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The effects of various chelating agents on the teratogenicity of lead nitrate in rats
TOXICOLOGY
AND
APPLIED
The
31,434+42(1975)
PHARMACOLOGY
Effects of Various Chelating Agents on the Teratogenicity of Lead Nitrate in Rats1 R. M. MCCLAIN
Department
Hoffmann-La of Experimental Received
AND
J. J. SIEKIERKA
Roche Incorporated, Research Division, Pathology and Toxicology, Nutley, New Jersey March
18, 1974;
accepted
August
07110
12,1974
The Effects of Various Chelating Agents on the Teratogenicity of Lead Nitrate in Rats. MCCLAIN, R. M. AND SIEKIERKA, J. J. (1975). Toxicol. Appl. Phuvn~~col. 31, 434-442. Lead nitrate alone or lead chelated with ethylenediaminetetraacetic acid CEDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), and penicillamine (PEN) was administered iv to pregnant rats on days 9, 1 I, or 16 of gestation (day 1 = vaginal plug) at a 1 : I molar ratio of chelating agent to lead at a dosage of 35 or 50 mg/kg with respect to lead nitrate. Lead nitrate alone caused resorptions and fetal malformations on day 9, resorptions on day 1I, and resorptions and hydrocephalus following treatment on day 16. The lead-chelate complex either reduced or equaled but did not enhance the overall embryo or fetal toxicity. There were no qualitatively different effects produced with chelated lead versus lead alone. The chelating agents were more effective after treatment on gestation day 9 that after days I I or 16. Ethylenediaminetetraacetic acid produced the greatest reduction in overall effect, with PEN and NTA intermediate, while IDA provided the least overall protective effects. The administration of chelating agents to pregnant animals can produce teratogenic effects. This effect may be due to interactions with trace metals affecting their distribution, excretion, function, or toxicity. Chelating agents may also interact with teratogens to augment or ameliorate their actions. There are many examples in the literature in which chelating agents may either decrease or increase the toxicity of various metals. Prolonged administration of EDTA produced malformations in rats (TuchmannDuplessis and Mercier-Parot, 1956) and dimercaprol produced malformations in mice (Nishimura and Takagaki, 1959). Swenerton and Hurley (I 97 I ) demonstrated that the addition of supplemental zinc in the maternal diet prevented EDTA malformations, suggesting that these malformations are due to a zinc deficiency. Matsomoto et al. (1967) demonstrated that penicillamine reduced the teratogenicity of methyl mercury in the rat. Hood and Pike (1973) found that the administration of BAL alleviated arsenateinduced teratogenesis in mice. Chernoff and Courtney (1970) reported that NTA could enhance the teratogenicity of cadmium chloride and methyl mercury in rats and mice; however, Nolen et al. (1972a.b) found that NTA administered to pregnant rats did not enhance-cadmium chloride or methyl mercury teratogenesis and may have brought about a slight reduction in the teratogenicity of these metals. Although chelating agents are used to alleviate lead intoxication by increasing the excretion of lead, it is also known that they can cause a transient worsening of the clinical I Presented 1972.
in part
at the 5th International
Copyright ~8 1975 by Academic Press, Inc. All rights ofreproduction in any form reserved. Printed in Great Britain
Congress 43-l
on Pharmacology,
San Francisco,
California,
CHELATING AGENTS AND LEAD TERATOGENICITY
435
signsof lead intoxication (Chisolm, 1968).Under certain experimental conditions they may enhance lead toxicity (Braun et al., 1946; Peters and Stocken, 1947; Germuth and Eagle, 1948; Tanabe, 1959). Thus, in this study an assessmentof the teratogenicity and fetal toxicity of 1: 1 molar ratios of lead to various chelating agents was made at three stagesof gestation. The chelating agents selectedrepresent a range of dissociation constants of the lead-chelate complex and included penicillamine (PEN) and three aminopolycarboxylic acids: ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and iminodiacetic acid (IDA). METHODS
Female Charles River CD rats (225-275 g) were mated overnight. The following morning was designated day 1 of gestation if a vaginal plug was found. Solutions for injection were prepared by dissolving the appropriate quantities of lead nitrate in 40 ml of distilled water’and of the chelating agents’ in 10 ml distilled water. Equimolar amounts of both lead and chelating agentswere mixed just prior to injection. A few drops of dilute HCl was added to the IDA mixture to prevent a slight precipitation. Animals under light ether anesthesia were injected via the saphenous vein (5 ml/kg) with lead nitrate or the lead-chelate complex on days 9. 11, or 16 of gestation at a dosage of 50 mg/kg with respect to lead nitrate. On day 22 of gestation rats were killed using excessCO,, and a cesareansection was performed. The numbers of live, dead, and resorbed fetusesand implantation siteswere recorded. Fetuseswere weighed, examined for external malformations, and then fixed in Bouin’s solution. The headsof fetusesfrom rats treated on day 16 of gestation were sectioned and examined for hydrocephalus. Hydrocephalus was scored as severe, moderate, or slight basedon the degreeof dilatation of each of the two lateral ventricles. In severe hydrocephalus the dilatation of the lateral ventricles nearly completely filled the distended cranium, resulting in compressionand extensive atrophy of neural tissue. Statistical analysis of data was performed using the Mann-Whitney Rank Sum Test (Goldstein, 1964). Significance levels were chosen at p < 0.05. RESULTS
