Mercury renal disposition has been studied following HgCl2 injection (5.0 mg/kg body wt., s.c.) in controls, diethylmaleate and N-acetylcysteine-treated rats. The different treatments were used to generate statistically different degrees of non-protein sulfhydryls concentration in kidneys. Diethylmaleate (4 mmol/kg body wt., i.p.) diminished kidney glutathione levels to 25% and N-acetylcysteine (2 mmol/kg body wt., i.p.) increased kidney non-protein sulfhydryls levels up to 75% compared with new controls. The amount of mercury in the kidneys, the mercury excretion rate in urine and the mercury plasma disappearance curves were calculated during 3 h post HgCl2 injection. BUN was measured in plasma at the same time period to determine the onset of kidney damage. The results indicate a higher HgCl2 renal clearance in N-acetylcysteine-treated rats compared to controls and less renal mercury accumulation. The data agree with diminished renal toxicity. On the other hand, renal mercury accumulation was higher and mercury renal clearance lower in diethylmaleate-treated animals, associated with higher renal toxicity. The results suggest that non-protein sulfhydryl levels (principally glutathione) might determine renal accumulation of mercury as well as its elimination rate and hence might enhance or mitigate the nephrotoxicity induced by the metal.

Physiological model for the pharmacokinetics of methyl mercury in the growing rat.

We describe a physiological pharmacokinetic model for methyl mercury and its metabolite mercuric mercury in the growing rat. Demethylation appears to occur in both host tissues and gastrointestinal flora with elimination dominated by biliary secretion of inorganic mercury and by transport of methyl mercury into the gut lumen followed by substantial bacterial metabolism. Biliary transport of both organic and inorganic mercury is modeled in terms of the known secretion of glutathione from the hepatic pool. At 98 days following an oral tracer dose of 203Hg-labeled methyl mercury chloride, 65% of the administered dose had been recovered in the feces as inorganic mercury and 15% as organic mercury. Urinary excretion is a minor elimination route, accounting for less than 4% of the dose as methyl mercury and 1% of the dose as inorganic mercury. Irreversible incorporation of the mercurials into hair is a significant route of elimination. Ten percent of the administered dose was contained in the hair shed during the 98 days and over 12% of the dose (almost 90% of the body burden) remained in the hair at the end of that time period. Apparent ingestion of hair by the rats during grooming represents a novel form of toxin recirculation. Transport of both chemical species between blood and tissues is bidirectional and symmetric with relatively slow movement into and out of the brain. Transport mechanisms for both mercurial species are discussed in the context of capillary transport physiology and the blood-brain barrier to small molecules and proteins.