Department of Environmental Health, University of Washington, 4225 Roosevelt Way NE, Suite 100, Seattle, Washington 98105
Received November 6, 2000; accepted January 30, 2001
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ABSTRACT |
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Key Words: 2,3-dimercapto-1-propanesulfonic acid (DMPS); chelation; mercury; methylmercury; mercuric ion; brain; blood; kidney.
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INTRODUCTION |
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A specific concern associated with methylmercury exposure in humans is the need for effective therapy in dealing with intoxication. In this respect, chelation therapy is the most commonly used and seen as the least invasive (Aposhian, 1983). Chelating agents compete with the in vivo binding site for the metal ion through the process of ligand exchange (Jones, 1994
). The toxic metal bound to the chelating agent is excreted from the body through the urine or feces. Among chelating agents currently available, the sodium salt of 2,3-dimercapto-1-propane-sulfonate (DMPS) has been found to be highly effective, particularly with respect to promoting mercury elimination following inorganic or elemental mercury exposure (Aaseth et al., 1995
; Aposhian 1983
; Garza-Ocanas et al., 1997
; Gonzalez-Ramirez et al., 1995
; Maiorino et al., 1996
). Remaining to be confirmed is the efficacy of DMPS in reduction of mercury body burden resulting from organic mercury exposure, and in particular, its value in the removal of inorganic Hg (Hg2+) arising from dealkylation of methylmercury in target tissues.
In the present studies, we evaluated the efficacy of DMPS in reducing Hg2+ and CH3Hg+ from kidney, brain, and blood mercury levels following prolonged methylmercury exposure in rats. As part of this assessment, we performed mass balance calculations to demonstrate the quantitative relationship between mercury removed from each tissue and that excreted in the urine. We also employed linear regression analysis to assess the strength of association between tissue mercury concentrations and urinary mercury levels in relation to DMPS treatments.
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MATERIALS AND METHODS |
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Animal treatment.
Immediately upon receipt, all animals were placed in individual, wire bottom, hanging cages and given deionized water and food (Wayne Rodent Blox rat chow) ad libitum. This diet has been shown to be free of mercury content. Lighting was set at a 12-h light/dark cycle, and the temperature of the animal housing facility was kept at 20°C. Following 1-week acclimation, the animals were separated into 2 groups (described below) and given either deionized water or drinking water containing 10 ppm MMH. This dose of MMH was selected from previous studies (Fowler and Woods, 1977; Woods and Fowler, 1977
) as sufficient to permit significant target tissue accumulation without evidence of overt neurotoxicity or renal injury during the course of the treatment regimen.
Study design.
Two prolonged methylmercury-exposure studies were undertaken. Study 1 was designed to evaluate the efficacy of DMPS dosage on mercury clearance from brain and kidney. In this study, 30 rats were divided into 2 groups of 18 and 12 each. The larger group of 18 animals was placed on a continuous regimen of drinking water containing 10 ppm MMH, whereas the remaining 12 animals were placed on deionized water (dH2O) as controls. After 9 weeks, all 30 rats were transferred to individual metabolism cages for 24-h urine collections. Immediately thereafter, the 18 animals that had received MMH for 9 weeks were divided randomly into 3 groups of 6 each. The first 2 groups of 6 animals received single ip injections of DMPS at either 100 mg/kg or 200 mg/kg, respectively, whereas the third group received saline injections. Concurrently, the 12 control rats that had been maintained for 9 weeks on dH2O were divided into 3 groups of 4 each and given comparable injections of 100 or 200 mg/kg DMPS or saline. Subsequently, all rats were returned to individual metabolism cages for post-treatment 24-h urine collections. All animals were then sacrificed, and tissues were collected for mercury assessments.
The second study (Study 2) was designed to assess the effect of consecutive DMPS treatments on mercury clearance from brain, kidney, and blood of MMH-exposed rats. In this study, 30 rats were placed on a continuous regimen of drinking water containing 10 ppm MMH for 6 weeks. The shorter MMH exposure time in Study 2 was selected based on the observation from Study 1 that the equilibrium between organic and inorganic mercury species in kidney cortex is achieved by 6 weeks of MMH exposure (described in Results). Hence, the prechelation concentrations of both Hg2+ and CH3Hg+ in renal cortex are sufficient following 6 weeks of MMH exposure to permit evaluation the efficacy of DMPS chelation in clearing both organic and inorganic mercury species from kidney, as well as from brain and blood.
