Synthesis and pharmacological study of a polymer which selectively binds mercury

TOXICOLOGYANDAPPLIEDPHARMACOLOGY42, 445--454 (1977)

Synthesis and Pharmacological Study of a Polymer Which Selectively Binds Mercury R. D. HARBISON, M. M. JONES, J. S. MACDONALD, T. H. PRATT, AND R. L. COATES Center in Toxicology, Vanderbilt University Medical Center, Nashville, Tennessee 37232 Received July 20, 1976; accepted June 15, 1977

Synthesis and Pharmacological Study of a Polymer Which Selectively Binds Mercury. HARBISON,R. D., JONES,M. M., MACDONALD,J. S., PRATT,T. H., ANDCOATES,R. L. (1977) Toxicol. Appl. Pharmacol. 42, 445-454. A polymeric chelating agent (MBP) prepared by condensation of a mercaptoethyl sulfide with a terephthaldicarboxaldehyde exhibited a high degree of specificity for mercury (II) as judged by its ability to reduce rapidly the free metal ion concentration in an aqueous solution. The polymer exhibited a low degree of toxicity (LD50 > 5 g/kg) when administered orally to mice, and it was capable of antagonizing acute methyl mercury-induced lethality. MBP at a level of 1% in food promoted a rapid clearance of mercury from the body after acute administration of methyl mercury. The biological half life of the metal was reduced from 10.0 days to 4.5 days. The concentration of mercury in various tissues including the brain was reduced 40 to 70% after administration of MBP when compared to contents obtained in animals not treated with MBP. Numerous examples of poisoning following exposure to methyl mercury have resulted in an appreciation of the difficulty involved in removing this substance from binding sites in biological systems (Friberg and Vostal, 1972; Miller and Clarkson, 1973). The approaches to antagonism of alkyl mercurial toxicity have generally utilized enhanced urinary excretion of the metal through the use of water-soluble chelating agents ( G o o d m a n and Gilman, 1975). It has been shown that low-molecular weight thiol c o m p o u n d s can increase tissue deposition of the metal (Berlin and Lewander, 1965; Foulkes, 1974). As has been suggested by others (Clarkson et al., 1973; Takahashi and Hirayama, 1972), a more valuable therapeutic approach to antagonism of methyl mercury toxicity would be to promote removal of the metal from the body through enhanced fecal rather than urinary excretion. Methyl mercury is excreted into bile at a rate of less than 0.5% over a 2-hr period (Klaassen, 1975). This relatively slow excretion and the fact that mercury undergoes extensive enterohepatic recirculation (Clarkson et al., 1973; Berlin and Ullberg, 1963; Norseth and Clarkson, 1971) accounts for the long biologic half-life o f methyl mercury. As pointed out by Clarkson et al. (1973), it should be possible to enhance the fecal excretion of methyl mercury by interrupting this enterohepatic recirculation of the metal through the use of a relatively inert compound of sufficient molecular weight and charge characteristics to bind mercury and prevent reabsorption of the complex. The necessary size characteristics of such a chelating agent can be satisfied through the use of a polymeric compound. The purpose of this investigation was synthesis of a Copyright ~) I977 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain

445 ISSN 0041 00SX

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HARBISON E T AL.

polymer with (1) a high degree of specificity for mercury, (2) a low degree of toxicity when administered orally, and (3) the ability to effectively reduce the body burden of mercury after administration of methyl mercury.