Day 9 Treatnlerlt The iv administration of lead nitrate (50 mg/kg) on day 9 of gestation resulted in resorption of 24 “/, of the embryos and malformations in 54 % of the surviving fetuses (Fig. 1). Table 1 lists the types and incidences of malformations produced with lead nitrate alone; the most prominent are defectsin the posterior third of the fetus, consisting of a fusion of the hind limbs (sirenomelia), urorectal and tail malformations. The administration of PbIDA, PbNTA, PbPEN, or PbEDTA significantly reduced the incidencesof malformations as compared to lead alone (Fig. 1 and Table 1). A reduction in the resorption rate was also observed with PbPEN, PbNTA, and PbEDTA; however, the difference with PbPEN was not statistically significant. The resorption rate with PbIDA was moderately higher than with lead alone; however, the overall effects on resorptions and malformations were lessthan that of lead alone. * The chelating agents used were the following: IDA, iminodiacetic acid disodium salt monohydrate; NTA, nitrilotriacetic acid disodium salt (Eastman Kodak Company); EDTA, ethylenediaminetetraacetic acid disodium salt (Fisher Scientific Co.); and PEN, penicillamine (Merck Sharp and Dohme).
436
MCCLAIN AND SIEKIERKA
Thus, essentially complete protection against the teratogenic effects of lead on day 9 of gestation was observed with NTA, PEN, and EDTA. Iminodiacetic acid, although reducing the effects of lead, provided the least protection.
TREATMENT
LEAD
LEA0
e IDA
LEAD 8 NTA
LEAD C, PEN
LEA0 8 EOTA
FIG. 1. The effects of chelating agents on the embryolethal and teratogenic effects of lead administered iv on day 9 ofgestation at a dosage of 50 mg/kg of lead alone and with an equimolar amount of chelating agent. An asterisk indicates a significant difference (p < 0.05) from lead nitrate alone. Stippled columns, percent resorbed; clear columns, percent malformed.
Dav 11 Treatment Treatment of pregnant rats on day 11 of gestation with lead nitrate (50 mg/kg, iv) resulted in a 100% embryolethal effect, an effect greater than that observed with administration on day 9 of gestation (Fig. 2). A reduced incidence of resorptions was observed with the IDA, NTA, or EDTA but not the PEN-lead chelate. Ethylene diaminetetraacetic acid provided the greatest protection (Fig. 2). No external malformations were observed in surviving fetuses, 10060 ‘; kf k 605 Y % 40 20
TREATMENT
LEAD
LEAD BIDA
LEAD 8 NTA
LEAD & PEN
LEAD e EOTA
FIG. 2. The effects of chelating agents on the embryolethal effects of lead administered iv on day 11 of gestation at a dosage of 50 mg/kg of lead nitrate alone and with an equimolar amount of chelating agent. An asterisk indicates a significant difference (p < 0.05) from lead nitratealone. Five animals per group were used. Stippled columns, percent resorbed.
Day 16 Treatment Treatment of pregnant rats on day 16 of gestation with lead nitrate (50 mg/kg, iv) resulted in a high incidence of resorptions with all surviving fetusesmalformed (Fig. 3),
CHELATING
AGENTS
AND
LEAD
TERATOGENICITY
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438
MCCLAIN AND SIEKIERKA
exhibiting moderate to severehydrocephalus as indicated in Table 2. A decreasein the resorption rate and incidence of malformed fetuseswas observed with PbIDA, PbPEN, and PbEDTA treatment (Fig. 3). The reduced resorptions observed with PbIDA were not statistically significant. Table 2 shows that no fetus within the PbEDTA group exhibited hydrocephalus and the severity of hydrocephalus was greatly reduced with
60
* ** rl $
* *
TREATkENT
LEAD
LEAD 8 IDA
LEAD 8 NTA
LEAD
a PEN
LEAD
8 EDTA
FIG. 3. The effects of chelating agents on lead-induced resorptions and malformations administered iv on day 16 of gestation at a dosage of 50 mg/kg of lead nitrate alone and with an equimolar amount of chelating agent. An asterisk indicates a significant difference (p < 0.05) from lead nitrate alone. StippIed columns, percent resorbed; clear columns, percent malformed.
too
20
TREATMENT
LEAD
LEAD
8. IDA
LEAD
8 NTA
FIG. 4. The effects of chelating agents on lead-induced resorptions and malformations, administered iv on day 16 of gestation at a dosage of 35 mg/kg of lead nitrate alone and with an equimolar amount of chelating agent. Stippled columns, percent resorbed; clear columns, percent malformed.
