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Determination and Metabolism of Dithiol Chelating Agents: The Zinc Chelate of the Dimethyl Ester of meso-2,3-DimercaptosuccinicAcid Increases Biliary Excretion of Cadmium and Platinum+ H. Vasken Aposhian,*J Deborah J. Levine,$ Mario Rivemil and Quintus Fernandoll University Department of Molecular and Cellular Biology, Department of Pharmacology and Toxicology, and Department of Chemistry, University of Arizona, Tucson, Arizona 85721 Received October 12, 1992
meso-2,3-Dimercaptosuccinic acid is an orally active chelating agent useful for the treatment of lead intoxication. Since it is believed to be extracellular in its distribution, analogs have been synthesized in a search for one that will enter the cell and successfully compete for firmly bound intracellular toxic heavy metals such as cadmium and platinum. The biological properties of the zinc chelate of DiMeDMSA, ( [Zn(DiMeDMSA)2I2-with tetramethylammonium or sodium as the counterion, have been investigated in the rat. In short-term (hourly) experiments, [Zn(DiMeDMSA)#- increased the biliary excretion of cadmium and platinum. In experiments of longer duration (days), severe renal toxicity was noted even in the absence of any exogenously administered cadmium chloride or cis-dichlorodia“ineplatinum(I1). The zinc content of the kidneys of rats receiving N ~ ~ [ Z I I ( D ~ M ~ D M was S A )found ~ I to be about 10-fold greater than rats receiving saline or meso-dimethyldimercaptosuccinicacid. Although it appears that the source of the zinc is the injected [Zn(DiMeDMSA)2I2-,at this time it is unknown as to whether the toxicity is due t o the Zn chelate molecule, per se, or Zn derived from the molecule after its degradative biotransformation. Introduction Cadmium is excreted very slowly from the body. Its biological half-lives in human muscles, kidney cortex, and livers have been estimated to be 10-30 years (1). Chronic exposure to it in humans occurs primarily in industrial settings and may result in lung and renal damage (2,3). Unfortunately, at the present time, there is not for humans an acceptable antidote for the renal dysfunction caused by chronic Cd exposure (4). Renal toxicity is seen also with the use of cis-dichlorodiammineplatinum(I1) (CDDP),l an effective agent for the treatment of a wide variety of human tumors including head, neck, and testicular carcinomas (5). Although mannitol diuresis coupled with iv prehydration has been utilized to minimize or decrease this acute nephrotoxicity (6),the cumulative renal toxicity remains the dose-limiting factor for CDDP. A chelating agent that would remove the platinum from the kidney without affecting the antitumor activity of CDDP might be of therapeutic interest. Inhibition of CDDP nephrotoxicity by diethyldithiocarbamate was reported in a rat model by Borch and Pleasants (7). The synthesis and, in experimental animals, the evaluation of many dithiocarbamate analogs ‘This paper is the 14th in a series entitled “Determination and Metabolism of Dithiol Chelating Agents”. * To whom correspondence should be addressed a t the University Department of Molecular and Cellular Biology, Life Sciences South Building, Room 440, University of Arizona, Tucson, AZ 85721. 3 University Department of Molecular and Cellular Biology. Department of Pharmacology and Toxicology. 1; Department of Chemistry. I Abbreviations: CDDP, cis-dichlorodiammineplatinum(I1); DMSA, meso-2,3-dimercaptosuccinicacid; BAL, British antilewisite or 2,3dimercapto-l-propanol; DiMeDMSA, dimethyl ester of meso-DMSA; [Zn(DiMeDMSA)2]2-,the zinc chelate of DiMeDMSA; DMPS, Na salt of 2,3-dimercapto-l-propanesulfonicacid.