METHODS

Synthesis of Polymeric Chelating Agent MBP Mercaptoethyl sulfide (20 ml, 0.15 mol, Aldrich Chemical Company) was dissolved in 100 ml of benzene; terephthaldicarboxaldehyde (10 g, 0.075 tool, Aldrich Chemical Company) was added to the solution. The solution was stirred and heated to 35°C to dissolve the dialdehyde. To this solution was added 10 ml of concentrated hydrochloric acid, and a rapid polymerization reaction ensued with the evolution of heat, causing the solution to boil vigorously. This vigorous boiling resulted in the formation of a very porous solid polymer, with a large surface to volume ratio. The polymer initially occupied a volume of 10 ml/g but was crushed by hand, and the solvent was removed by filtration. The polymer was washed three times with 100-ml portions of benzene, then one portion of 100 ml of acetone. The solid was crushed further by hand and extracted with acetone in a sophlet extractor for 24 hr, then dried in vacuo: yield 30 g (97%). Elemental analysis of the polymer was performed by Galbraith Laboratories of Knoxville, Tennessee. Anal. Calculated for CI6HzzS4: C, 47.29; H, 5.45; S, 47.24. Found: C, 47.27; H, 5.50; S, 47.39. MBP thus prepared was ground in a ball mill for 24 hr to reduce the particle size to <200 mesh.

Chemical Characterization of the Polymer The basic procedure used to investigate interaction of the polymer with metal ions involved measurement of the concentration of the metal ion as a function of time in a suspension of the polymer. A suspension of the polymer was prepared by shaking a sample of the polymer with 50 ml of 0.1 M NaC104 solution for 3 hr, after which it was made up to a total volume of 100 ml by the addition of more sodium perchlorate solution. At this point, 1.0 ml of 1.0 x 10-2 M metal ion perchlorate solution was added, and at various times thereafter, aliquots were taken, filtered, and analyzed for the metal ion concentration. Mercury (II), nickel (II), and cobalt (II) concentrations were determined using standard spectrophotometric procedures (Flaschka and Barnard, 1972). Copper (II) and lead (II) concentrations were determined with specific ion electrodes and a Radiometer pH/mV meter. The experiments were carried out at 25 ° C. Samples of polymer were filtered, dried, and analyzed after 20 hr in the mercuric perchlorate, nickel perchlorate, and cobalt perchlorate solutions. Polymer samples from the cupric perchlorate and lead perchlorate solutions were collected after 7 days.

Rate of Absorption of Mercury by the Polymer The rate of uptake of mercury (II) by the polymer was determined by removing a sample of the polymer, from time to time, from a rapidly stirred slurry of the polymer in a 1 x 10-3 M Hg(C104) 2 solution. Three grams of the polymer were stirred in 100 ml of 1 x 10 -3 M mercuric perchlorate solution to which a few drops of detergent solution had

M E R C U R Y - B I N D I N G POLYMER

447

been added. Samples of the slurry were removed from time to time and analyzed for their mercury content.

Rate of Removal of Mercury from Aqueous Solutions by the Polymer The rate of reduction in the concentration of mercury (II) in an aqueous solution exposed to MBP was also measured. These data were obtained by the electrometric study described below. A suspension of the polymer was prepared by stirring 2.0 g of MBP into 178 ml of water and 20 ml of 1.0 M sodium perchlorate with a few drops of liquid detergent added. The temperature of the suspension was maintained at 25 + 0.1°C, and the solution pH was adjusted to ~ 1 with concentrated perchloric acid. Then 2.0 ml of 0.01 M mercuric perchlorate solution was introduced into the suspension, and the free mercuric ion concentration was monitored as a function of time with a Beckman Research model pH meter equipped with saturated calomel and J-tube mercury electrodes. The uptake of mercuric ion from the solution was followed for approximately 24 hr after which the polymer was collected by filtration, washed, dried in vacuum, and analyzed for mercury content.

Antagonism Studies Male, Swiss-origin mice (Harlan Industries, Cumberland, Indiana) were housed in groups of five and were allowed access to food (Wayne Lablox) and tap water ad libitum. Methyl mercuric chloride (ICN, K & K Laboratories, Irvine, California) was dissolved in sterile normal saline (0.9%) and administered as a single intraperitoneal injection of various dosages. The polymer (ground to a particle size less than 200 mesh) was administered by oral gavage as a uniform suspension in distilled water. The LD50 for MBP was > 5 g/kg. When the polymer was administered in the food, the same size particles were mixed with powdered food (pulverized Wayne Lablox) to achieve a final concentration of 1%. The effect of the polymer on acute CH3HgCl-induced lethality was determined by administration of the various materials beginning 4 hr after administration of the organic mercurial. Antagonists were administered each 24 hr after this initial dose for 4 consecutive days for a total of five administrations. When the chelating agents were administered in the food, animals were allowed access to food ad libitum 4 hr after administration of methyl mercury. Animals were observed daily for a period of 3 weeks.