To determine the effects of multiple DMPS treatments on tissue mercury levels, animals were given up to 3 DMPS injections over a period of 37 days prior to sacrifice. For this study, the 30 MMH-exposed rats were divided into 2 groups of 18 and 12 animals, respectively. The first group of 18 rats received a single ip injection of 100 mg/kg DMPS, whereas the remaining 12 were given a saline injection, as controls. All 30 animals were then transferred to individual metabolism cages for 24-h urine collections. Rats were denied food but were provided dH2O ad libitum during the urine collection period. Following urine collections, 6 animals from the DMPS-treatment group and 4 rats from the control group were sacrificed, and brains, kidneys, and blood were retrieved for mercury analyses. Seventy-two hours after the first injection, the remaining 12 DMPS-treated rats were given a second 100mg/kg DMPS injection, while the remaining 8 saline-treated rats were given a second saline injection. After 24 h, 6 of the DMPS-treated rats and 4 of the control rats were sacrificed and tissues collected. Seventy-two h after the second injection the remaining animals were given a third DMPS or saline treatment. Twenty-four h after the third injection all remaining rats were sacrificed and tissues collected. Between DMPS treatments, animals were held in metabolism cages without food but with dH2O for 24-h urine collections and then returned to their hanging cages and permitted food and water ad libitum for 48 hours. No animals were deprived of food for more than 24 hours during the treatment period. In all studies, animals were anesthetized by carbon dioxide (CO2) and then sacrificed by decapitation. Blood was obtained by cardiac puncture into heparinized tubes prior to sacrifice. Brains and kidneys were harvested surgically immediately following sacrifice. All tissues were preserved at 80°C until mercury analysis.
Collection of urine samples.
Animals were placed in hanging metabolism cages for 24 h with free access to drinking water (containing either MMH or dH2O) but not food. The metabolism cages were fitted with metal funnels attached to the bottom with a plastic ping-pong ball placed at the hole of the funnel to allow urine but not feces to pass through. Aluminum foil-covered, polypropylene 125-ml volumetric flasks were placed under the funnels to collect the urine without allowing evaporation. At the end of 24 h the urine volume was measured. The urine was then acidified with a drop of 6 N HCl and frozen at 20°C until mercury analysis.
Mercury determination.
Urinary mercury was measured using a modified version of the digestion method by Corns et al. (1994). 2.5 ml of HCl and 2-ml bromate/bromide solution was added to 2.5-ml urine sample and allowed to sit overnight in a 20-ml glass scillation vial. Hydroxylammonium chloride (20%) was then added to decolorize the sample and to stop the digestion process. The fully digested sample was transferred to a 50-ml borosilicate glass volumetric flask, and distilled water was added to a total volume of 50 ml. The total (organic and inorganic) mercury content of the sample was then analyzed by cold vapor atomic fluorescence spectroscopy (CVAFS) using a PSA Merlin Mercury Analysis System (Questron Corp., Mercerville, NJ). For inorganic mercury (Hg2+) determinations, a 0.5-ml sample of urine was digested overnight in 2 ml of HNO3. The sample was then diluted to a volume of 20 ml with dH2O, and the total Hg2+ content was measured using CVAFS. The organic mercury content of the sample was determined by the difference in the total and inorganic mercury values.
The total and inorganic mercury concentrations in kidney and brain tissues were analyzed by CVAFS following digestion and preparation of tissues as described by Atallah and Kalman (1993). The organic mercury content of tissue samples was again determined by the difference in the total and inorganic mercury concentrations.
Total and organic mercury concentrations in blood samples were directly measured by ethylation-GC-CVAFS after alkaline digestion-solvent extraction, as described by Liang et al., 2000. Inorganic mercury was calculated as the difference between total and organic mercury.
For each of the above cited procedures, validation of Hg2+ and CH3Hg+ analysis was confirmed by concomitant measurement of control urine or tissue samples containing a range of known concentrations of Hg2+, CH3Hg+ or total mercury (Hg2+ + CH3Hg+) derived from standard reference materials. Mean recoveries of Hg2+ and CH3Hg+ from spiked tissues were on the order of 95100% with no detectable cross contamination of mercury species, consistent with findings reported by original authors. All procedures employed specific quality control protocols, including pre-analysis of all reagents and materials used in the analyses, precluding potential Hg2+ or CH3Hg+ contamination from sources other than biological samples under evaluation.