as CDDP antidotes by Jones and his associates (8,9)have been promising. Effective treatment for intoxication by some heavy metals depends upon the accessibility of the chelating agent to the intracellular sites where the heavy metal ion is deposited and bound. For example, Pt and Cdare bound to intracellular metallothionein (10, 11). To mobilize such intracellular, firmly-bound metals, an ideal chelating agent should readily enter the cell and effectively compete with metallothionein for the bound metal. The situation with Cd and Pt is further complicated by each one inducing the synthesis (11,121 and the latter inducing overexpression of metallothionein (13). The interactions of chelating agents with Cd and metallothionein have been discussed in an excellent review by Jones and Cherian (4). Several water-soluble chelating agents, effective as antidotes for other metals, have been ineffectivein treating chronic Cd or Pt intoxication, perhaps because they do not readily enter the cell. For example, meso-dimercaptosuccinic acid (DMSA), an effective antidote for lead toxicity in humans (14, 15), is relatively ineffective in reversing the nephrotoxicity caused by CDDP or chronic Cd exposure (16-18). DMSA apparently does not enter cells (19). A t the pH of the body, its two carboxyl groups are ionized (20, 21). Such a highly charged molecule is limited in entering the cell and mobilizing metals that are firmly bound to intracellular ligands. Compounds which appear to be effectivein experimental animals for chelating and mobilizing intracellularly-bound cadmium are not highly charged and have some degree of lipophilicity. Examples of these are British antilewisite (2,3-dimercapto-l-propanol, BAL) (221, dithiocarbamates (23,24),N-benzyl-D-glutamine dithiocarbamate (25),and several esters of DMSA (20,21,26-28) (for a review see refs 4 and 29). But some of the dithiocarbamates (i.e.,
0893-228x/93/2706-0208$04.00/00 1993 American Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 209
DiMeDMSA Zinc Chelate 2-
2 [XI
X=Na+ or X= (Me)4N+
Figure 1. The Zn chelate of DiMeDMSA: Na+ or (MehN+ counterion.
ethyl- and methyldithiocarbamates) cause redistribution of Cd, resulting in increased concentrations of Cd in the brain (23). BAL, which has many adverse side effects, also increases the Cd concentration in the kidney. Many of these agents, therefore, are only of limited or questionable use for treatment of chronic Cd intoxication. Another goal in the design of a chelating agent for treating Cd or CDDP toxicity might be the diversion of the metal to an excretory route less vulnerable than the kidney. Biliary excretion is such a route and is known to be of importance for the elimination of many metals (30). A chelating agent that might divert Cd or Pt from the kidney to the bile, thus encouraging its excretion in the feces, might be useful therapeutically. The dimethyl ester of DMSA (DiMeDMSA) when given to rats increased the biliary excretion of deposited cadmium (20). This indicated that it entered liver cells. The synthesis of the Zn chelate of DiMeDMSA ([Zn(DiMeDMSA)zlZ-)has been reported recently (31). In this chemical analog of meso-DMSA, one atom of Zn is chelated with two molecules of DiMeDMSA (Figure 1). Because of the advantages of CaNazEDTA over EDTA (32),the initial aim of the present research was to examine some of the biological properties of the zinc chelate of DiMeDMSA, [Zn(DiMeDMSA)z]2-,in particular whether it would increase the biliary excretion of Cd and Pt. This paper is a sequel to the one (31)describing the chemical synthesis of this novel analog of DMSA.