Distribution and Whole Body Retention of Mercury To determine the quantity of mercury in the body as well as the organ distribution at various times after the administration of the metal, CH~°3HgC1 (ICN, Irvine, Calif.)was added as a tracer to the injection solution. At various times after administration of the mercurial, mice were anesthetized with ether and blood was drawn by cardiac puncture. Animals were then sacrificed by further ether inhalation and tissue samples were taken and solubilized with Soluene (Packard Inst. Co., Downers Grove, Illinois), and aliquots were counted in a Packard Tri-Carb liquid scintillation spectrometer. Whole body concentrations of Hg were determined by sacrificing mice with ether, shaving the animals to remove most of the body hair, and homogenizing the intact carcass in distilled water in a Waring blender, Model 1120. Several aliquots of the whole mouse homogenate were then solubilized with Soluene and counted as indicated above.

448

HARBISON E T A L .

In both of these studies, appropriate standards were prepared and counted to enable correction for background and decay of the isotope.

Statistics Statistical evaluations between sample means were made by two-tailed grouped Student's t test. Level of significance chosen in all cases w a s p ~<0.05.

RESULTS MBP (Fig. 1) exhibited a high degree of selectivity for Hg (II) (Table 1). The polymer was able to bind 1205 ppm of Hg.

~'S-(CH 2)2_S_(C H2)2_ $

/ ~n

MBP FIG. 1. Chemical structure of mercury-binding polymer. TABLE 1 ABSORPTIVE SELECTIVITY OF MBP FOR VARIOUS METAL IONS a

Metal ion Hg 2+ Co 2+ Ni 2+ Cd 2+ Cu 2+ Zn 2+ Mg 2+ Ca 2+ Pb 2+

Metal content of resin (ppm) 1205 2 37 49 35 22 3 6 11

A 1-g sample of the resin was stirred with I00 ml of a 1 x 10-4 M solution of the metal salt for 1 hr. The resin sample was then removed, washed, and analyzed. Results of uptake studies with MBP are presented in Fig. 2. The most notable feature of the data is the very rapid initial rise in mercury content of the resin and its rapid attainment of an "equilibrium" value. F r o m other studies, in which much longer immersion times were used, it is probable that the mercury slowly diffuses into interior binding sites where it is bound quite firmly. As a result, a sample of polymer which has been immersed in a mercury solution for 10 days or 2 weeks, will generally have a mercury content of the order of 2% or more. The uptake of Hg 2+ from solution by MBP is shown in Fig. 3 where solution potential is plotted vs time. The concentration of free mercuric ion in solution at any time is calculated by the Nernst equation as: log [Hg 2+] = E -- 0.614/0.0296.

449

MERCURY-BINDING POLYMER

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I

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TFME (hrs) F l a . 2. Uptake o f mercury from an aqueous solution by MBP. Concentration of mercury as Hg(CIO4) 2 was l x 10 -3 M at 2 5 ° C .

As seen in Fig. 3, by the time the electrode system could reach a stable reading following the addition of mercuric ion to the solution (1 to 3 min), the mercuric ion concentration had already fallen from 1 x 10 -4 to ~1 x l0 -l° M. Essentially, all of the mercuric ion introduced into the suspension was taken up by the polymer in this short time. The mercury analyses of a sample of polymer handled as described above along with two samples treated with mercuric ion at a neutral pH show an average mercury content of 2040 + 28 ppm. Using this value, a 2-g sample of the Hg2+-treated polymer contains 2.0 x 10 -5 tool of Hg 2+. The suspension, 200 ml in total volume, was initially prepared to be 1 x 10 -4 M in Hg 2+, offering exactly 2.0 x 10 .5 mol of Hg ~+ for uptake. This suggests that the capacity of the polymer for binding Hg 2+ has not been reached in these experiments. The most striking feature is the surprising rapidity with which a very low concentration of Hg 2+ is produced in the solution. This represents what the resin can achieve under favorable conditions. It shows how a concentration gradient unfavorable to the continued reabsorption of mercury from the gut can be established. soc

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FIG. 3. Rate o f removal of mercury from an aqueous solution in contact with MBP.

