Determination and metabolism of dithiol chelating agents

TOXICOLOGY AND APPLIED PHARMACOLOGY Im,96-

Determination

106 (1989)

and Metabolism

of Dithiol Chelating Agents

VIII. Metal Complexes of meso-Dimercaptosuccinic Acid’

MARIO RIVERA,* WEI ZHENG,~ H. VASKEN APOSHIAN,$ AND QUINTUS *Department

of Chemistry,

VDepartment Cellular Biology,

Received

of Pharmacology and Toxicology, and *Department University ofArizona, Tucson, Arizona 85721

December

21,1988;

accepted

April

FERNANDO* of Molecular

and

22, 1989

Determination and Metabolism of Dithiol Chelating Agents. VIII. Metal Complexes of mesoDimercaptosuccinic Acid. RIVERA, M., ZHENG, W., APOSHIAN, H. V., AND FERNANDO, Q. ( 1989). Toxicol. Appl. Pharmacol. 100,96- 106. Metal complexes of meso-dimercaptosuccinic acid (DMSA) with Pb2+, Cd2+, and Hg2+ were studied by potentiometric and infrared methods. This dimercapto metal-binding agent was found to form complexes whose structures are dependent on the metal ion to be complexed. In the cases of Pb2+ and Cd’+, one oxygen and one sulfur act as the donor atoms; in the case of Hg*+, two sulfur atoms act as the donors. The solubilities of all metal chelates were found to be pH dependent. Complexes of cadmium and lead are insoluble in the pH range 1.O to 7.1, but are solubilized when the noncoordinated sulfhydryl and carboxylic acid groups are ionized. The mercury complex is insoluble in the pH range 1.O to 3.0. It dissolves when one of the noncoordinated carboxylic acid groups is ionized. The dimethyl ester of meso-DMSA (DiMe-meso-DMSA) was synthesized and its acid dissociation constants were determined (pK, = 6.38 and pK2 = 8.00). Esterification of the carboxyl groups of meso-DMSA changes its coordination properties in that the two sulfur atoms of DiMe-mesoDMSA are used to coordinate with Hg 2+, Cd2+ , or Pb2+. Esterification of meso-DMSA also changes its biological properties. DiMe-meso-DMSA, when given to rats 3 days after Cd administration, greatly increased the excretion of Cd via bile. In contrast, meso-DMSA was devoid of such activity. 0 1989 Academic Press, Inc.

meso-Dimercaptosuccinic acid (DMSA) is an effective metal-binding agent that has been used successfully to increase the urinary excretion of lead in children (Graziano et al., 1988) and adults (Friedheim et al., 1978; Graziano et al., 1985). Because of its potential for becoming the drug of choice for deleading children with elevated levels of lead in their blood, it has been given orphan drug classification by the Food and Drug Administration. Although the biological properties of this dimercapto metal-binding agent have re-

ceived increasing attention (see reviews by Aposhian, 1983; Aaseth, 1983) very little is known about its physicochemical properties. Lenz and Martell ( 1965) determined equilibrium constants for soluble metal chelates of meso-DMSA and interpreted them in terms of the probable structures of the complexes. Whether metal ions such as Pb*+, Hg*+, or Cd*+ coordinate with the two sulfur atoms or with a sulfur atom and an oxygen atom of DMSA has not been clarified. This paper presents infrared and potentiometric evidence for the following: First, the coordination sites in meso-DMSA for Pb*+, Cd*+, or Hg*+ are oxygen and/or sulfur atoms. Second, the coordination sites depend

’ This research was supported in part by NIEHS Grant ES-03356 and NC1 Grant 49252.

0041-008X/89

$3.00

Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

96

meso-DIMERCAPTOSUCCINIC

on the type of metal ion that is to be coordinated. Third, when the carboxylic groups of meso-DMSA are esterified with methanol, the two sulfur atoms become the coordination sites, and the coordination sites are independent of the coordinated metal ion. Finally, esterification not only changes the coordination sites, but the resulting dimethyl ester, DiMe-meso-DMSA, increases the biliary excretion of Cd, a property consistent with intracellular distribution and markedly different from that of meso-DMSA.