Experimental Section Chemicals. Cadmium chloride was purchased from J. T. Baker (Phillipsburg, NJ), and cadmium-109 was purchased from DuPont (Claremont, CA). Urethane and cis-platinum(I1) diammine dichloride were purchased from Sigma Chemical Co. (St. Louis, MO). meso-DMSA was a gift from Johnson and Johnson Baby Products (Skillman, NJ). DiMeDMSA and its zinc chelate were synthesized by the method of Rivera and Fernando (31). All reagents used in these experiments were of analytical grade, HPLC grade, or the best available pharmaceutical grade. Animals. Male Sprague-Dawley rats (150-170 g) and male Swiss-Webster mice (18-22 g) were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). The animals, after arrival, were allowed to acclimate for at least 7 days in a temperature-controlled facility, with a 12-h light/dark cycle. Teklad Rat Blox purchased from Teklad (Madison, WI) and water were available ad libitum. Chelating Agents. [(Me)4Nlz[Zn(DiMeDMSA)~l (31)in solid form was dissolved in 0.9% saline and injected iv into rats a t a dose of 0.01 mmolikg body wt and a t a rate of 0.1 mL/min. For some experiments, the mononuclear complex [ZnLJ- was synthesized as an ion association complex with sodium instead
of tetramethylammonium to investigate whether or not the counterion was causing toxicity. [Zn(DiMeDMSA)zlZ-with sodium as the counterion was not available for use in solid form. Naz[Zn(DiMeDMSA)z] was prepared immediately before use by dissolving 2.0 mmol of DiMeDMSA in 8 mL of an 8% NaHC03-0.9% saline solution. The solution, under Nz, was stirred for 15min and 0.429 M ZnClz (2.3 mL) was added to give 0.097 M Naz[Zn(DiMeDMSA)z]. This was injected iv a t a rate of 0.1 mL/min into rats a t a dose of either 0.01 or 0.15 mmol/kg body wt. DiMeDMSA was dissolved under nitrogen in 5% NaHC030.9% saline solution to give a0.097 M solution. This was injected iv a t a rate of 0.1 mL/min and a t a dose of 0.15 mmol/kg body wt.
Biliary Excretion of Platinum. CDDP (2 mg/mL of saline) was injected into the tail vein of rats (210-225 g body weight) a t a dose of 8 mg/kg body wt. Control animals were injected with saline. Twenty-four hours later, the rats were anesthetized with urethane (1.0g/ kg body wt) ip and the jugular vein was cannulated using PE-50 polyethylene tubing. The vein was infused with saline (1.5 mL/h) during the experiment. Rats were kept on an electric blanket, to maintain their core temperature at 36 0.5 "C during the experiment. The bile duct was cannulated with PE-10 polyethylene tubing. The tubing was placed high into the bile duct close to where it joins the duodenum and deep enough to avoid contamination with any excretions from the pancreas. One hour after a steady bile flow was established, the chelate was injected iv and the bile collected every 30 min for 2.5-5 hours. Pt levels in the bile samples were determined using a ThermoJarell Ash Model 12E atomic absorption spectrophotometer under standard operating parameters for Pt. A hollow cathode lamp was used with a lamp current of 10 mA. The wavelength was set a t 265.9 nm. The slit width used was 160 pm. The bandpass was 0.5 nm. The integration time was 8 s. The AA was equipped with a controlled-temperature furnace atomizer 188 and SmithHieftje 12 background correction. Samples were digested in 14 N HN03: 12 N HC1 (2:l) for 24 h a t 50 "C. The volume then was adjusted with double distilled water to ten times the original sample volume. Each sample was analyzed in triplicate. Biliary Excretion of Cadmium. Rats (210-225 g body wt) were injected ip with 1 mg of Cd/kg body wt, as CdC12, and 75 pCi of logCd/kg body wt. Three days later, the rats were anesthesized with urethane (1.0 g/kg) ip. The jugular vein and bile duct were cannulated as above. Chelating agents were injected 1 h after a steady bile flow was established. Bile was collected as above for 4 h at 30-min intervals. Radioactivity was measured using an LKB type 1282 CompuGamma counter. L D ~in o Mice of [ ( M ~ ) ~ N ] z [ Z ~ ( D ~ M ~ D M or SNaz[ZnA)~] (DiMeDMSA)z]. To compare the toxicities of the Zn chelate having different counterions, LDbovalues were determined in male mice. Ten mice per dose were used. The doses of [(Me)4Nlz[Zn(DiMeDMSA)zl tested were 0-1.0 mmol/kg body wt. The compound was dissolved in 0.9% saline at a concentration of 62.9 mg of [Zn(DiMeDMSA)2l2-/mL. The Naz[Zn(DiMeDMSA)z] was synthesized immediately before use. Five groups of mice (10 per group) with doses from 0 to 0.8 mmol/kg body wt were studied. The chelates were injected ip, using a volume of 0.3 mL/20 g body wt. The animals were oberved for 14 days, and the LDb0 was determined using Fieller's theorem (32). Toxicity of Naz[Zn(DiMeDMSA)z] in Rats. Eight male Sprague-Dawley rats (140 g body wt) were housed in individual metabolic cages and allowed to acclimate for 1 day. Doubledistilled drinking water and food were available ad libitum. Each rat was injected ip with 1 mg of Cd/kg body wt, as CdC12,and 25 pCi of lo9Cdin a volume of 0.1 mL1100 g body wt. Three days later, the rats were divided into two groups and injected twice a day, sc, with either 0.15 mmol/kg Naz[Zn(DiMeDMSA)z] or saline. Solutions were prepared immediately prior to injection. Urine and fecal samples were collected daily from each rat, and the radioactivity was determined using an LKB type 1282 CompuGamma counter.