450

HARBISON E T AL.

Antagonism Studies Several different dosages of CH3HgC1 were administered ip to different groups of mice; mice in each group received a single dose. F o u r hours following methyl mercury administration, treatment with the chelating agent was begun and continued at each 24hr interval for 4 days for a total o f five administrations. The L D 5 0 of methyl mercuric chloride alone and after treatment with various chelating agents was calculated (Table

2). All the chelating agents examined except E D T A effectively reduced the acute lethality of methyl mercuric chloride as indicated by the L D 5 0 and potency ratio. Administration of M B P either po or in the food resulted in a parallel shift of the rather steep methyl mercury lethality curve to the right; a significant degree of protection against methyl mercury-induced toxicity was afforded by treatment with this polymer. TABLE 2 ANTAGONISM OF METHYL MERCURY-INDUCED

LETHALITY a

Chelating agent Compound None MBP MBP BAL N-Acetyl-DLpenicillamine CaNa2EDTA

Dosage and route of administration

LD50

Upper limit

-500 mg/kg po 1% in food 25 mg/kg im 50 mg/kg po

10.0 13.4 12.7 12.5 11.2

11.3 16.4 15.5 14.9 13.2

8.8 11.0 10.5 10.5 9.5

-0.75 0.78 0.80 0.89

1% in food

10.0

12.2

7.9

0.98

Lower limit Potency ratio

" CH3HgC1 was administered as a single ip dose in sa!ine to five dosage groups (10 mice/group): 2.5, 5. 7.5, 10, and 15 mg of CH3HgCI by body weight. Injection volume was 10 ml/kg. Treatment with chelating agents was begun 4 hr after CH3HgC1 administration and continued at each 24-hr interval from the first dose for 4 consecutive days; total = five administrations. Where chelating agent was administered in food, animals were allowed access to food ad libitum beginning 4 hr after CH3HgCI administration. MBP and N-acetyl-DLpenicillamine were administered in saline; BAL was diluted in corn oil. LD50 values, upper and lower limits of LD50 values, and potency ratios were determined after a 3-week observation period (Day 1 - CH3HgCI administration) using the method of Finney (1962) as adapted to a computer program by Spratt (1966). The program used includes a test for parallelism; i.e., potency ratio values are not calculated unless calculated LD50 curves are parallel within a tolerance of 0.10 probit unit.

Distribution and Whole Body Retention The ability of the polymeric chelating agent to alter the distribution of previously administered methyl mercury was investigated. The results are presented in Fig. 4. Methyl mercuric chloride was administered as a single ip dosage of 1 mg/kg which included CH~°3HgCI added as tracer. Mice were allowed access to powdered food ad libitum containing M B P at a level o f 1% beginning 4 hr after administration of the mercurial. Administration of MBP resulted in a significant reduction in mercury content of the various tissues examined at 10 days after treatment with methyl mercuric chloride. A percentage of reduction of 55.4, 43.5, 75.2, 39.8, 63.5, 67.8, and 52.6 was seen for whole blood, brain, liver, kidney, spleen, fat, and muscle, respectively. A

MERCURY-BINDING POLYMER

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FIG. 4. Distribution of mercury in various tissues 10 days after treatment with CH~HgC1. CH~°3HgCI, 1 mg/kg (2.5 mCi/mg Hg), was administered ip. Animals treated with MBP were allowed access to powdered food containing the polymer at 1% (w/w) beginning 4 hr after administration of CH3HgC1. Blood, plasma, and tissue samples were taken 10 days following CH3HgCI treatment. Bars represent mean _+ SD of at least 30 animals. MBP treatment significantly reduced (p < 0.0l) Hg concentrations in all tissues examined.