PbPEN treatment since most ventricles showedonly a slight dilatation. The severity of hydrocephalus in affected fetuses in the PbIDA group was only moderately reduced, while PbNTA did not differ from lead nitrate treatment alone. The effects of PbIDA and PbNTA were also determined at a lower dosageof lead (35 mg/kg. iv, on day 16 of gestation), A high incidence of resorbed and malformed fetuseswas observed with lead alone (Fig. 4 and Table 3). A slight reduction in resorptions wasnoted with PbNTA (Fig. 4); however, the severity of hydrocephalus in affected
AGENTS
8’ 12d (75)P 4 (25) 0 (0)
8
Lead 6
OF CHELATING
17 17 (50) 14 (41) 3 (9)
29
Lead + IDAb 6
ON LEAD-INDUCED
2
4 8 (100) 0 (0) 0 (0)
4
Lead + NTAb 6
HYDROCEPHALUS
IN
41 4 (5) 17(21) 61 (74)
67
Lead + PENb 6
FETAL RATS’
a Lead nitrate was administered iv on day 16 of gestation at a dosage of 50 mg/kg with or without an equimolar amount of chelate. * See Table 1. c Number of fetuses exhibiting hydrocephalus. d Number of lateral ventricles scored as indicated. e Percentage of lateral ventricles scored as indicated.
Hydrocephalus Severe Moderate Slight
No. of fetuses examined:
Treatment group No. of dams dosed :
EFFECTS
TABLE
0 (0) 0 (0)
56
Lead + EDTAb 4
e F ci ;;1
5
5 ?z
p
440
MCCLAIN
AND
SIEKIERKA
fetuseswas not appreciably different from lead alone. PbIDh treatment resulted in a slightly decreasedincidence of resorbed fetusesand reduced severity of hydrocephalus. These differences were not statistically significant. TABLE EFFECTS
OF CHELATING
Treatment group No. of dams dosed
No. of fetusesexamined Hydrocephalus Severe
Moderate Slight
3
AGENTS ON LEAD-INDUCED IN FETAL RATP
Lead 9
Lead+ IDA” 7
33 33’ 33d (50)’ 22 (33) II (17)
HYDROCEPHALUS
53 48 17 (18) 33 (34) 46 (48)
Lead + NTA” IO 52 50 52 (53) 38 (38) 10 (10)
L? Lead nitrate was administered iv on day 16 of gestation at a dosage of 35 mg/kg with or without an equimolar amount of chelate. * See Table
1.
( Number of fetuses exhibiting hydrocephalus. d Number of lateral ventricles scored as indicated. e Percentage of lateral ventricles scored as indicated.
In summary. the iv administration of lead nitrate complexed with various chelating agents resulted in lessembryotoxic and fetotoxic effects than that produced by lead nitrate alone. The sametypes of malformations were observed with the lead-chelate complexes aswith lead alone. Although the effectivenessin reducing resorptions or the severity of malformations varied with individual chelates on the various days of gestation studied, it appearsthat EDTA produced the greatest reduction with PEN and NTA intermediate and IDA resulting in the least overall protective effects.
DISCUSSION
In this study a direct assessmentof the embryo and fetal toxicity of preformed leadchelate complexeswasmade at three different stagesof gestation. On the 3 days of gestation studied. lead produced definite and reproducible endpoints against which the toxicity of the chelate complexescould be determined (McClain and Becker, 1975).The results clearly indicate that under theseconditions lead-chelate complexeseither reduced or equaled but did not enhance the embryotoxic and fetotoxic effects of lead alone. While it is theoretically possiblefor lead asa chelate complex to reach an otherwise inaccessiblesite in the embryo or fetus to elicit a qualitatively different toxic response,this does not appear to be the casein these studies since all malformations when observed were characteristic of the lead ion. Some factors that should be considered in interpreting theseresults are the route of administration, the molar ratio of the metal to chelating agent, the relative dissociabiiity of theselead-chelate complexes, and the stageof gestation during administration of the complexes.