Statistical analyses.
Data are presented as means ± standard error of the mean (SEM). Statistical analyses were conducted using Student's t-test with 1-tailed distribution. p values less than 0.05 were considered significant. Correlational analysis was performed using the Excel function (Microsoft, Redmond, WA) and expressed as the correlation coefficient.
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RESULTS |
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Consecutive DMPS Injections Promote Successive Urinary Elimination of Both Organic and Inorganic Mercury Species
We conducted Study 2 to assess the effects of consecutive DMPS treatments on mercury clearance from kidney, brain and blood of MMH-exposed rats and to measure the corresponding changes in urinary mercury levels. In these studies 30 rats were exposed to MMH in drinking water for 6 weeks, followed by 1, 2, or 3 consecutive DMPS treatments (100 mg/kg, ip) at 72-h intervals, as described in Materials and Methods. As shown in Figure 1, consecutive DMPS treatments were highly effective in promoting successive release of both organic and inorganic forms of mercury into the urine. The inorganic and organic mercury concentrations in the urine of MMH-exposed animals prior to DMPS injection were 1.13 and 1.17 µg/ml, respectively, compared with 0.01 and 0.00 µg/ml, respectively, in rats exposed only to dH2O. Following a single DMPS injection, the total mercury concentration in 24-h urine samples increased 18-fold, with 6- and 12-fold increases in inorganic and organic species, respectively, compared with that observed in urine of MMH-exposed rats receiving a saline injection. A second DMPS injection administered 72 h following the first was equally effectively in promoting mercury excretion, the total urinary mercury concentration increasing to 27 times that observed in MMH-exposed animals receiving 2 consecutive saline injections. A third DMPS injection given 72 h after the second promoted additional excretion of both organic and inorganic mercury species, the total urinary mercury concentration increasing another 18-fold that seen in urine of saline-injected controls. Quantitatively, mean total urine mercury concentrations following the first, second, and third DMPS injections were 24.8, 25.6, and 14.4 µg/ml, respectively. The sum of total mercury excreted in the urine following all 3 DMPS injections was 64.8 µg/ml, compared with 4.4 µg/ml after 3 consecutive saline injections. Creatinine adjustment of urinary mercury concentrations showed a slightly more pronounced mercury excretion trend associated with the number of DMPS injections when compared with that of the noncreatinine adjusted results, although no trend in creatinine excretion in the urine following DMPS administration was seen (not shown). DMPS treatments did not affect mean total urine 24-h excretion rates, which remained constant (12.2 ± 1.3 ml/24 h) throughout the treatment regimen.
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DISCUSSION |
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The dissociation of methylmercury into organic and inorganic species in the kidney during prolonged exposure to MMH in rats has been previously described (Woods et al., 1991). Of note, an equilibrium between Hg2+ and CH3Hg+ concentrations in the kidney cortex is readily achieved in a dose- and time-dependent manner during the course of continuous MMH exposure, and this equilibrium is maintained by the kidney over a wide range of total mercury concentrations to approximately 100 µg/g cortex. Inasmuch as both Hg2+ and CH3Hg+ may participate in renal toxicity, it is notable that DMPS is equally effective in depleting both species from kidney tissue. Analysis of the data presented here shows a direct correlation between the increases in the urinary concentrations of both organic and inorganic mercury fractions and the decline in the concentrations of each species in kidney following DMPS treatment. These findings are consistent with the very high ligand exchange rates (109/s) of both Hg2+ and CH3Hg+ for thiol groups (Martin, 1986
), and suggests comparable availability of organic and inorganic Hg species for exchange with DMPS in kidney cells.
Consistent with the effects on renal mercury content, DMPS effectively reduced blood total mercury concentrations over the course of consecutive treatments. Unlike in the kidney, however, less than 5% of total mercury in the blood was present as Hg2+. The effect of DMPS in lowering total blood mercury was restricted entirely to the organic mercury constituent, which decreased significantly following the second and third consecutive DMPS injections. Previous studies (Planas-Bohne, 1981) have demonstrated that DMPS is moderately effective in removing methylmercury from blood following administration to rats. However, the effects of DMPS on the inorganic Hg fraction derived from demethylation of methylmercury in blood cells was not reported. This is the first report to our knowledge to assign the effect of DMPS on blood mercury to the organic fraction.