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210 Chem. Res. Toricol., Vol. 6, No.2, 1993 A control study in which the rata were not given cadmium was
alsoperformed. Six rata (140-150g) were acclimated in metabolic cages as above and were injected sc with Naz[Zn(DiMeDMSA)d, DiMeDMSA, or saline. The urines were collected and observed for any changes. The animals were necropsied a t the time of death or the appropriate time. Determinationof Zn and Cd in the Kidney. Four groups of three male Sprague-Dawley rata (200g) were injected sc with saline or equimolar concentrations (0.15mmol/kg) of Naz[Zn(DiMeDMSA)z], DiMeDMSA, or ZnClz. When a rat of the Zn chelate group died, one animal from each of the other was necropsied. Kidneys were removed from the animalsand digested in concentrated HN03 (1g of tissue in 3 mL of HN03) a t 85 OC for 1 h. The digestion mixture was cooled, HzO2 was added, and the samples were heated again for 1 h. Double-distilled water was added to a final volume of 10 mL. Ammonium phosphate (10%) was used as a matrix modifier. Cd levels in the kidney were determined using a Thermdarell Ash Model 12E atomic absorption spectrophotometer equipped with Smith-Hieftje 12 background correction and a controlled-temperature furnace atomizer 188. The light source used was a hollow cathode lamp set with a current of 3 mA. The wavelength was set a t 228.8nm. The slit width was 320rm,and the bandpasa was 1 nm. Standards were made up a t 0,2.5,5.0,7.5, and 10.0ppb of cadmium. Kidney samples were run in triplicate. The usual precautions for metal analysis, such as acid-washed glassware, were followed. The digested solution of a bile sample was diluted an additional 10-fold with double-distilled water before analysis for the total amount of zinc in the kidney. Zinc was determined using flame atomic absorption. The light aource was a hollow cathode lamp. The wavelength was set a t 213.9nm. The flame was an oxidizing, fuel lean, blue flame. Zinc standards were diluted to make up solutions a t 0, 0.1,0.2,0.3,0.4,and 0.5 ppm. Kidney samples were run in triplicate. Statistics. Data for bile studies are given as means f standard error. Statistical analyses were carried out using standard ANOVA procedures.
