decreased effectiveness of chelation therapy with time after acute cadmium poisoning

TOXICOLOGY AND APPLIED PHARMACOLOGY 63, 173-180 (1982)

Decreased Effectiveness of Chelation Therapy with Time after Acute Cadmium Poisoning’

LOUIS R. CANTILENA, JR.,' ANDCURTIS D. KLAASSEN

Department of Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66103

Received June 12, 1981; accepted October 30. 1981

Decreased Effectiveness of Chelation Therapy with Time after Acute Cadmium Poisoning. CANTILENA, JR., L. R., AND KLAASSEN, C. D. (1982). Toxicol. Appl. Pharmacol. 63, 173- 180. The effect of increasing the time interval between cadmium (Cd) exposure and chelation therapy was studied in male Swiss Webster mice. The following chelating agents were administered ip at 0, 2, 12, 36, and 72 hr after giving radioactive Cd (1 mg Cd/kg, iv): diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), and 2,3- dimercaptosuccinic acid (DMSA). Daily elimination of Cd into urine and feces was determined for 5 days after which time the mice were killed, and the concentration of Cd was determined in various organs. In most of the tissues examined, including kidney and liver, only administration of chelators immediately after Cd significantly reduced tissue Cd concentrations. The mice treated immediately after Cd administration excreted between 50 and 75% of the Cd into urine in the first 24 hr compared to 0.1% excreted by controls. Treatment at later times (2 to 72 hr) significantly increased Cd excretion but the magnitude of the effect was far less than that seen in mice treated immediately after Cd. In a separate group of mice, the time course for the induction of metallothionein synthesis was determined by administering 1 mg Cd/kg iv at various times before death. Metallothionein concentration increased within 2 hr after Cd administration and reached a maximum within 8 hr. The results show that the length of time before initiating chelation therapy for Cd poisoning greatly influences the effectiveness of the therapy. Hepatic metallothionein may be responsible for this phenomenon.

Although the element cadmium (Cd) has diethylenetriaminepentaacetic acid (DTPA), been known to be a toxic substance to man ethylenediaminetetraacetic acid (EDTA), for more than 100 years (Prodan, 1932), and 2,3-dimercaptosuccinic acid (DMSA) treatment of Cd poisoning has not proven were the most effective in increasing animal to be an easy task. Several early studies using survival after a lethal dose of Cd, in en- chelators such as 2,3-dimercaptopropanol hancing urinary excretion of Cd, and in de- (BAL) or ethylenediaminetetraacetic acid creasing the concentration of Cd in various (EDTA) were unable to describe a safe and tissues. DTPA was consistently the most ef- effective protocol for the treatment of Cd fective compound. The chelating agents in poisoning (Gilman et al., 1946; Friberg, that study were administered ip immediately 1956). following an iv injection of Cd.

Recently, a study of seven chelating agents of various chemical types for use in acute Cd poisoning has been reported (Cantilena and Klaassen, 198 1). Of the seven agents,

'Supported by USPHS Grant ES-01142. * Supported by NIH Training Grant ES-07079.

Metallothionein synthesis occurs within 24 hr after an ip injection of a sublethal dose of Cd in mice (Probst et al., 1977). The effect of BAL on tissue concentrations and biliary excretion of Cd before and after the synthesis of metallothionein has been studied in the rat (Cherian, 1980). The results of

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174 CANTILENA AND KLAASSEN

that study showed that BAL effectively reduced the concentration of Cd in liver only when given within 24 hr of Cd. However, excretion of Cd into bile could be increased by BAL when given as much as 14 days after the metal.

A decrease in the effectiveness of chelation therapy when the time interval between administration of Cd and chelator is increased has also been observed for some aminocar- boxylic acid chelators (Eybl et al., 1965). However, this decreased efficacy of chelation therapy has not been well correlated with the induction of metallothionein synthesis.