METHODS Potenliometrv Potentiometric measurements of hydrogen ion concentration were performed in a 150-m] titration vessel provided with a magnetic stirrer and a tightly fitting rubber stopper. The latter was equipped with inlet and outlet tubes for nitrogen. a buret for delivery of NaOH, and a combination glass-saturated calomel electrode. To an aqueous suspension of meso-DMSA. sufficient NaOH was added to form the species HZD’- (where H4D represents meso-DMSA). A solution of the metal nitrate or chloride of known concentration was added slowly to give a 1:1 mole ratio of metal to H2D’-. The liberated hydrogen ions were monitored potentiometrically by the addition of a standard solution of NaOH. The hydrogen ion:DMSA mole ratio was plotted against the measured pH. All potentiometric studies were carried out in a nitrogen atmosphere to prevent oxidation of the thiol groups to disulfides.

Synthesis oj’the DimethJ,l Ester of meso-DMSA A suspension of meso-DMSA in I M methanolic HCl was refluxed for 1 hr and slowly cooled under nitrogen to ambient temperature. The methanol was evaporated to one-third of its original volume and the resulting solution chilled to 0°C. A white precipitate formed almost immediately. The solution was maintained at O’C for 2 hr. The precipitate was filtered, dried, and recrystallized from chloroform. The NMR spectrum of the ester was determined with a Bruker WM-250 spectrometer using CDCll as solvent. The infrared spectrum of the compound in KBr was determined with a Perkin-Elmer 987 spectrophotometer.

ACID-METAL

COMPLEXES

97

Determination of Acid Dissociation Constants of DiMemeso-DMSA The pK, values of the two sulfiydryl groups were determined potentiometrically with the aid of a glass-saturated calomel electrode. Since DiMe-meso-DMSA is not water soluble, a 50% (v/v) solution of water-methanol was used to minimize the hydrolysis of the ester. The glass electrode was calibrated in the 50% water-methanol mixture since the dielectric constant of a mixture of 50% water-methanol is different from that of water. This was accomplished by the potentiometric titration of a standard solution of perchloric acid with a standard solution of NaOH in a 50% (v/v) water-methanol mixture at a constant ionic strength of 0.1. The measured pH of the solvent mixture was lower by 0.30 than the pH calculated from the hydrogen ion concentration at every point in the titration. A weighed amount of DiMe-meso-DMSA was dissolved in methanol in the vessel that was used for the potentiometric titrations. When the ester was dissolved, an equal volume of water was added together with sufficient sodium perchlorate to maintain the ionic strength at 0.1. The titration vessel was then placed at 25°C in a constant-temperature water bath and the potentiometric titration performed with a solution of carbon dioxidefree NaOH. For every volume increment of NaOH solution added. the same volume of methanol was added to maintain a 50% (v/v) aqueous methanol solution throughout the titration. Syntheses ql‘Meta1 Compkxes sf meso-DMSA Syntheses were carried out in a Schlenck line under nitrogen to prevent oxidation of the mercapto groups to disulfides. All solvents were freed from oxygen by freezethaw cycles in which the solvent was solidified by cooling with liquid nitrogen followed by evacuation of the flask: the flask was then filled with nitrogen gas. The cycle was repeated three times. All solvents were transferred from one flask to another with the aid ofa cannula to minimize contact with oxygen in the air. C4H40,S2Pb.2H,0. To a solution of 5.28 mmol ot the disodium salt of meso-DMSA in 30 ml of water, a stoichiometric amount of lead nitrate in 25 ml of water was added dropwise from an addition funnel. A yellou precipitate formed almost immediately. Once the addition of lead nitrate was completed, the suspension was stirred for 30 min, filtered under nitrogen, and washed with water (15 ml) and with methanol (15 ml). The compound was filtered and dried in vacua. The dried compound is stable in air. The yellow compound obtained was suspended in dimethylsulfoxide (DMSO) to dissolve any unreacted DMSA, filtered, and washed with water and then with methanol: the last traces of solvent were