Results [Zn(DiMeDMSA)2I2-Increased Biliary Excretion of Cadmium-109. [Zn(DiMeDMSA)zlZ(0.01mmol/kg) with tetramethylammoniumas the counterion, when given iv to rata which had been injected 3 days previously with "Wd, increased the biliary excretion of Cd (Figure 2) as compared to the saline-injected control (p< 0.005). The biliary excretion of Cd peaked 1 h after injection of [Zn(DiMeDMSA)2IZ(Figure 2). TetramethylammoniumIon Increases the Toxicity of [Zn(DiMeDMSA)2IZ. The highest concentrations of (Me4N)z[Zn(DiMeDMSA)zI that could be administered iv without the animal dying during the experiment was 0.01 mmol/kg body wt. The amount of tetramethylammonium ion present in a 0.01 mmol/kg body w t dose of (MedN)2[Zn(DiMeDMSA)z]was close to ita LDa. Because of this, [Zn(DiMeDMSA)zlS was prepared as an ion association complex with sodium as the counterion in place of tetramethylammonium. The toxicity of [Zn(DiMeDMSA)21Z- was different depending on which counterion was present. The LDm for (Me4N)z[Zn(DiMeDMSA)2] was 0.34 mmol/kg body wt, when given ip to male mice. The 95% confidence interval was 0.26-0.41 mmol/kg body wt. (Mice were used for the toxicity studies because of the limited supply of [Zn(DiMeDMSA)zP.) When sodium was used as the counterion, the toxicity of [Zn(DiMeDMSA)zlZ- decreased. The injection of up to 0.80 mmol of Naz[Zn(DiMeDSMA)zl/kg body wt, ip, did not result in any mice lethalities. It was concluded, therefore, that the dose of
0.000 0
i
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Figure 2. Biliary excretion of "Cd after intravenous injection of saline or equimolar amounta of Naz[Zn(DiMeDMSA)z] or (Me,N)z[Zn(DiMeDMSA)d. The biliaryexcretionsof 'OgCdafter the administration of the Zn chelate containing either counterion were not statistically different from each other, but they were different when compared to the controls (p< 0.006). Three rata were in each group. In this and other figures,the arrow indicates the time the treatment was injected and error bars indicate standard error.
[Zn(DiMeDMSA)zl%might be increased safely by changing the counterion from tetramethylammoniumto sodium. The Cd mobilizing activity of the Zn chelate was nqt dependent on the tetramethylammonium counterion (Figure 2). There was no statistically significant difference in the biliary excretion of Cd when [Z~(D~M~DSMA)ZPat a dose of 0.01 mmol/kg body wt contained either tetramethylammonium or sodium as the counterion. [Zn(DiMeDSMA)2]%Increased Biliary Excretion of Platinum. When rata were given CDDP ip, followed 24 h later by the iv administration of 0.01 mmol of [ Z ~ ( D ~ M ~ D S M A ) body Z I ~ -wt, / ~ there ~ was a significant increase in the biliary excretion of Pt aa compared to controls (Figure 3). At this dose, the biliary excretion was independent of the counterion in the preparation (Figure 3). According to two-way ANOVA analysis, there was no significant difference in biliary excretion of Pt after injection of [Zn(DiMeDSMA)2l2-(0.01mmol/kg bodywt) prepared using the tetramethylammonium counterion or the sodium counterion. The biliary excretion of Pt was increased by Naz[Zn(DiMeDSMA)a] in a dose-dependent manner (Figure 3). Comparison of meseDiMeDMSA with Ita Zn Chelate as to the Biliary Excretion of Platinum and Cadmium. CDDP was administered iv, and 24 h later, equimolar doses of neso-DiMeDSMA or Naz[Zn(DiMeDMSA)zI were administered iv. The biliary excretion of Pt after Naz[Zn(DiMeDMSA)nl was approximately twice that found after meso-DiMeDMSA administration
Chem. Res. Toxicol., Vol. 6, No.2, 1993 211
DiMeDMSA Zinc Chelate 2.5
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E
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Figure 3. Biliary excretion of Pt after administration of [Zn(DiMeDMSA)zI”. CDDP (8mg/kg body wt) was injectediv. Twenty-four h o w later, Naz[Zn(DiMeDMSA)z], (Me,N)z[Zn(DiMeDMSA)z], or saline was injected iv. There were 3 rata in each group.