The purpose of this investigation was to study the effect of increasing the time interval between administration of Cd and ini- tiation of chelation therapy on the effectiveness of the chelators in mice. The chelating agents examined were those previously shown to be very effective when administered immediately after Cd (Cantilena and Klaassen, 1981), namely DTPA, EDTA, and DMSA. Effectiveness of chelation therapy was determined by measuring the ability of the chelators to increase the urinary excretion of Cd and decrease the concentration of Cd in several tissues. The efficacy of the chelators was then correlated with the time course for induction of the synthesis of metallothionein after iv administration of Cd.

METHODS

General. Male Swiss Webster mice (Sutter, Spring- field, MO.) weighing 25 to 45 g were used. The mice received commercial chow and water od libitum and were exposed to a 12-hr light (6:OO AM to 6:00 PM) and 12-hr dark (600 PM to 600 AM) cycle. Ambient tem- perature was maintained between 21 and 23°C at all times.

Preparation of chelator solutions. The calcium, tri- sodium salt of diethylenetriaminepentaacetic acid (DTPA) was prepared by adding 3: 1:l mole ratio amounts of NaOH, DTPA, and Ca(OH),, respectively, in that order to distilled water. The final solution was 0.18 M CaNa,DTPA. Calcium disodium ethylenediaminetetraacetic acid (EDTA) was dissolved in distilled water at 0.40 M. The chelator 2,3-dimercaptosuccinic acid (DMSA) was dissolved in 10% NaHCO, at 0.41 M. All three chelators were administered ip in a volume

of 10 ml/kg and were obtained from Sigma Chemical Company. (St. Louis, MO.).

Cadmium distribution and excretion study. The effect of each chelating agent on the distribution and excretion of Cd was determined. Radiolabeled Cd (‘V!d, carrier free, New England Nuclear, Boston, Mass.) was added to a solution of CdClr (Fisher Scientific Co., Fair- lawn, N.J.) and injected iv into the tail vein at 1 mg Cd/kg in a volume of 10 ml/kg. Approximately 50 pCi/ kg was administered. Twenty mice were used for each experiment in which the animals received saline, DTPA, EDTA, or DMSA ip at 0, 2, 12, 36, or 72 hr after Cd injection. The dose of the chelators was approximately one-fourth of their respective LD50 values (Cantilena and Klaassen, 1981). The doses were 0.90 g DTPA/kg, 1.5 g EDTA/kg, and 0.74 g DMSA/kg.

Immediately after Cd administration, the mice were placed in stainless steel metabolic cages, and urine and feces were collected every day for 5 days. Five days ( 120 hr) after Cd administration the mice were removed from the metabolic cages and anesthetized with ether; 1 ml of blood was obtained by cardiac puncture. The mice were then killed, and the following organs were removed: testes, heart, lung, pancreas, spleen, small intestine, kidney, stomach, bone plus marrow, brain, liver, and muscle. The femur and gastrocnemius were considered rep- resentative of bone and muscle, respectively. The organs were weighed immediately after their removal. The stomach and small intestine were washed thoroughly with warm tap water prior to weighing. The amount of Cd in the collected feces and urine and in tissues and blood was calculated using the wet weight of the tissues and measuring the amount of radioactivity with a Pack- ard Model 5 130 Autogamma scintillation counter.

Estimation of metallothionein concentrations. Mice were given nonradiolabeled CdC& (1 mg Cd/kg, iv) at 0, l/2, 1, 2, 4, 8, 12, 24, 36, 48, and 72 hr before the liver was removed under ether anesthesia and homog- enized in 6 vol of 1.15% KC1 at 4°C. Metallothionein was estimated by the method of Piotrowski et al. (1973) with a slight modification (Kotsonis and Klaassen, 1977a). Mercuric chloride (400 pg Hg) labeled with “‘Hg (2 mCi/mol, New England Nuclear, Boston, Mass.) was incubated with 3.5 ml of liver homogenate (25”C, 10 min). One milliliter of 10% trichloroacetic acid (TCA) was then added, and the tubes were mixed on a vortex and centrifuged (3OOOg, 10 min). The amount of ‘03Hg in the supernatant fluid was determined with a Packard Model 5130 Autogamma scintillation counter and is an estimate of the metallothionein concentration.