RIVERA

98

removed with a vacuum pump. Pb found, 49.06%; Pb calculated, 48.95%. C,H,O&Cd. 2H2 0. The synthesis was carried out in a manner similar to that described for the Pb2+ analog. Cadmium nitrate was used as a source of Cd*+. The cadmium-DMSA complex was obtained as a white solid. The dried white compound is not air sensitive. Cd found, 34.05%; Cd calculated, 34.22%. C4H404S2Hg.2H,0. meso-DMSA (10 mmol) was placed in a Schlenk flask filled with nitrogen. A solution of 20 mmol of triethylamine in 35 ml of methanol was added with the aid of a cannula into the flask containing meso-DMSA to solubilize it as an ion pair with triethylammonium ion. A solution of HgC12 (10 mmol in 30 ml of methanol) was then added dropwise. The white precipitate that formed initially completely dissolved in about 15 min. Addition of about 60 ml of water to the solution resulted in precipitation of a white solid, which was filtered, washed with water, and dried in vacua. Hg found, 48.29%; Hg calculated, 48.13%. Infrared Spectra Infrared spectra of all compounds were obtained with a Perkin-Elmer 983 spectrophotometer. All compounds were suspended in KBr and pressed to form pellets. Animals Male Sprague-Dawley rats (150 g) were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). They were kept in a temperature-controlled facility, with a 12-hr light/dark cycle, and fed ad libitum Teklad rat diet purchased from Teklad (Madison, WI). Rats were allowed to acclimate for 1 week after arrival. Administration

ET AL. from the pancreas. During the experiment, rats were infused with saline via the jugular vein at a rate of 1.5 ml/ hr. After a steady bile flow rate was established, bile samples were collected at 30-min intervals for 6.5 hr. Rats were kept on an electric heating pad so that their core temperature was maintained at 36 + 0.5”C during bile collection. The radioactivity of the bile samples was counted in a LKB type- 1282 CompuGamma Counter.

Assay for Biliary GSH and Dithiols Bile samples were collected at 10-min intervals directly into a vial containing 0.1 ml of 80 mrvt monobromobimane (mBBr) and 1.8 ml of 0.1 M ammonium bicarbonate buffer (pH 8.4) to derivatize the thiol groups and to prevent the spontaneous oxidation of GSH to GSSG. Vials were wrapped with aluminum foil to avoid exposure to light. After collection, the volume of each bile sample was measured and the samples were extracted with 4 ml of methylene chloride to remove excess mBBr. The aqueous phase was then diluted lo-fold and examined by HPLC as described below.

HPLC Analysis HPLC analysis of dithiol-BB derivatives was performed using the method of Maiorino et al. (1986). Samples were fractioned on a 250 X 4.6-mm Ultrasphere IP C- I8 reversed-phase column, using a Beckman Model 157 fluorescence detector with 356-nm excitation and 450 f 20-nm emission filters. Separations were performed at room temperature and at a rate of 1 ml/min. The mobile-phase gradient system was described in the procedure reported by Maiorino and Aposhian (1989).

of Chelating Agents

RESULTS The compounds that were injected intravenously were dissolved in 80% ethanol and administered via the jugular vein at a rate of 0.1 ml/min. Biliary Excretion of Cadmium Rats (200-220 g) were injected intraperitoneally with 1 mg Cd (3.08 X 10’ cpm of ‘09Cd)/kg. Three days later, the rats were anesthetized with urethane 1.Og/kg, ip. The bile duct and jugular vein were cannulated with PE-50 polyethylene tubing (Clay Adams). The tubing was inserted into the bile duct, close to the side where the duct joins the duodenum and deeply enough (about 1- 1.5 cm) to avoid contamination with any exocrine excretion

Coordination of meso-DMSA to HP Two Sulfur Atoms

via

When an aqueous suspension of mesoDMSA (H,D) is titrated with NaOH, complete solubilization of the ligand occurs after 1 mol of NaOH has been added. The ionic species H3D- is formed. When a second mole of NaOH is added to the solution of the ampholyte H3D-, the species H@- is formed. The pH of the resulting solution is in the rap-

meso-DIMERCAPTOSUCCINIC

ACID-METAL

99

COMPLEXES

12;00-

9.00

-

‘

O.OOobo! mole H*/ mole

Lipand

FIG. I. Potentiometric titration of (a) I mmol DiMemeso-DMSA + 1 mmol HgCL, (b) 1 mmol mesoDMSA, and (c) I mmol meso-DMSA + 1 mmol HgCIZ.