(Figure 4). The difference was significant (p < 0.005). But each molecule of Naz[Zn(DiMeDMSA)nl contains two molecules of meso-DiMeDMSA. If Naz[Zn(DiMeDMSA)zl were to yield two DiMeDMSA molecules by biotransformation, the excretion of twice as much platinum in the bile would be understandable. Since [Zn(DiMeDMSA)zP contains two molecules of DiMeDMSA per atom of Zn, the action of mesoDiMeDMSA at a molecular dose twice that of the Zn chelate, on the biliary excretion of Cd, was determined (Figure 5). [Zn(DiMeDMSA)#- a t 0.15 mmol/kg body wt appeared to be more effective than DiMeDMSA at 0.30 mmol/kg body wt as far as increasing the biliary excretion of cadmium, p < 0.005 (Figure 5). Renal Toxicity of [Zn(DiMeDMSA)#-. In order to determine if [Zn(DiMeDMSA)#- would increase the urinary excretion of Cd, rata were injected fiist with 1mg of Cd/kg body wt, and 3 days later with daily doses of 0.15 mmol of Naz[Zn(DiMeDMSA)z]/kg body wt, sc. After 2 days of treatment with [Zn(DiMeDMSA)zl2, the animals began to exhibit bloody urines. Soon after, these rata became anuretic and died 1day later. Rata given [Z~(D~M~DMSA)Z]”, only without prior injection of Cd, also were found to have blood in their urine and they died within the day. These rata were necropsied, and massive necrosis of the proximal convoluted tubule epithelium in the kidneys was observed.2Such a lesion would account for the anuria and death. No such lesions were found in the rata receiving meso-DiMeDMSA or control rata; their kidneys appeared normal. Personal communication from Dr. Gregory A. Bradley.
Figure 4. Biliary excretion of Pt after administration of equimolar concentrations (0.15 mmovkg body wt) of NadZn(DiMeDMSA)z] or meso-DiMeDMSA, iv. There were 3 rata in each group. Each compound significantly increased biliary Pt excretion aa compared to controls (p < 0.006).
Zinc and Cadmium Content of the Kidney after Injection of Chelating Agents. Other chelating agenta, for example, BAL (221, increase renal Cd, causing renal damage. When the Cd content of the kidneys was determined using atomic absorption spectrophotometry, the amount of Cd in the kidneys of rata receiving saline, DiMeDMSA, or NadZn(DiMeDMSA)z]was 3.2,2.95, and 3.0 pg of Cd/g, respectively. Neither one of the chelating agenta, therefore, was bringing Cd to the kidneys under these experimental conditions. To determine whether administration of NadZn(DiMeDMSA)zI increased the zinc content of the kidney, the following experiment was performed. Rata were injected with equimolar doses of Naz[Zn(DiMeDMSA)zI, DiMeDMSA, or ZnCl2 (0.15 mmol/kg body wt, sc) daily. The rata injected with Naz[Zn(DiMeDMSA)d died withii 3 days of injections. At the time individual animals injected with Naz[Zn(DiMeDMSA)d died, one animal from each of the other groups was sacrified and their kidneys were examined. The kidneys of animals which had been treated with Naz[Zn(DiMeDMSA)zl were found to be discolored and enlarged. The kidneys of all other groups appeared normal. Zinc analysis, however, showed that the zinc concentration in the kidneys of the rata injected with [Zn(DiMeDMSA)#- was about 10 times greater than in any of the other groups of rata (Figure 6). The zinc concentrations in the kidneys of the rata injected with DiMeDMSA, ZnClz, or saline were not significantly different. It would appear that Naz[Zn(DiMeDMSA)zl carried zinc from body stores directly to the kidney. But such a conclusion appears to be incorrect for two reasons. First of all, when an equimolar amount of ZnClz or [Zn(DiMeDMSA)#- was injected, the resulting levels of
212 Chem. Res. Toricol., Vol. 6, No.2, 1993
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Figure 6. Zinc concentration in the kidney of rata given saline, DiMeDMSA, Naz[Zn(DiMeDMSA)z], or ZnClz. There were 3 rata in each group. Subcutaneousinjections (0.15mmol/kgbody wt) were given daily until the rata receiving Naz[Zn(DiMeDMSA)z] died. At such times, rata from the other groups were sacrificed.