Statistics. Data were analyzed by a one-way analysis of variance. When the analysis indicated that a significant difference existed, the treated groups were compared to control by Duncan’s new multiple range test (Steel and Torrie, 1976). Since there were unequal vari- ances in some of the urinary excretion data, it was nec-

CHELATION THERAPY FOR CADMIUM 175

essary to use the nonparametric Kruskal-Wallis test (Conover, 197 1) followed by the appropriate multiple comparison test (Dunn, 1964). The level of significance was set at P i 0.05.

RESULTS

The effect of the chelating agents on daily urinary elimination of Cd for each day after administration of Cd is shown in the five panels (top to bottom) of Fig. 1. The scale

of the ordinate (pg Cd excreted/kg body weight) is not the same in all panels. The excretion of Cd from mice receiving either saline (control) or one of the three chelators at various times after Cd administration (0, 2, 12, 36, or 72 hr) is shown from left to right in each panel. Urinary excretion data for groups which had not yet received chelator treatment are omitted for clarity.

During the first 24 hr after Cd adminis-

I o hr 2hr 12 hr 36hr 72hr

1 T DAY 5

FIG. 1. The effect of DTPA (DT), EDTA (E), and DMSA (DM) administered at the indicated times after Cd (0 to 72 hr) on the urinary elimination of the metal. The daily excretion of Cd of treated groups is shown for 5 consecutive days after Cd (Days 1-5). Bars indicate SE, n = 5 to 8, * indicates statistically different from control (p < 0.05).


tration (Day 1, Fig. l), the first three groups (0, 2, and 12 hr) received chelation treatment. Saline-treated controls in these three groups excreted approximately 1 pg Cd/kg during this period. Treatment with DTPA significantly increased urinary excretion of Cd in all three groups during Day 1. The DTPA-treated mice excreted 500, 50, and 2.5 gg Cd/kg when treated at 0, 2, and 12 hr, respectively. Treatment with either EDTA or DMSA at 0 or 2 hr after Cd also markedly increased urinary elimination of Cd.

Excretion of Cd into urine during Day 2 is shown in the second panel from the top of Fig. 1. Mice which received either DTPA or DMSA immediately after Cd (0 hr) had an increased urinary elimination of Cd on the second day after administration. Mice which received chelation therapy 2 hr after Cd did not excrete more Cd than their respective control. The animals treated 36 hr after Cd received their respective chelator 12 hr into the Day 2 collection period. Treat- ment with DTPA, but not with EDTA or DMSA, significantly increased the urinary elimination of Cd at this time.

The third panel from the top of Fig. 1 shows the urinary elimination of Cd during the third day after Cd administration. No group received treatment on the third day. Mice which received either DTPA or DMSA 0 hr after Cd still excreted more Cd than the control group. However, during this period the mice were excreting only 1 to 2.5 pg Cd/kg compared to the 500 pg Cd/kg they eliminated during Day 1.

The urinary elimination of Cd during Day 4 is shown in the fourth panel from the top of Fig. 1. Significant increases in the amount of Cd eliminated by mice given DTPA or DMSA immediately after Cd (0 hr) were observed. During Day 4, the 72-hr group received chelation therapy. The mice treated at this time with DTPA or EDTA excreted more Cd into the urine than their respective controls.

The bottom panel of Fig. 1 shows the

urinary elimination of Cd during Day 5. After 5 days, mice which received DTPA or DMSA immediately after Cd (0 hr) contin- ued to excrete more Cd than controls. Sim- ilarly, mice which received DTPA 72 hr after Cd excreted more Cd than controls.