idly rising portion of the titration curve (curves b. Figs. l-3). When an equimolar amount of Hg’+ is added to this solution, the pH drops to 3.2 (curve c, Fig. 1). This pH drop occurs because Hg”- is complexed, and as a result, protons are released. An insoluble white precipitate is formed. If the two sulfur atoms are coordinated to the Hg2+ ion, the liberated protons would be expected to protonate the carboxylic acid groups since the equilibrium pH of

1.00 mole H’ i mole

I

2.00

3.00

Ligond

FIG. 3. Potentiometric titration of (a) 1 mmol DiMemeso-DMSA + 1 mmol Cd(NO+, (b) I mmol mesoDMSA. and (c) I mmol mes+DMSA + I mmol Cd(NO,)z

the suspension is 3.2. If this were the case, continued addition of two additional moles of NaOH should give a result similar to the result obtained when a suspension of the ligand alone is titrated (curve b, Fig. 1). This is indeed what occurs, and is strong evidence that the two sulfur atoms are coordinated to the Hg2+ ion. Coordination of mesa-DMSA to Pb’+ or Cd’+ via One Oxygen Atom and One Su& Atom

mole

W’

l mole

Ligwid

FIG. 2. Potentiometric titration of (a) 1 mmol DiMemes+DMSA + 1 mmol Pb(NO&, (b) I mmol mesoDMSA, and (c) I mmol meso-DMSA + 1 mmol Pb(NO&.

Addition of an equimolar concentration of Pb2+ or Cd2+ to a solution of the species HzD2- results in completely different behavior upon titration with NaOH. When 1 mole of NaOH is added to a solution containing a suspension of 1 mole of the solid yellow complex of Pb”+ with DMSA, the pH of the solution, which is in equilibrium with the solid, continues to increase from 2.4 to 6.2 (curve c, Fig. 2). In this pH region, one carboxylic acid group in the ligand is neutralized, identical behavior is observed with the Pb-DMSA complex. Upon addition of a second mole of NaOH, the pH of the solution increases from

100

RIVERA

6.2 to 10.9 (curve c, Fig. 2). It is unlikely that a carboxylic hydrogen is neutralized in this pH region; a sullhydryl hydrogen in the PbDMSA complex is neutralized between pH 6.2 and 9.2. This is strong evidence that meso-DMSA is coordinated to Pb2+ via one sulfur atom and one oxygen atom. When 1 mol of NaOH is added to a solution containing a suspension of 1 mol of the solid white complex of Cd2+ with DMSA (curve c, Fig. 3), the pH increases from 2.3 to 5.5, as in the case of its lead analog. Addition of a second mole of NaOH causes the pH to increase from 5.5 to 10.8. Hence, it may be concluded that mesu-DMSA is coordinated to Cd2+ via one sulfur atom and one oxygen atom, as in the lead complex.

ET AL.

b/

C

/

Coordination of DiMe-meso-DMSA to H$+, Pb2+, or Cd2+ via Two Surfur Atoms When a solution of Hg2+, Pb2+, or Cd2+ in a 50% water-methanol mixture is added in equimolar amounts to a solution of DiMemeso-DMSA, the pH drops to 1.6 (curves a, Figs. l-3). In the cases of Hg2+ and Cd*+ a white precipitate is formed; a yellow precipitate is formed in the case of Pb2+. The DiMemeso-DMSA complexes have limited solubility over the pH range 2- 12. When a solution containing a suspension of the Hg2+, Cd2+, or Pb*+ complex of DiMe-meso-DMSA is titrated potentiometrically, 2 mol of NaOH are necessary per mole of metal complex to raise the pH from 1.6 to 10.7. This behavior demonstrates that DiMe-meso-DMSA coordinates to Hg2+, Pb2+, and Cd*+ ions by using two sulfur atoms as the donors. In the infrared spectrum of meso-DMSA (Fig. 4a). The peak at 17 15 cm-’ corresponds to the C=O stretching of a protonated carboxylic group. In the infrared spectra of the Pb*+ and Cd2+ complexes of meso-DMSA (Figs. 4b and 4c), the peak at 1700 cm-’ corresponds to the C=O stretching frequency of a protonated carboxylic group. In addition to

