SEPTEMBEIUOCTOBER 1994 VOLUME 7,NUMBER 5 0 Copyright 1994 by the American Chemical Society
Art ides Efficacy of Combined Chelation in Lead Intoxication1 S. K. Tandon,*Surendra Singh, and Vinod K. Jain Industrial Toxicology Research Center, Lucknow 226001, India Received November 23, 1993@
The combination of a bone lead mobilizer, viz., CaNazEDTA, and a soft tissue depleter such a s meso-2,3-dimercaptosuccinicacid (DMSA) or the sodium salt of 2,3-dimercapto-l-propanesulfonic acid (DMPS) was investigated for its efficacy to enhance the excretion of lead, to reduce its body burden, and to reverse certain lead-induced biochemical alterations by either of them alone in rats. The administration of CaNa2EDTA or DMSA was more effective than that of DMPS, and the combination of CaNazEDTA and DMSA was more efficient than that of CaNa2EDTA and DMPS or the individual chelators in enhancing urinary/fecal excretion of lead, in reducing hepatic, renal, and femur concentration of lead, and in restoring lead-induced inhibition of blood 6-aminolevulinic acid dehydratase and increase in blood zinc protoporphyrin and urinary 6-aminolevulinic acid levels. The brain lead was depleted by DMSA only, but the combined chelation failed to improve upon the ability of DMSA. However, chelation therapy increased diuresis and tissue depletion of zinc.
Introduction One of the disadvantages of chelation therapy in lead intoxication is the redistribution of lead into soft tissues or redeposition in bone following release from bone, its main depot in the body. The removal of lead from different intracellular deposits also poses problems, as most of the chelating agents do not pass through the cellular membrane. The combination therapy involving at least two chelating agents with different characteristics or modes of action might offer a solution to these problems to a certain extent (1-3). The combined
* Address correspondence and reprint requests to this author a t the Chemical Toxicology Section, Industrial Toxicology Research Center, Post Box 80, Mahatma Gandhi Rd.,Lucknow 226001, U.P., India. Abstract published in Advance ACS Abstracts, July 1, 1994. ‘This is paper No. XXXVII in the series “Chelation in Metal Intoxication”. @
treatment with a hydrophilic and a lipophilic chelating agent has been found to be more effective than a single chelator in childhood lead poisoning (4). Thus, combining treatment with a soft tissue lead mobilizer such as meso2,3-dimercaptosuccinic acid (DMSAY and a bone lead mobilizer such as CaNazEDTAmight prevent redistribution or redeposition of lead mobilized from bone, while enhancing depletion of soft tissue lead (5). Two currently used thiol chelators, viz., DMSA and the sodium salt of 2,3-dimercapto-l-propanesulfonic acid (DMPS), are safe and more effective than CaNazEDTA (6). One of the advantages with DMSA or DMPS is the possibility of their ability to enter into the cell, although to a limited extent (7).
* Abbreviations: DMSA, meso-2,3-dimercaptosuccinicacid; DMPS, the sodium salt of 2,3-dimercapto-l-propanesulfonic acid; ALAD, 6-aminolevulinic acid dehydratase; ALA, 6-aminolevulinic acid; ZPP, zinc protoporphyrin.
0893-228x/94~2707-0585$04.50/0 0 1994 American Chemical Society
586 Chem. Res. Toxicol., Vol. 7, No. 5, 1994
Tandon et al. X
I CONTROL
DMSA
CaN3EDTA
DMPS
11
r
11
CaNa2EDTA
CaNoZFDTA
DMSA
DMPS
+
t
Figure 1. Influence of chelating agents or their combinations on urinary excretion of lead in lead-exposed rat. Each bar represents mean f SE (6values). *p < 0,001 vs normal animal (horizontal line). < 0.001 vs Pb-exposed control. xp < 0.001, y p < 0.01 vs CaNaZEDTAElMSAiDMPS. +p < 0.001 vs CaNazEDTA DMSA.
+
It was considered of interest to investigate whether CaNa2EDTA treatment in combination with DMSA or DMPS improves upon the efficacy of either of them alone and, if so, which combination was more effective in the therapy of experimental lead intoxication as evaluated by the excretion and tissue depletion of lead and the reversal of lead-induced biochemical alterations. The chelation was conducted in two standard 5-day treatment courses, separated by a 5-day period of no treatment.
Materials and Methods Chemicals. Lead(I1) acetate trihydrate (AnalyticalReagent) was purchased from BDH (Bombay, India), CaNazEDTA.GH20 was purchased from E. Merck (Darmstadt, Germany), and DMSA, DMPS, and 8-aminolevulinicacid (ALA)were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents used were of analytical grade. Animals and Treatment. Forty-two male albino rats (155 f 5 g) from Industrial Toxicology Research Center's colony were maintained on standard pellet diet (Lipton's India Limited; metal contents of feed, in ppm dry weight Cu 10.0, Mn 55.0, Co 5.0, Fe 70.0, and Zn 45.0) and water ad libitum. Thirty-six rats had free access to lead acetate (0.1%) in drinking water, and the remaining six rats were given unleaded water for 8 weeks (normal). The lead-exposed animals were randomly divided equally into six groups and treated daily for 5 days as follows: Group I: distilled water (4 mUkg), orally through gastric gavage (control);Group 11: CaNazEDTA (0.3 mmoy4 mL' kg), ip. Group 111: DMSA (0.3 mmoW mL/kg), ip. Group N: DMPS (0.3 mmoy4 m u g ) , ip. Group V CaNazEDTA as in group I1 and DMSA as in group 111, contralaterally. Group VI: CaNazEDTA as in group I1 and DMPS in group IV,contralaterally. The animals were kept individually in metabolic cages for 24-h urine and feces collection on days 1and 2 of the treatment. After the first 5-day course of chelation, the animals were left without any treatment for 5 days. ThereaRer, the animals were given a second 5-day course of treatment with the chelating agents and 24-h urine and feces collected for 2 days as before. After the final dose of chelators, 24-h urine samples were collected for certain biochemical estimations. All the animals were decapitated and blood, liver, kidney, brain, and femur removed. Biochemical Assays. Standard procedures were used to measure the activity of blood d-aminolevulinic acid dehydratase (ALAD)(81, zinc protoporphyrin (ZPP)(91, urinary &aminolevulinic acid (ALA)(IO),and total proteins (11).