Discussion The present results show that the Zn chelate of mesoDiMeDMSA mobilized Cd and Pt in the rat and increased the excretion of these metals via the bile (Figures 2 and 3). The biliary excretion of platinum increased with increasing doses of [Zn(DiMeDMSA)#- when sodium was the counterion (Figure 3). But it should be realized that, after iv injection of [Zn(DiMeDMSA)#-, the bile experiments lasted for only a short period of time (hours). Subsequent experiments, however, in which the urine was collected, lasted longer (days), and renal toxicity was observed. After administration of Naz[Zn(DiMeDMSA)zl the biliary excretion of Pt was twice that found after administration of equimolar amounts of meso-DiMeDMSA (Figure 4). Although equimolar amounts of mesoDiMeDMSA and [Zn(DiMeDMSA)#- were injected, if [Zn(DiMeDMSA)zl” were unstable in vivo, it might be expected to release two molecules of meso-DiMeDMSA. When the injected dose of meso-DiMeDMSAwas doubled, the biliary excretion of cadmium, after [Zn(DiMeDMSA)2I2-injection, extended over a longer period of time (Figure 5). This suggests that NadZn(DiMeDMSA)zI, on the basis of these short-term biliary experiments, has no great advantage over DiMeDMSA for increasing the biliary excretion of Cd. It does suggest, however, that the zinc chelate may be acting aa a prodrug in that its extended
action might be due to its being biotransformed to mesoDiMeDMSA. Although DMSA is an effectiveantidote for lead toxicity (14,15),its ionized carboxylgroups are believed responsible for preventing its crossing cell membranes. Thus, it is unlikely to reach metals which are firmly bound intracellularly. Esterification of the two carboxyl groups of DMSA increased its lipophilicity. meso-DiMeDMSA, for example, is excreted in the bile (19),an indication that it has penetrated the hepatocyte cell membrane and has intracellular distribution. Several methods were used in attempts to detect and quantitate the [Zn(DMeDMSA)#in the bile, including HPLC, GLC, and TLC analyses. We were, however, unsuccessful, since [Zn(DiMeDMSA)#was unstable during the various procedures used for derivatization, isolation,and/or analysisof biliary samples. Most chelating agents do not have an absolute specificity for heavy metal ions. While they chelate and increase the excretion of the unwanted toxic metal, they may also cause essential endogenous metals to be excreted, sometimes creating a deficiency of that metal in the body. A classic example has been EDTA, which, if used for treatment of lead intoxication,will severely deplete the body of calcium. For this reason, CaNazEDTA has been used for treatment of lead intoxication instead of EDTA (33),even though it does increase the urinary excretion of zinc and copper. An analgous rationale was the reason for the synthesis and testing of [Zn(DiMeDMSA)212-. Zinc, it was reasoned, might displace Pt or Cd in the cell and the Pt and/or Cd would be chelated and removed. The zinc on the other hand would remain in the body, reducing the possibility of a zinc depletion. Although the renal Zn content was
DiMeDMSA Zinc Chelate elevatedafter [Zn(DiMeDMSA)zl2- administration (Figure 6), renal toxicity resulting in death occurred. The toxicity of [Zn(DiMeDMSA)212-,as measured by LD50 studies, was greater when tetramethylammonium ion was used in the place of sodium ion as the counterion. Subsequent experiments found Naz[Zn(DiMeDMSA)z] to be toxic to the kidneys. The same dose of the sodium form of [Zn(DiMeDMSA)2]2-(0.15 mmol/kg body wt) was used ip for the urinary excretion studies as in the iv bile studies. But the time period over which the former study was carried out was longer than that for the latter. After only 2 days of injections (4 doses), blood was observed in the urine of the rats receiving Naz[Zn(DiMeDMSA)21. Blood in the urine and a subsequent anuria indicated the possibility of kidney dysfunction. But when the Zn chelate was given without previous administration of cadmium, similar renal toxicity was noted. These rats were found to have severe necrosis of the convoluted tubules, while the rats receiving DiMeDMSA did not show any renal pathology and appeared similar