4ooo

3ooo

zoo0

mm

lM0

800

400

FIG.4. Infrared spectra of (a) meso-DMSA, (b) mesoDMSA-PbZf complex, (c) meso-DMSA-Cd*+ complex, and (d) meso-DMSA-Hg’+ complex.

this peak, two bands are found that correspond to asymmetric and symmetric stretching frequencies at 1537 and 1366 cm-r, respectively. These correspond to a coordinated carboxylate group. In contrast, the infrared spectrum of the Hg*+ complex (Fig. 4d) shows only the band that corresponds to the protonated carboxylic acid at 1692 cm-‘. These results corroborate the results obtained in the potentiometric studies.

~WW-DIMERCAPTOSUCCINIC

ACID-METAL 12.00

101

COMPLEXES

1 . .

9.00

,,H

-

3000

2000

1600

1200

600

6.00.

/**

400 Volume

FIG. 5. Infrared spectrum of DiMe-meso-DMSA.

of

N-OH

Id)

FIG. 7. Potentiometric titration of 0.01089 meso-DMSA with 0.1463 M NaOH.

Dimethyl

l

J

-

4000

.

M

DiMe-

Ester of meso-DMSA

The compound obtained by the esterification of methanol with meso-DMSA was analyzed by NMR and by IR spectroscopy. The absence of bands in the IR spectra of the ester (Fig. 5) that correspond to the O-H stretching frequency of the carboxylic acid group indicates that the ester is not contaminated with unreacted DMSA. The NMR spectrum of the ester is shown in Fig. 6. Methyl protons show a singlet at d = 3.80, methine protons show a

1

quartet at 6 = 3.65, and S-H protons show the same pattern at 6 = 2.24. The multiplicity of these signals arises from the coupling of methine and sullhydryl protons. Acid Dissociation DMSA

Constants (11DiMe-meso-

The titration curve for DiMe-meso-DMSA was obtained in a 50% (v/v) water-methanol mixture (Fig. 7). Since titration of two sulfhydry1 protons results in two distinct buffer regions, the acid dissociation constants of the sulfhydryl groups were calculated independently of each other: pK, was found to be 6.38 and pK;, 8.00. The basis for these calculations follows. The dissociation of DiMe-meso-DMSA can be represented as A-

H2Y 2 HY- + H+ ii. HY- 2 y’- + H+ where H?Y represents DiMe-meso-DMSA. J 6

5

4

3

2

FIG. 6. NMR spectrum of DiMe-meso-DMSA terochloroform, referenced to TMS.

1

in deu-

K = [H+I[HY-1 aI WhYI

(‘1

k’ = [H+lW’l a? [HY-]

(2)

102

RIVERA

O-O.5

0.5-l.Al.-l.5

1.5-2.

ET AL.

2.-3.

3.-4.*4.-4.5

4.5-5.55.5-6.5

HOUR FIG. 8. DiMe-mesc-DMSA increases biliary excretion of cadmium-109. Rats were injected ip with 1 mg Cd (4.2 X 10’ cpm of “‘Cd)/kg. Three days later, the bile duct and jugular vein were cannulated. DiMemeso-DMSA and meso-DMSA were dissolved in 80% ethanol. Chelating agents (0.10 mmol/kg) or 80% ethanol (control) was administered via the jugular vein as indicated by the arrows.

The following equation was used to determine K,, and K,, from the potentiometric titration data: 4, or K,, “’

IHfl i (V, + v) =

(hca (Vo

-

vcb)

+

v

+ [H+] - [OH-] I _

[Hf]

+

[OH-]

(3)

where all concentration terms are expressed in moles per liter, C, is the analytical concentration of the acid (DiMe-meso-DMSA), V. is the initial concentration of acid, V is the volume of base added, and Cb is the concentration of base. The acid dissociation constants were obtained from Eq. (3) by a linear least-squares method. DiMe-meso-DMSA tion of Cadmium

Increases Biliary

Excre-

DiMe-meso-DMSA, when given iv to rats that had received ‘09Cd 3 days previously, increased the biliary excretion of lo9Cd compared with DMSA and the controls (Fig. 8).