significant increase of 96.8% was seen in plasma Hg content after administration of MBP in the food. The presence of MBP in food promoted a more rapid clearance of mercury from whole blood and from the body (Figs. 5 and 6). At whole body contents of mercury less than 10% of the original body burden, the polymer had no effect on the rate of clearance of metal from the body despite continued treatment. Before this point of noeffect (i.e., 20 days), however, the biologic half-life of methyl mercury was 10.0 days in animals receiving methyl mercury alone, while the half-life was 4.5 days in the MBPtreated group. Thus, the polymer was capable of significantly reducing tissue concentrations and increasing elimination of methyl mercury from mice.

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F]o. 5. Whole blood concentrations of mercury on various days following administration of CH3HgCI. CH]°3HgC1, 1 mg/kg (2.4 mCi/mg Hg), was administered ip. Animals treated with MBP were allowed access to powdered food containing the polymer at 1% (w/w) beginning 4 hr after CH3HgCI administration. Blood samples were obtained by cardiac puncture. Bars represent mean + SD of at least 10 animals.

HARBISON E T A L .

452

To demonstrate that the polymer exerted this effect on tissue contents of mercury while remaining in the gastrointestinal tract, the distribution of [14CJMBP was examined. The labeled polymer was synthesized as described above using [carbonyllgC]terephthaldicarboxaldehyde (specific activity, 3 mCi/mmol; obtained from Amersham/Searle). The specific activity of the final product was found to be 0.052 ¢tCi/mg of polymer. When this labeled polymer was administered to the animals, either 500 mg/kg po or 1% in the food, radioactivity could not be detected in any of the tissues examined at any time period up to 2 weeks following the start of administration. When a single oral dose was administered, the polymer could be followed through the gastrointestinal tract.

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60

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20

40

FIG. 6. Whole body concentrations of mercury on various days following administration of CH3HgC1. CH]°3HgCI, 1 mg/kg (2.4 mCi/mg Hg), was administered ip. Animals treated with MBP were allowed access to powdered food containing the polymer at 1% (w/w) beginning 4 hr after CH3HgCI administration. Bars represent mean +_SD of at least 10 animals.

Less than 40% of the initial activity was detected in the ileum 4 days after administration with no activity seen in this segment of the intestine 15 days after administration. DISCUSSION One of the primary difficulties encountered in chelation therapy is exacerbation of the toxicity of the metal through the use of chelating agents. This normally occurs as a result of the broad spectrum of metals, including essential metals, that are complexed or because of the inherent toxicity of the chelating agent or the ligand-metal complex itself. The solid thioether-based polymer, MBP, avoids this limitation through a high degree of selectivity for Hg as well as a low degree of toxicity. Although it is rather difficult to obtain values for the stability constant of the Hg complex due to the very limited water solubility of the polymer, it can be inferred from studies with other compounds containing the same mercaptal moiety that this value is as high as 1025 (Jones et al., 1975). The proposed mechanism of action of the mercury binding polymer is an interruption of the enterohepatic circulation of methyl mercury. Although relatively small quantities of methyl mercury undergo biliary secretion (Klaassen, 1975), the high affinity of the