CHELATING
AGENTS
AND
LEAD
TERATOGENICITY
441
Since chelating agents can increase the oral absorption of metals, their use in acute oral lead intoxication is contraindicated (Harvey, 1965). Sapieka (1954) demonstrated excellent oral absorption of PbEDTA, and it has been shown that chelated lead will dissociate to a certain extent in vivo (Rubin and DiChiro, 1959; Hammond and Aronson, 1960). Since lead is poorly absorbed orally in the rat and no teratogenicity can be elicited by the oral route (McClain and Becker, unpublished observations), the iv route of administration was used so that results would not be influenced by variable absorption from the gut. Conceivably, a large increase in the oral absorption of lead caused by a chelating agent with sufficient dissociation in vivo could intensify the effects of lead: however, this aspect was not investigated in this study. In these studies a 1 : 1 molar ratio of metal to chelate was employed. Schubert (196 I) states that an alteration of the metal-chelate ratio can vary the toxic effects of metals. Tanabe (1959) found an intensification of lead toxicity when CaEDTA and lead acetate were injected simultaneously unless there was a sufficient molar excess of CaEDTA. Braun et al. (1946) and Germuth and Eagle (1948) demonstrated decreased survival in rats and rabbits due to insufficient doses of BAL. Peters and Stocken (1947) demonstrated that the 1 : 1 lipid soluble complex of BAL-marpharside was more toxic than the arsenical alone whereas the water-soluble 2 : 1 molar ratio of BAL-marpharside showed protective effects. In the present studies, presumably, a larger ratio of each chelating agent to lead would result in even greater protective effects; however, it is uncertain what effects molar ratios of less than 1: 1 may be. There was a rough correlation of effectiveness with dissociation constants. Ethylenediaminetetraacetic acid formed the most stable complex and provided the best protection, followed by PEN and then NTA. Although the IDA complex appeared to dissociate more readily irz zko than the other agents (McClain and Siekierka, 1975) more protective effects were afforded by IDA on day 1 I than PEN and more on day 16 than NTA. Differences were apparent not only in the relative effectiveness of the various chelating agents in protecting against the toxic effects of lead but also with the individual chelating agents administered on individual days of gestation. The most effective chelating agent was EDTA, providing essentially complete protection on all 3 days of gestation studied. Penicillamine was effective on days 9 and 16 of gestation while no protection on day 11 was observed since both Pb and PbPEN were 100 “/, embryolethal. Moderate protective effects were exhibited by IDA on all 3 days studied and NTA, while providing complete protection on day 9, did not protect against the CNS damage on day 16. The reasons for differences arising with a Iead-chelate complex on different days of gestation are not known. The simultaneous effects of chelating agents on maternal blood concentrations, maternal distribution and excretion, diffusibility into the embryo prior to allantoic placentation, placental permeability and transfer, and distribution within the embryo and fetus are very complex interactions. The results of these experiments represent the summed total of these various possible effects. Some of these factors have been further investigated by us and are reported in a subsequent publication (McClain and Siekierka, 1975). In conclusion, the results of this study with respect to the embryo and fetus are in agreement with the generally observed protective effects of chelating agents in heavy metal toxicity.
442
MCCLAIN
AND
SIEKIERKA
REFERENCES H. A., LUSKY, L. M. AND CALVERY, H. 0. (1946). The efficacy of 2.3-dimercaptopropanol (BAL) in the therapy of poisoning by compounds of antimony, bismuth, chromium, mercury, and nickel. J. Phurmacol. Exg. Ther. 87, 119-125. CHERNOFF, N. AND COURTNEY, K. D. (1970). Maternal and fetal effects of NTA, NTA and cadmium, NTA and mercury, NTA and nutritional inbalance in mice and rats. Progress report from the National Institute of Environmental Health Sciences, Dec. 1,1970, Research Triangle Park, North Carolina. CHISOLM, J. J. (1968). The use of chelating agents in the treatment of acute lead intoxication in childhood. J. Pediat. 73, l-38. GERMUTH, F. G., JR. AND EAGLE, H. (1948). The efficacy of BAL (2,3-dimercaptopropanol) in the treatment of experimental lead poisoning in rabbits. J. Phartmcol. Exp. Thu. 92, 397410. GOLDSTUN, A. (1964). Biostatistics, An hltroductory Text, pp. 55-59. Macmillan, New York. HAMMOND, P. B. AND ARONSON, A. L. (1960). The mobilization of lead in cattle: A comparative study of various chelating agents. Am. N. Y. Acad. Sci. 88, 498-511. HARVEY, S. C. (1965). Heavy Metals. In The Phurnmological Basis of Therapeutics (L. S. Goodman and A. Gilman, Eds.), 3rd ed., p. 971. Macmillan, New York. HOOD, R. D. AND PIKE, C. T. (1973). BAL alleviation ofarsenate-induced teratogenesisinmice. Teratology 6, 235-238. MATSOMOTO, H., SUZUKI, A., MORITA, C., NAKAMURA, K. AND SAEKI, S. (1967). Preventive effect of penicillamine on the brain defect of fetal rat poisoned transplacentally with methyl mercury. Li/k Sri. 6, 2321-2326. MCCLAIN, R. M. AND BECKER, B. A. (1975). Teratogenicity, fetal toxicity and placental transfer of lead nitrate in rats. Toxicol. Appl. Pharmacol. 