Within 30 min of DiMe-DMSA administration, biliary excretion of Cd increased 54fold. A second injection of DiMe-DMSA 3 hr after the first elicited a 49-fold increase in biliary excretion within 30 min. DMSA did not influence the biliary excretion of Cd, confirming previous results (Zheng et al., unpublished). The action of DiMe-meso-DMSA in increasing the biliary excretion of Cd was not due to an increase in bile flow rate (Table 1). Biliary GSH Is Not Increased by DiMe-mesoDMSA Bile samples from a rat given DiMe-mesoDMSA (0.20 mmol/kg iv) were analyzed by HPLC and the results compared with the HPLC profile of bile from a control rat that had received only alcohol. No increase in biliary GSH was found (Fig. 9). Is DiMe-meso-DMSA

Excreted in the Bile?

Dithiols are labile compounds. They are easily oxidized to form disulfides with them-

meso-DIMERCAPTOSUCCINIC

ACID-METAL

103

COMPLEXES

TABLE 1 INCREASEIN THE BILIARY EXCRETION OF Cd BY DiMe-meso-DMSA Is NOT DUE TO INCREASEIN THE RATE OF BILE FLOW’ Response From 30 min before to time of injection Bile flow rate (ml/30 min) Biliarv Cd (cpmlml/30 mitt)

0.314

From time of injection to 30 min after

'_ 0.067

% Increase

0.347 kO.076 8788+1054

324kSl

10 2612

a Data represent F + SE. Experimental conditions as described in the legend to Fig. 8, except that 1 mg Cd (3.08 X 10’ cpm of ‘@‘Cd)/kg was injected ip.

selves or mixed disulfides with other thiol compounds such as GSH or proteins. For example, meso-DMSA, when given po to humans, is biotransformed to a mixed disulfide in which two molecules of cysteine are individually linked, by disulfide bonding, to the sulfur atoms of meso-DMSA (Maiorino et al., 1989). After injection of DiMe-meso-DMSA iv, the bile was examined using bromobimane derivatization, HPLC, and fluorescence detection. Unchanged or unaltered DiMe-meso-DMSA was found in the bile within 30 min of injection (Fig. 10). The peak concentration occurred within 15-30 min. In

previous experiments, neither unaltered nor altered mesu-DMSA was detected in the bile after administration of meso-DMSA. Unaltered meso-DMSA in extremely small amounts, however, appeared in the bile within 30 min of administration of DiMemeso-DMSA (Fig. 10). DISCUSSION Valuable qualitative information can be obtained if potentiometric titrations are performed on a system containing a suspended precipitate in equilibrium with the solution. The results of the experiments presented in this paper demonstrate the following: When solutions containing equimolar amounts of H2D-* and Hg(I1) are mixed, the compound that precipitates is the neutral species HgH2D: 0

0

II

II

-0-C-CH-CH-C-O-+Hg*+ I I SH SH .

IIIN

FIG. 9. DiMe-meso-DMSA does not increase biliary GSH. Rats (200-220 g, n = 3) were anesthetized ip with I .O g urethane/kg. DiMe-mesuDMSA (0.20 mmol/kg) or 80% ethanol (equal volume) was injected via the jugular vein at zero time. The bile sample was collected directly into a bromobimane-containing solution.