The effect of administration of DTPA, EDTA, or DMSA immediately after Cd on its elimination into feces is shown in Fig. 2. Treatment with DTPA, EDTA, or DMSA decreased the fecal elimination of Cd by approximately 50% during Day 1 (top panel) and approximately 50 to 70% during Day 2 (bottom panel). The excretion of Cd into feces by control mice diminished with time from 80 pg Cd/kg during Day 1 (Fig. 2) to 2 pg Cd/kg during Day 5 (data not shown). The excretion data of the 0-hr group for Day 1 (top panel) and Day 2 (bottom panel) are the only fecal excretion data shown because all other groups (2, 12, 36, or 72 hr) and

CONT DTPA

FIG. 2. The effect of the indicated chelators on the fecal elimination of Cd. Chelating agents were administered immediately after Cd. Fecal elimination during the first 2 days post-Cd is shown. Bars indicate SE, n = 5 to 8, l indicates significantly different from control

(p < 0.05).


days (3 to 5) showed no statistically significant effects.

The effect of chelation treatment at various times after Cd administration on the concentration of Cd in the kidney, the main target organ of Cd toxicity (Friberg et al., 1974), is shown in Fig. 3. Administration of DTPA, EDTA, or DMSA immediately after the Cd (0 hr) resulted in a significant decrease in the concentration of Cd in the kidney 5 days later. DTPA, EDTA, and DMSA administrations produced 70, 60, and 30% decreases in renal Cd content, respectively. Chelator treatment at later times did not significantly affect the concentration of Cd in the kidney.

The time course for the effectiveness of the chelators in removing Cd from the liver, the main storage site for Cd in the body (Kotsonis and Klaassen, 1977b), is shown in Fig. 4. Treatment with DTPA, EDTA, or DMSA at 0 hr resulted in a significant decrease (75 to 80%) in the concentration of Cd in the liver; administration of chelators at later times had no significant effect. Sim- ilar results were obtained in the testes (Fig. 5) and in the brain (Fig. 6) in that only the

FIG. 4. The effect of administration of the indicated

chelators at various times (0 to 72 hr) after Cd exposure on the concentration of Cd in the liver. Bars indicate

SE, n = 5 to 8, * indicates significantly different from control.

administration of the chelators immediately after Cd (0 hr) resulted in a significant decrease in the concentration of Cd in these tissues. This trend was observed for the other tissues examined (data not shown) in that statistically significant decreases in tissue Cd concentrations were only noted in mice that

tlhr 36hr 7?.rh I

FIG. 3. The effect of administration of the indicated chelators at various times (0 to 72 hr) after Cd exposure

on the concentration of Cd in the kidney. Bars indicate SE, n = 5 to 8, * indicates significantly different from control.

FIG. 5. The effect of administration of the indicated

chelators at various times (0 to 72 hr) after Cd exposure on the concentration of Cd in the testes. Bars indicate SE, n = 5 to 8, * indicates significantly different from control.


0.06

0.03

FIG. 6. The effect of administration of the indicated chelators at various times (0 to 72 hr) after Cd exposure on the concentration of Cd in the brain. Bars indicate SE, n = 5 to 8, * indicates significantly different from control.

received chelation therapy immediately after Cd.

The time course for induction of metallothionein is shown in Fig. 7, The concentration of metallothionein begins to increase 1 hr after iv administration of Cd and

reaches half of the maximal level seen with this dose of Cd by 2 hr. Maximum metallothionein concentrations were attained within 4 hr after Cd administration and re- mained at that level for the duration of the experiment.

DISCUSSION

Successful therapy for intoxication by heavy metals such as with lead and mercury involves formation of a relatively stable chelator-metal complex which is transported via the circulation to the kidney and excreted. This therapy decreases the body burden and therefore the toxicity of the metal.