31, 72-82. MCCLAIN, R. M. AND SIEKIERKA, J. J. (1975). The placental transfer of lead chelate complexes in the rat. Toxicol. Appl. Pharmacol. 31, 443-451. NISHIMURA, H. AND TAKAGAKI, S. (1959). Developmental anomalies in mice induced by 2,3dimercaptopropanol (BAL). Amt. Rec. 135, 261-267. NOLEN, G. A., BUEHLER, E. V., GEIL, R. G. ANDGOLDENTHAL,E. I. (1972a). Effects oftrisodium nitrilotriacetate oncadmium and methyl mercury toxicity and teratogenicity in rats. Toxicol. Appl. Pharmacol. 23. 222-237. NOLEN, G. A., BOHNE, R. L. AND BUEHLER, E. V. (1972b). Effects of trisodium nitrilotriacetate trisodium citrate and a trisodium nitrilotriacetate-ferric chloride mixture on cadmium and methyl mercury toxicity and teratogenesis in rats. Toxicol. Appl. Pharmacol. 23,238-250. PETERS, R. A. AND STOCKEN, L. A. (1947). Preparation and pharmacological properties of 4-hydroxymethyl-2-(3’-amino-4’-hydroxyphenyl)-I : 3 dithia-2-arsa cyclopentane (marpharside-BAL compound). Biochem. J. 41, 53-56. RUBIN, M. AND DICHIRO, G. (1959). Chelates as possible contrast media. Am. N. Y. Acad. Sci. 78, 764-792. SAPIEKA, N. (1954). Lead EDTA complex, a water soluble contrast medium. South African Med. J. 28, 759-762. SCHUBERT, J. (1961). Biological aspects of metal binding. II. suppl. 10, Fed. Proc. Fed. Amer. Sot. Exp. Biol. Suppl. 20, 203. SWENERTON, H. AND HURLEY, L. S. (1971). Teratogenic effects of a chelating agent and their prevention by zinc. Science 173, 62-64. TANABE, Y. (1959). Investigations of the metabolism of 8-aminolevulinic acid and porphobilinogen in lead poisoning. III. On the effects of Ca-EDTA upon the intermediary metabolism of porphyrin, Jap. J. Nat. Health 28, 560-567. TUCHMAN-DUPLESSIS, H. AND MERCIER-PAROT, L. (1956). Influence d’un corps de chelation, I’acide ~thylenediaminet&a&tique sur la gestation et le d&elopement foetal du rat. C. R. Acad. Sri. Paris 243, 1064-1066. BRAUN,
AND
APPLIED
The
31,434+42(1975)
PHARMACOLOGY
Effects of Various Chelating Agents on the Teratogenicity of Lead Nitrate in Rats1 R. M. MCCLAIN
Department
Hoffmann-La of Experimental Received
AND
J. J. SIEKIERKA
Roche Incorporated, Research Division, Pathology and Toxicology, Nutley, New Jersey March
18, 1974;
accepted
August
07110
12,1974
The Effects of Various Chelating Agents on the Teratogenicity of Lead Nitrate in Rats. MCCLAIN, R. M. AND SIEKIERKA, J. J. (1975). Toxicol. Appl. Phuvn~~col. 31, 434-442. Lead nitrate alone or lead chelated with ethylenediaminetetraacetic acid CEDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), and penicillamine (PEN) was administered iv to pregnant rats on days 9, 1 I, or 16 of gestation (day 1 = vaginal plug) at a 1 : I molar ratio of chelating agent to lead at a dosage of 35 or 50 mg/kg with respect to lead nitrate. Lead nitrate alone caused resorptions and fetal malformations on day 9, resorptions on day 1I, and resorptions and hydrocephalus following treatment on day 16. The lead-chelate complex either reduced or equaled but did not enhance the overall embryo or fetal toxicity. There were no qualitatively different effects produced with chelated lead versus lead alone. The chelating agents were more effective after treatment on gestation day 9 that after days I I or 16. Ethylenediaminetetraacetic acid produced the greatest reduction in overall effect, with PEN and NTA intermediate, while IDA provided the least overall protective effects. The administration of chelating agents to pregnant animals can produce teratogenic effects. This effect may be due to interactions with trace metals affecting their distribution, excretion, function, or toxicity. Chelating agents may also interact with teratogens to augment or ameliorate their actions. There are many examples in the literature in which chelating agents may either decrease or increase the toxicity of various metals. Prolonged administration of EDTA produced malformations in rats (TuchmannDuplessis and Mercier-Parot, 1956) and dimercaprol produced malformations in mice (Nishimura and Takagaki, 1959). Swenerton and Hurley (I 97 I ) demonstrated that the addition of supplemental zinc in the maternal diet prevented EDTA malformations, suggesting that these malformations are due to a zinc deficiency. Matsomoto et al. (1967) demonstrated that penicillamine reduced the teratogenicity of methyl mercury in the rat. Hood and Pike (1973) found that the administration of BAL alleviated arsenateinduced teratogenesis in mice. Chernoff and Courtney (1970) reported that NTA could enhance the teratogenicity of cadmium chloride and methyl mercury in rats and mice; however, Nolen et al. (1972a.b) found that NTA administered to pregnant rats did not enhance-cadmium chloride or methyl mercury teratogenesis and may have brought about a slight reduction in the teratogenicity of these metals. Although chelating agents are used to alleviate lead intoxication by increasing the excretion of lead, it is also known that they can cause a transient worsening of the clinical I Presented 1972.