+

0

0

II

II

HO-C-,CH-CJ-C-OH S

(4)

S

’

Hg ’

In this neutral complex, HgH2D, the carboxylate groups are protonated in the presence of

104

RIVERA

ET AL.

protons that are released upon complex formation [Eq. (4)]. Addition of a solution of NaOH to an aqueous suspension of HgH2D neutralizes the carboxylic acid groups in the pH region 3.2-l 1.5 (curve c, Fig. 1): 0 0 II II HO-C-CH-CH-C-OH+OH/ \ S

Addition of a solution of NaOH to an aqueous suspension of the species PbHzD or CdH2D neutralizes a carboxylate group in the pH region 2.4 to 6.2 (curves c, Figs. 2 and 3) with generation of the insoluble species PbHD- or CdHD-: 0

e

0 B

II

C-CH-CH-C-OH+OH-

/

S

0

’ Hg ’ 0

0

II

II

\

HO-C-,$H-C\H-C-0-+H,O

SH

\M/’ 0

(5)

)_

I

0

II

\

C-CC\H-y-C-0-+H,O

(8)

/

S

0

S ’ Hg ’

0

0

II

II

HO-C-,CH-C\H-C-0-+OHs

__

s

0

0

II

II

-O-C-,y--C\H-C-0-+H,O

0

(6)

S S ’ Hg ’

When solutions containing equimolar amounts of H2D2- and Pb(I1) or [Cd(II)] are mixed, the compound that precipitates is the neutral species PbHzD [or CdH2D]. In the presence of protons released upon complex formation, one carboxylic group and one sullhydryl group are protonated: 0

II

-CH-C-0-+M*+ I I SH SH 0

-3 0

\

C-Ct-+-OH

(7)

/ 0

\M/’

SH

Continued addition of NaOH to the aqueous suspension of PbHD- or CdHD- neutralizes a sulfhydryl group in the pH region 6.2 to 11.5 (curves c, Figs. 2 and 3), with formation of the soluble compound PbDv2 or CdD’-:

’ Hg /

-O-i-,,

S ‘M’

SH

M = Pb” or Cd*+

0

II

\

C-CH-CH-C-0-+OH1 \ I 0 S SH ‘M’ 0 0 \ C-CF-YH-!-0-+H,O

__

(9)

/ 0

slMIS

When solutions containing equimolar amounts of DiMe-meso-DMSA (H,Es) and Hg(II), Pb(II), or Cd(I1) are mixed, the compound that precipitates is MEs, where M rep resents Hg(II), Pb(II), and Cd(U). The protons released upon complex formation are neutralized by the addition of a solution of NaOH to the aqueous suspension of MEs (Eq. 10). This occurs between pH 1.5 and 11.5 (curves a, Figs. l-3). Thus, it is not unreasonable to predict that the chelate structures of meso-DMSA are those shown in Fig. 11.

meso-DIMERCAPTOSUCCINIC 0 II C-OMe+M’+

MeO-C-CH-CH I SH

H

H

H

H

I

I

I

fl

O

o=c-c-c-CZEO

I

__

105

COMPLEXES

o=c-c-c-c=0 I s H

~0

I SH

I s H

I o-

I ;/” Cd

0 II MeO-C-CH-CH-C-OMe+2H’ / \ s S ‘M’

0

H

II

+

H

o=c-c-c-c=0

H

H

I s

I s

o=c-c-c-c=0

I

I

v

I

I

“H

O-

I .o

\

Pb

(10) 2H++20H-

ACID-METAL

FIG. Il. Structures of meso-DMSA-metal

! 0

/ w

chelates.

2H,O

M2+ = Pb Cd or Hg

When given iv to rats, DiMe-meso-DMSA greatly increased the elimination of Cd via bile from the liver (Fig. S), the organ in which the greatest amount of Cd deposited is found (Lucis el al., 1969). This action is not due to DiMe-meso-DMSA’s increasing the rate of bile flow (Table 1). Neither is it related to a change in biliary glutathione content (Fig. 9). Since it is found in the bile after its iv ad-

‘T

3-

-I

MIN

FIG. 10. DiMe-meso-DMSA and meso-DMSA are found in the bile after DiMe-meso-DMSA administration. DiMe-meso-DMSA (0.20 mmol/kg) was administered via the jugular vein at zero time. See legend to Fig. 9 for methods.