Metal Estimations. Standard procedures were used to estimate lead (12)and zinc (13) in urine, feces, blood, liver, kidney, brain, and femur. The measured samples were digested completely in a mixture of concentrated nitric acid and perchloric acid (6:1), and the residue was dissolved in a known volume of 5% HNO3. The flame atomic absorption spectrometer (Perkin-Elmer 5000) was used t o record absorption at 283.3 and 213.9 nm for lead and zinc, respectively, in samples against suitable standards processed identically. The standard stock solutions (1mg mL-', as Pb or Zn) were prepared by dissolving analytical reagent-grade Pb (CH&00)2*3H20 (BDH, Poole, U.K.)or ZnS0~7H20(Merck, India) in 5% HN03 using double glass distilled water. Six working standard solutions within the linear range for each element were prepared by appropriate dilutions of the stock. StatisticalAnalysis. Student's "f' test was used to calculate the significance between normal animals and lead-exposed control. The effects of chelating agents were evaluated by twoway analysis of variance (randomized block design) for each parameter separately. The independent variables considered were six treatment groups (lead, lead CaNa2EDTA, lead DMSA, lead DMPS, lead + CaNazEDTA DMSA, and lead CaNazEDTA DMPS) in a single block and six replicates in six different blocks. The individual treatment difference between two groups was assessed by computation of least significant difference (14).
+
+
+
+
+
+
Results The administration of CaNa2EDTA or DMSA produced more urinary excretion of lead than DMPS, and the combination of CaNa2EDTA and DMSA was more effective than that of CaNazEDTA and DMPS in enhancing this property of the individual chelators. These effects were more prominent during the first 5-day course of treatment than the second course (Figure 1). The increase in fecal excretion of lead was observed only on the second day of the first course by all the chelators alone. However, combined treatments significantly mobilized lead in feces, on both days of two courses. The combination of CaNa2EDTA and DMSA was more efficient than that of CaNa2EDTA and DMPS (Figure 2). Both CaNazEDTA and DMSA were more effective than DMPS in decreasing lead concentrations of blood, liver, kindey, and femur, while brain lead was reduced by DMSA only. The combined treatments enhanced the efficacy of individual chelating agents to reduce hepatic,
Chem. Res. Toxicol., Vol. 7, No. 5, 1994 687
Chelation in Lead Intoxication 90-
X
80-
0'Treotment Day
70-
a2
60-
50)r
g
LO-
\
0
a
30-
*T
2010-
-
Course1
I1
CONTROL
I
I1
I1
CON02 EDTA
DMSA
I
I
11 DMPS
CaNO2EDTA
+
CaNo~EOlA + DMF'S
DMSA
Figure 2. Influence of chelating agents or their combinations on fecal excretion of lead in lead-exposed rat. Each bar represents mean f SE (6 values). *p < 0,001 vs normal animal (horizontal line). "p < 0,001, bp < 0.05 vs Pb-exposed control. "p < 0.001, y p < 0.01, *p < 0.05 vs CaNa2EDTA/DMSA/DMPS. tp < 0.001, ttp < 0.01 vs CaNazEDTA DMSA.
+
Table 1. Effect of Chelating Agents or Their Combinations on the Body Burden of Lead in Lead-Exposed Rap u d e of fresh tissue
treatment normal animal control
femur
blood Olg/lOO mL)
liver
kidney
brain
9.83 f 0.42 136.67 f 3.33b 80.83 f 3.96' 73.83 f 4.04c 107.50 f 6.29 68.33 f 4.01 76.66 f 4.40
2.35 f 0.32 13.38 f 0.57b 8.23 f 0.57c 6.01 f 0.68' 10.40 f 0.63d 4.43 f 0.33 6.93 f 0.42'
3.52 f 0.29 15.58 f 0.40b 12.72 f 0.65d 7.92 f 0.47" 14.73 f 1.14 4.28 f 0.27e 9.88 f 0.94fh
0.29 f 0.03 0.84 f O.OSb 0.69 f 0.04 0.53 f 0.06b 0.73 f 0.06 0.52 f 0.08 0.57 f 0.06
7.43 f 0.63 41.32 f 0.81b 19.13 f 0.88' 26.0 f 0.57c 37.25 f 0.96d 10.77 f 1.15e 15.92 f 1.278~~
CaNazEDTA DMSA DMPS CaNazEDTA + DMSA CaNazEDTA + DMPS Mean f SE (6 values). b p < 0.001 vs normal animal. ' p < 0.001. d p < 0.01 vs Pb-exposed control. e p < 0.001. f p < 0.01. 8 p vs CaNa2EDTA/DMSA/DMPS.h p < 0.001. ' p < 0.01 vs CaNazEDTA + DMSA.