in part
at the 5th International
Copyright ~8 1975 by Academic Press, Inc. All rights ofreproduction in any form reserved. Printed in Great Britain
Congress 43-l
on Pharmacology,
San Francisco,
California,
CHELATING AGENTS AND LEAD TERATOGENICITY
435
signsof lead intoxication (Chisolm, 1968).Under certain experimental conditions they may enhance lead toxicity (Braun et al., 1946; Peters and Stocken, 1947; Germuth and Eagle, 1948; Tanabe, 1959). Thus, in this study an assessmentof the teratogenicity and fetal toxicity of 1: 1 molar ratios of lead to various chelating agents was made at three stagesof gestation. The chelating agents selectedrepresent a range of dissociation constants of the lead-chelate complex and included penicillamine (PEN) and three aminopolycarboxylic acids: ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and iminodiacetic acid (IDA). METHODS
Female Charles River CD rats (225-275 g) were mated overnight. The following morning was designated day 1 of gestation if a vaginal plug was found. Solutions for injection were prepared by dissolving the appropriate quantities of lead nitrate in 40 ml of distilled water’and of the chelating agents’ in 10 ml distilled water. Equimolar amounts of both lead and chelating agentswere mixed just prior to injection. A few drops of dilute HCl was added to the IDA mixture to prevent a slight precipitation. Animals under light ether anesthesia were injected via the saphenous vein (5 ml/kg) with lead nitrate or the lead-chelate complex on days 9. 11, or 16 of gestation at a dosage of 50 mg/kg with respect to lead nitrate. On day 22 of gestation rats were killed using excessCO,, and a cesareansection was performed. The numbers of live, dead, and resorbed fetusesand implantation siteswere recorded. Fetuseswere weighed, examined for external malformations, and then fixed in Bouin’s solution. The headsof fetusesfrom rats treated on day 16 of gestation were sectioned and examined for hydrocephalus. Hydrocephalus was scored as severe, moderate, or slight basedon the degreeof dilatation of each of the two lateral ventricles. In severe hydrocephalus the dilatation of the lateral ventricles nearly completely filled the distended cranium, resulting in compressionand extensive atrophy of neural tissue. Statistical analysis of data was performed using the Mann-Whitney Rank Sum Test (Goldstein, 1964). Significance levels were chosen at p < 0.05. RESULTS
Day 9 Treatnlerlt The iv administration of lead nitrate (50 mg/kg) on day 9 of gestation resulted in resorption of 24 “/, of the embryos and malformations in 54 % of the surviving fetuses (Fig. 1). Table 1 lists the types and incidences of malformations produced with lead nitrate alone; the most prominent are defectsin the posterior third of the fetus, consisting of a fusion of the hind limbs (sirenomelia), urorectal and tail malformations. The administration of PbIDA, PbNTA, PbPEN, or PbEDTA significantly reduced the incidencesof malformations as compared to lead alone (Fig. 1 and Table 1). A reduction in the resorption rate was also observed with PbPEN, PbNTA, and PbEDTA; however, the difference with PbPEN was not statistically significant. The resorption rate with PbIDA was moderately higher than with lead alone; however, the overall effects on resorptions and malformations were lessthan that of lead alone. * The chelating agents used were the following: IDA, iminodiacetic acid disodium salt monohydrate; NTA, nitrilotriacetic acid disodium salt (Eastman Kodak Company); EDTA, ethylenediaminetetraacetic acid disodium salt (Fisher Scientific Co.); and PEN, penicillamine (Merck Sharp and Dohme).
436
MCCLAIN AND SIEKIERKA
Thus, essentially complete protection against the teratogenic effects of lead on day 9 of gestation was observed with NTA, PEN, and EDTA. Iminodiacetic acid, although reducing the effects of lead, provided the least protection.
TREATMENT
LEAD
LEA0
e IDA
LEAD 8 NTA
LEAD C, PEN
LEA0 8 EOTA
FIG. 1. The effects of chelating agents on the embryolethal and teratogenic effects of lead administered iv on day 9 ofgestation at a dosage of 50 mg/kg of lead alone and with an equimolar amount of chelating agent. An asterisk indicates a significant difference (p < 0.05) from lead nitrate alone. Stippled columns, percent resorbed; clear columns, percent malformed.