ministration, DiMe-meso-DMSA must enter the hepatocytes. This property of entering cells may be one reason that it can mobilize Cd deposited in liver. On the other hand. meso-DMSA has an extracellular distribution, does not enter hepatocytes and does not increase the biliary excretion of cadmium (Zheng et al., 1989). After administration of DiMe-meso-DMSA, a small amount of meso-DMSA was found in the bile (Fig. 10). Previous work, however unequivocally demonstrated that meso-DMSA, when given iv. is not found in the bile (Zheng et al., 1989), indicating that DiMe-meso-DMSA undergoes biotransformation to some extent in hepatocytes or bile canaliculi. meso-DMSA is emerging as the drug of choice for treatment of lead intoxication (Graziano et al., 1985, 1988), because of its low toxicity (Aposhian et al., 1983). oral usefulness, and chelation activity. Its low toxicity is due in part to its inability to enter the cell. DiMe-meso-DMSA enters the cell. The structures proposed for the metal chelates of meso-DMSA and supported by the evidence in this paper are shown in Fig. 11. It should be noted that a structure for a HgDMSA chelate different from that for a PbDMSA chelate has not been proposed previously. It has always been assumed that dimercapto chelates were formed by coordination with two sulfur atoms. Our groups are in the midst of collaborative efforts to determine

106

RIVERA

whether the structures of the chemically synthesized chelates (Fig. 11) are the same as those found in, for example, the urine of rabbits given Hg or Pb followed by meso-DMSA treatment. ACKNOWLEDGMENT We thank Johnson and Johnson Baby Products, Inc., for providing us with meso-DMSA.

REFERENCES AASETH, J. (1983). Recent advances in the therapy of metal poisonings with chelating agents. Hum. Toxicol. 2,257-272. APOSHIAN, H. V. (1983). DMSA and DMPS-Water soluble antidotes for heavy metal poisoning. Annu. Rev. Pharmacol. Toxicol. 23,193-2 15. APOSHIAN, H. V., Hsu, C., AND HOOVER, T. D. (1983). DL- and meso-dimercaptosuccinic acid: In vitro and in vivo studies with sodium arsenite. Toxicol. Appl. Pharmacol.

69,206-2

I 3.

FRIEDHEIM, E., GFUZIANO, J. H., POPOVAC, D., AND KAuL, B. (1978). Treatment of lead poisoning by 2,3dimercaptosuccinic acid. Lancet 2, 1234- 1236.

ET AL. GRAZIANO, J. H.. L~LACONO, N. J., AND MEYER, P. A. (1988). A dose-response study of oral 2,3-dimercaptosuccinic acid (DMSA) in children with elevated blood lead concentrations. J. Pediatr. 113,751-757. GRAZIANO, J. H., SIRIS, E. S., LOLACONO, N., SILVERBERG, S. J., AND TURGEON, L. (1985). 2,3-Dimercaptosuccinic acid as an antidote for lead intoxication. Clin. Pharmacol. Ther. 37,43 l-438. LENZ, G. R., AND MARTELL, A. E. (1965). Metal chelates of mercaptosuccinic and cu,a’-dimercaptosuccinic acids. Inorg. Chem. 4,378-384. Lucrs, 0. J., LYNK, M. E., AND Lucrs, R. (1969). Tumover of cadmium 109 in rats. Arch. Environ. Health l&307-3 10. MAIORINO, R. M., AND AFQSHIAN, H. V. (1989). Determination and metabolism of dithiol chelating agents: IV. Urinary excretion of meso-2,3-dimercaptosuccinic acid and mercaptosuccinic acid in rabbits given meso2,3-dimercaptosuccinic acid. Biochem. Pharmacol. 38,1147-1154. MAIORINO. R. M., BRUCE, D. C., AND APOSHIAN, H. V. (1989). Determination and metabolism of dithiol chelating agents. VI. Isolation and identification of the mixed disulfides of meso-2,3-dimercaptosuccinic acid with L-cysteine in human urine. Toxicol. Appl. Pharmacol.

91,338-349.

MAIORINO, R. M., WEBER, G. L., APOSHIAN, H. V. (1986). Fhtorometric determination of 2,3-dimercaptopropane- 1-suIfonic acid and other dithiols by precolumn derivatization with bromobimane and column liquid chromatography. J. Chromatogr. 374,297-3 IO.