The results of the present study have shown that DTPA, EDTA, and DMSA are capable of increasing the urinary elimination of Cd when administered at various times following Cd exposure. These agents were chosen because they were found to be the only effective agents of seven chelators sur- veyed for use in Cd intoxication when given immediately after Cd administration (Can- tilena and Klaassen, 1981). The results of

L 2 4 8 12 24 36 48 72

Hems

FIG. 7. The time course of induction of hepatic metallothionein following Cd exposure iv). Bars indicate SE, n = 6.

(1 mg Cd/kg,


that study showed that DTPA was the most effective chelator of those examined. In the present study, DTPA was also found to be the most effective chelating agent studied. Only treatment with DTPA at 12 or 36 hr after Cd exposure significantly increased the urinary elimination of the metal. The magnitude of this increased elimination was greatly attenuated by increasing the time interval between Cd injection and administration of the chelator. For example, mice that received DTPA at 0, 2, 12, 36, and 72 hr after Cd excreted approximately 515, 52, 3.6, 3.8, and 3.9 pg Cd/kg, respectively, throughout the 5-day period. The marked decline in chelator effectiveness occurred within the first 12 hr after Cd exposure. Administration of any of the three chelators 12 hr or later after Cd resulted in elimination of only 0.3% of the administered Cd compared to an approximate 50% by mice given chelation therapy immediately after Cd.

The concentration of Cd in the various tissues was decreased only when the chelators were administered immediately after the metal. A delay of even 2 hr between Cd exposure and chelator administration pre- vented a significant decrease in tissue concentrations of Cd from occurring. The mice receiving chelation therapy 2 hr after Cd had a 500% increase in the urinary elimination of Cd but only 5% of the administered dose of Cd was excreted. This finding suggests that the percentage of the dose of Cd eliminated more accurately predicts the effect of chelators on tissue concentration of Cd than does the percentage increase in urinary elimination of the metal.

A decrease in the effect of certain ami- nocarboxylic acid chelators on the removal of Cd from various tissues has been previously reported (Eybl et al., 1965; Niemeir, 1967). Our results agree with these earlier findings and more closely delineate the time course for the decrease in chelator effectiveness. The time dependence for induction of metallothionein after iv Cd administration

was determined in order to explain the dim- inution of chelator effectiveness for Cd.

The time course for hepatic metallothionein induction after ip exposure to Cd has been reported for Swiss Webster mice (Probst et al., 1977). In that study, maximal metallothionein concentrations were reached 36 to 48 hr after Cd exposure. The present study, using the same strain of mice, showed that maximal metallothionein concentrations were reached 4 to 8 hr after iv exposure to Cd (Fig. 7). A five fold increase in hepatic metallothionein was observed in both studies. The difference in route of administration of Cd could explain the discrepancy in the time course for the induction of metallothionein observed.

The induction of hepatic metallothionein described in the present investigation cor- relates well with the time course for decreas- ing effectiveness of chelation therapy for Cd intoxication. When any of the three chelators were administered 2 hr after Cd when metallothionein concentrations were half of the maximum reached in this study, a IO- fold decrease in the effect of the chelators on the urinary excretion of Cd was seen compared to administration of the chelators at 0 hr (Day 1, Fig. 1). When metallothionein levels were maximal (beyond 4 hr), the effect of chelation therapy on urinary excretion of Cd, although statistically significant, was greatly reduced. Chelation therapy did not significantly decrease tissue Cd concentration when metallothionein levels were half maximal or greater (Figs. 3-6).

The induction of the synthesis of hepatic, and possibly other, metallothioneins appears to prevent the successful reduction of the body burden of Cd by the chelating agents DTPA, EDTA, or DMSA after a single administration. Cd is probably transferred to a deeper pharmacokinetic pool and becomes less available for interaction with chelators. It would thus appear that in therapy for Cd intoxication chelating agents of this type should be given as soon as possible after ex-


posure to the metal. These results suggest that hepatic metallothionein serves, at least in part, as a component of the physiological factors responsible for the decreased avail- ability of Cd for chelation therapy with time.

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decreased effectiveness of chelation therapy with time after acute cadmium poisoning

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