Dav 11 Treatment Treatment of pregnant rats on day 11 of gestation with lead nitrate (50 mg/kg, iv) resulted in a 100% embryolethal effect, an effect greater than that observed with administration on day 9 of gestation (Fig. 2). A reduced incidence of resorptions was observed with the IDA, NTA, or EDTA but not the PEN-lead chelate. Ethylene diaminetetraacetic acid provided the greatest protection (Fig. 2). No external malformations were observed in surviving fetuses, 10060 ‘; kf k 605 Y % 40 20
TREATMENT
LEAD
LEAD BIDA
LEAD 8 NTA
LEAD & PEN
LEAD e EOTA
FIG. 2. The effects of chelating agents on the embryolethal effects of lead administered iv on day 11 of gestation at a dosage of 50 mg/kg of lead nitrate alone and with an equimolar amount of chelating agent. An asterisk indicates a significant difference (p < 0.05) from lead nitratealone. Five animals per group were used. Stippled columns, percent resorbed.
Day 16 Treatment Treatment of pregnant rats on day 16 of gestation with lead nitrate (50 mg/kg, iv) resulted in a high incidence of resorptions with all surviving fetusesmalformed (Fig. 3),
CHELATING
AGENTS
AND
LEAD
TERATOGENICITY
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438
MCCLAIN AND SIEKIERKA
exhibiting moderate to severehydrocephalus as indicated in Table 2. A decreasein the resorption rate and incidence of malformed fetuseswas observed with PbIDA, PbPEN, and PbEDTA treatment (Fig. 3). The reduced resorptions observed with PbIDA were not statistically significant. Table 2 shows that no fetus within the PbEDTA group exhibited hydrocephalus and the severity of hydrocephalus was greatly reduced with
60
* ** rl $
* *
TREATkENT
LEAD
LEAD 8 IDA
LEAD 8 NTA
LEAD
a PEN
LEAD
8 EDTA
FIG. 3. The effects of chelating agents on lead-induced resorptions and malformations administered iv on day 16 of gestation at a dosage of 50 mg/kg of lead nitrate alone and with an equimolar amount of chelating agent. An asterisk indicates a significant difference (p < 0.05) from lead nitrate alone. StippIed columns, percent resorbed; clear columns, percent malformed.
too
20
TREATMENT
LEAD
LEAD
8. IDA
LEAD
8 NTA
FIG. 4. The effects of chelating agents on lead-induced resorptions and malformations, administered iv on day 16 of gestation at a dosage of 35 mg/kg of lead nitrate alone and with an equimolar amount of chelating agent. Stippled columns, percent resorbed; clear columns, percent malformed.
PbPEN treatment since most ventricles showedonly a slight dilatation. The severity of hydrocephalus in affected fetuses in the PbIDA group was only moderately reduced, while PbNTA did not differ from lead nitrate treatment alone. The effects of PbIDA and PbNTA were also determined at a lower dosageof lead (35 mg/kg. iv, on day 16 of gestation), A high incidence of resorbed and malformed fetuseswas observed with lead alone (Fig. 4 and Table 3). A slight reduction in resorptions wasnoted with PbNTA (Fig. 4); however, the severity of hydrocephalus in affected
AGENTS
8’ 12d (75)P 4 (25) 0 (0)
8
Lead 6
OF CHELATING
17 17 (50) 14 (41) 3 (9)
29
Lead + IDAb 6
ON LEAD-INDUCED
2
4 8 (100) 0 (0) 0 (0)
4
Lead + NTAb 6
HYDROCEPHALUS
IN
41 4 (5) 17(21) 61 (74)
67
Lead + PENb 6
FETAL RATS’
a Lead nitrate was administered iv on day 16 of gestation at a dosage of 50 mg/kg with or without an equimolar amount of chelate. * See Table 1. c Number of fetuses exhibiting hydrocephalus. d Number of lateral ventricles scored as indicated. e Percentage of lateral ventricles scored as indicated.
Hydrocephalus Severe Moderate Slight
No. of fetuses examined:
Treatment group No. of dams dosed :
EFFECTS
TABLE
0 (0) 0 (0)
56
Lead + EDTAb 4
e F ci ;;1
5
5 ?z
p
440
MCCLAIN
AND
SIEKIERKA
fetuseswas not appreciably different from lead alone. PbIDh treatment resulted in a slightly decreasedincidence of resorbed fetusesand reduced severity of hydrocephalus. These differences were not statistically significant. TABLE EFFECTS
OF CHELATING
Treatment group No. of dams dosed
No. of fetusesexamined Hydrocephalus Severe
Moderate Slight
3
AGENTS ON LEAD-INDUCED IN FETAL RATP
Lead 9
Lead+ IDA” 7
33 33’ 33d (50)’ 22 (33) II (17)
HYDROCEPHALUS
53 48 17 (18) 33 (34) 46 (48)
Lead + NTA” IO 52 50 52 (53) 38 (38) 10 (10)
L? Lead nitrate was administered iv on day 16 of gestation at a dosage of 35 mg/kg with or without an equimolar amount of chelate. * See Table
1.