The dissociation of methylmercury into organic and inorganic species in the brain during prolonged exposure of rats to MMH was also noted in the present study, although the formation of Hg2+ from MMH was substantially lower than that observed in kidney. In this respect, the changes in total brain mercury content observed during the course of MMH exposure appeared to largely reflect the CH3Hg+ constituent, similar to that observed in blood. Berlin (1986) reported that approximately 510% of methylmercury is demethylated to Hg2+ in the brain, consistent with this observation. Of note, DMPS was effective in decreasing total brain mercury content in MMH-exposed rats only following 3 consecutive treatments. Similar findings have been reported with respect to elemental mercury exposure (Cikrt et al., 1996). The initial increase in brain mercury content seen following a single DMPS injection is of interest, inasmuch as it suggests that DMPS may facilitate redistribution of CH3Hg+ from blood to brain until blood mercury levels are concomitantly depleted by subsequent DMPS treatments, or until DMPS accumulates in sufficient concentrations in the CNS to effect significant mercury chelation. Notably, the partitioning of DMPS into the CNS is relatively slow due to its polarity and limited lipid solubility (Jones, 1994
), consistent with this idea. The failure of DMPS to cause significant depletion of Hg2+ from the brain may reflect the slow rate of demethylation of methylmercury in the CNS and consequent low concentrations of Hg2 +relative to those of CH3Hg+ available for exchange with DMPS. Alternatively, the efficacy of DMPS in extracting CH3Hg+ may reflect the greater availability of organic mercury following distribution from the blood to the CNS, as compared with that of Hg2+, which most likely is formed following compartmentalization of methylmercury to specific regions of the CNS that may be poorly accessible to DMPS. The latter possibility raises concerns regarding the efficacy of DMPS or similar chelating agents in the remediation of neurotoxicity associated with methylmercury, since principal adverse effects may be attributable to Hg2+ accumulation following partitioning of methylmercury to specific CNS foci during prolonged exposure (Aschner and Aschner, 1990
; Friberg and Mottet, 1989
). In contrast to the present findings, Aposhian et al. (1996) reported that DMPS did not alter brain mercury levels in rats treated by ip injection with HgCl2. This distinction may reflect differences in the capacity of DMPS complexes of Hg2+ versus those of CH3Hg+ to partition from blood into the CNS.
Although total mercury tissue levels increased significantly during the course of MMH exposure, the mercury concentration in the urine increased only slightly during this period (Table 1 and Fig. 1
). These findings are consistent with the view that prechelation urinary excretion is not well correlated with mercury body burden (Aposhian et al., 1995
). In contrast, urinary mercury levels subsequent to DMPS chelation have been shown to constitute a better approximation of Hg body burden, particularly in the case of elemental mercury exposure (Echeverria et al., 1998
). Mass balance calculations performed in the present investigation extend these findings to organic mercury exposure, since the amount of mercury excreted in the urine following consecutive DMPS treatments accounted for essentially all of the mercury found in blood and the 2 principal target tissues, kidney, and brain. Notably, essentially all of the inorganic mercury excreted postchelation was of renal origin, since DMPS treatment did not significantly reduce Hg2+ concentrations in either blood or brain. These findings support the view that postchelation urinary mercury levels are an accurate measure of mercury body burden attributable to prolonged methylmercury exposure.
In conclusion, the present studies demonstrate the efficacy of DMPS in depleting mercury body burden and confirm previous observations regarding the utility of postchelation urinary mercury levels as an accurate measure of body burden attributable to prolonged methylmercury exposure. The finding that DMPS readily depletes both Hg2+ and CH3Hg+ from kidney, but only CH3Hg+ from brain and blood, suggests that the capacity of DMPS to remove Hg species reflects the tissue-specific dealkylation rate of MMH and, hence, the relative amount of species present. These findings may explain the relative inefficacy of DMPS and similar chelators in the remediation of neurotoxicity associated with organic mercury exposure, in which Hg2+, a principal mediator of toxicity, is present only in low concentrations.
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ACKNOWLEDGMENTS |
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NOTES |
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