MERCURY BINDING POLYMER

453

polymer for mercury results in a substantial extraction of the metal from the total quantity delivered to the gastrointestinal tract and a rapid reduction in the concentration of "free" or diffusible metal. This is suggested by the increased rate of clearance of the metal from the body as well as the reduced Hg content of the various organs examined after treatment of animals with the polymer. Further, the rapid rate of clearance of Hg from blood as contrasted with the rate of whole body elimination in the absence of the polymer suggests that the enhanced rate of clearance of the metal produced by the polymer is not simply a function of action of the chelating agent on Hg in the blood. The similarity of whole body Hg contents observed at 20 and 40 days post-treatment with methyl mercury is further evidence in support of the concept of interruption of enterohepatic circulation. At these time periods, less than 10% of the original body burden remains in the animal, and the blood concentrations of the metal are quite low. Thus, very small quantities of mercury will be undergoing biliary secretion at this time, and one would expect an inert polymer to have a proportionally lesser effect on clearance of the metal. The significant degree of protection from the acute lethal effects of methyl mercury afforded by the polymer is most probably a result of the enhanced rate of clearance of the metal from the body. It is interesting to note that the polymer is at least as effective in this regard as BAL or N-acetyl-DL-penicillamine, yet it is without the adverse reactions commonly encountered in the use of the latter two chelating agents. An important point to note in this regard is that administration of the polymer significantly reduces the brain content of mercury while it has been shown that BAL increases the mercury concentration in this organ (Berlin and Lewander, 1965). Mercury-binding polymer possesses the necessary chemical configuration for a high degree of specificity for mercury as well as a low oral toxicity. This polymer is an effective antagonist of acute methyl mercury toxicity and is capable of increasing the rate of clearance of mercury from the body. ACKNOWLEDGMENTS The authors wish to express their appreciation to Mrs. Julianna Bendt for her skillful technical assistance. This work was supported in part by USPHS Grants Nos. ES 01018, ES 00267, and ES 00782.

REFERENCES BERLIN, M., AND LEWANDER, T. (1965). Increased brain uptake of mercury caused by 2,3-

dimercaptopropanol (BAL) in mice given mercuric chloride. Acta Pharmacol. Toxicol. 22, 1-7. BERLIN, M., AND ULLBERG, S. (1963). Accumulation and retention of mercury in the mouse: III. An autoradiographic comparison of methylmercuric dicyandiamide with inorganic mercury. A rch. Environ. Health 6, 610-616. CLARKSON, T. W., SMALL, H., AND NORSETH, T. (1973). Excretion and absorption of methyl mercury after polythiol resin treatment. Arch. Environ. Health 26, 173-176. FINNEY,D. J. (1962). ProbitAnalysis, 3rd Ed. Cambridge University Press, London. FLASCHKA, H. A., AND BARNARD, A. J. (1972). Chelates in Analytical Chemistry. Marcel Dekker, New York.

454

HARBISON E T

AL.

FOULKES, E. C. (1974). Excretion and retention of cadmium, mercury, and zinc by rabbit kidney. A mer. J. Physiol. 277, 1356-1360. FRIBERG, L., AND VOSTAL, J. (eds.) (1972). Mercury in the Environment. An Epidemiological and Toxicological Appraisal. Chemical Rubber Co. Press, Cleveland, Ohio. GOODMAN, L. S., AND GILMAN, E. (eds.) (1975). The Pharmacological Basis of Therapeuties, 5th Ed. MacMillan Co., Toronto, Canada. JONES, M. M., BANKS, A. J., AND BROWN, C. H. (1975). Stability constant studies on some new thioether carboxylic acid chelating agents. J, Inorg. Nucl. Chem. 37, 761-765. KLAASSEN, C. D. (1975). Biliary excretion of mercury compounds. Toxieol. Appl. Pharmacol. 33, 356-365. MILLER, M. W., AND CLARKSON, T. W. (eds.) (1973). Mercury, Mercurials, and Mercaptans. Charles C. Thomas, Springfield, Illinois. NORSETH, Y., AND CLARKSON, T. W. (1971). Intestinal transport of 2°3Hg labeled methyl mercury chloride: Role of biotransformation in rats. Arch. Environ. Health 22, 568-577. SPRATT, J. L. (1966). Computer program for probit analyses. Toxieol. Appl. Pharmacol. 8, 110112. TAKAHASHI, H., AND HIRAYAMA,K. (1972). Accelerated elimination of methyl mercury from animals. Nature (London) 232, 201-202