to controls. Why is Naz[Zn(DiMeDMSA)zl so toxic to the kidney, and what is the mechanism of its toxicity? Some chelating agents (like BAL) are known to cause a redistribution of cadmium to the kidney, causing renal failure (22). The blood in the urine and the subsequent anuria after injection of Naz [Zn(DiMeDMSA)z] are both signs of acute renal failure. Necropsies of the animals injected with toxic amounts of Na2[Zn(DiMeDMSA)slshowed that the kidney was the major organ that was targeted by this drug. Although the liver was slightly discolored, none of the other organs were significantly different than controls. It was hypothesized that endogenous cadmium may have been mobilized and redistributed to the kidney by Na2[Zn(DiMeDMSA)z], causing the renal failure. (The control rats injected with DiMeDMSA showed no signs of renal toxicity.) The renal toxicity of Naz[Zn(DiMeDMSA)zl was at first thought to be due to the redistribution of endogenous cadmium to the kidney. Other chelating agents, for example, BAL, increase renal Cd, causing renal damage (22). When the kidneys were analyzed by atomic absorption, however, no significant difference in Cd content was found between the rats given Naz[Zn(DiMeDMSA)2], meso-DiMeDMSA, ZnC12, or saline. There was, however, a 10-fold increase of zinc in the kidneys of rats injected with Na2[Zn(DiMeDMSA)z], when compared to rats of the other three groups (Figure 6). This suggests that the increased zinc in the kidney may have been derived from the Naz[Zn(DiMeDMSA)z] instead of from endogenous zinc brought to the kidney by the chelator. It would appear that this renal toxicity is due to Na2[Zn(DiMeDMSA)z], per se, or to its releasing its zinc mainly in the kidney. The amount of zinc in the analyzed kidney was comparable to the zinc content of the injected Naz[Zn(DiMeDMSA)z]. The zinc from the injection ZnCl2 did not increase the zinc concentration in the kidney, suggesting that it was distributed throughout the body and not mainly in the kidney (Figure 6). Whether the kidney toxicity is due to Naz[Zn(DiMeDMSA)2]per se or to Zn released from it in the kidney is not known at present. It is pertinent to note that another chelate of DMSA, the antimony chelate, was designed and synthesized by Friedheim (34) many years ago with the expectation that it would increase the cellular uptake of antimony for the treatment of schistosomiasis. This was shown to be so (34).
Chem. Res. Toxicol., Vol. 6, No. 2, 1993 213 The increase in biliary Pt and Cd excretion occurred in short-term experiments in which biliary excretion of the metal ions was measured within 3 h of the ivadministration of the Zn chelate. When the experiments were lengthened in order to measure daily urinary excretion of the metals, renal toxicity and death resulted. Our laboratory and others often have used short-term biliary excretion as the first screen for chelating agents. When the results of our biliary excretion studies using the Zn chelate are compared with the urinary excretion experiments, the pitfalls and importance of choosing the appropriate assay system for screening metal chelation activity are apparent. Biliary experiments, however, in the past have yielded and in the future will continue to yield valuable but incomplete information. It should be realized that even though biliary cadmium excretion increased after iv administration of [Zn(DiMeDMSA)zl2-,the increase was not of the magnitude seen with some dithiocarbamate derivatives such as sodium N-benzyl-D-glutamine dithiocarbamate (35). In summary, although [Zn(DiMeDMSA)z12-,the zinc chelate of DiMeDMSA, increased the biliary excretion of cadmium and platinum in short-term (hourly) experiments, when used in longer (daily) experiments designed to measure urinary excretion of cadmium, severe renal toxicity resulted. The renal toxicity appears to be related to a 10-fold increase in the zinc level of the kidney. Acknowledgment. This work was supported by NIEHS Grant ES03356. We thank Dr. Gregory A. Bradley, of the Department of Veterinary Science, for his examination and evaluation of the pathology in the rat kidneys.
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