( Number of fetuses exhibiting hydrocephalus. d Number of lateral ventricles scored as indicated. e Percentage of lateral ventricles scored as indicated.
In summary. the iv administration of lead nitrate complexed with various chelating agents resulted in lessembryotoxic and fetotoxic effects than that produced by lead nitrate alone. The sametypes of malformations were observed with the lead-chelate complexes aswith lead alone. Although the effectivenessin reducing resorptions or the severity of malformations varied with individual chelates on the various days of gestation studied, it appearsthat EDTA produced the greatest reduction with PEN and NTA intermediate and IDA resulting in the least overall protective effects.
DISCUSSION
In this study a direct assessmentof the embryo and fetal toxicity of preformed leadchelate complexeswasmade at three different stagesof gestation. On the 3 days of gestation studied. lead produced definite and reproducible endpoints against which the toxicity of the chelate complexescould be determined (McClain and Becker, 1975).The results clearly indicate that under theseconditions lead-chelate complexeseither reduced or equaled but did not enhance the embryotoxic and fetotoxic effects of lead alone. While it is theoretically possiblefor lead asa chelate complex to reach an otherwise inaccessiblesite in the embryo or fetus to elicit a qualitatively different toxic response,this does not appear to be the casein these studies since all malformations when observed were characteristic of the lead ion. Some factors that should be considered in interpreting theseresults are the route of administration, the molar ratio of the metal to chelating agent, the relative dissociabiiity of theselead-chelate complexes, and the stageof gestation during administration of the complexes.
CHELATING
AGENTS
AND
LEAD
TERATOGENICITY
441
Since chelating agents can increase the oral absorption of metals, their use in acute oral lead intoxication is contraindicated (Harvey, 1965). Sapieka (1954) demonstrated excellent oral absorption of PbEDTA, and it has been shown that chelated lead will dissociate to a certain extent in vivo (Rubin and DiChiro, 1959; Hammond and Aronson, 1960). Since lead is poorly absorbed orally in the rat and no teratogenicity can be elicited by the oral route (McClain and Becker, unpublished observations), the iv route of administration was used so that results would not be influenced by variable absorption from the gut. Conceivably, a large increase in the oral absorption of lead caused by a chelating agent with sufficient dissociation in vivo could intensify the effects of lead: however, this aspect was not investigated in this study. In these studies a 1 : 1 molar ratio of metal to chelate was employed. Schubert (196 I) states that an alteration of the metal-chelate ratio can vary the toxic effects of metals. Tanabe (1959) found an intensification of lead toxicity when CaEDTA and lead acetate were injected simultaneously unless there was a sufficient molar excess of CaEDTA. Braun et al. (1946) and Germuth and Eagle (1948) demonstrated decreased survival in rats and rabbits due to insufficient doses of BAL. Peters and Stocken (1947) demonstrated that the 1 : 1 lipid soluble complex of BAL-marpharside was more toxic than the arsenical alone whereas the water-soluble 2 : 1 molar ratio of BAL-marpharside showed protective effects. In the present studies, presumably, a larger ratio of each chelating agent to lead would result in even greater protective effects; however, it is uncertain what effects molar ratios of less than 1: 1 may be. There was a rough correlation of effectiveness with dissociation constants. Ethylenediaminetetraacetic acid formed the most stable complex and provided the best protection, followed by PEN and then NTA. Although the IDA complex appeared to dissociate more readily irz zko than the other agents (McClain and Siekierka, 1975) more protective effects were afforded by IDA on day 1 I than PEN and more on day 16 than NTA. Differences were apparent not only in the relative effectiveness of the various chelating agents in protecting against the toxic effects of lead but also with the individual chelating agents administered on individual days of gestation. The most effective chelating agent was EDTA, providing essentially complete protection on all 3 days of gestation studied. Penicillamine was effective on days 9 and 16 of gestation while no protection on day 11 was observed since both Pb and PbPEN were 100 “/, embryolethal. Moderate protective effects were exhibited by IDA on all 3 days studied and NTA, while providing complete protection on day 9, did not protect against the CNS damage on day 16. The reasons for differences arising with a Iead-chelate complex on different days of gestation are not known. The simultaneous effects of chelating agents on maternal blood concentrations, maternal distribution and excretion, diffusibility into the embryo prior to allantoic placentation, placental permeability and transfer, and distribution within the embryo and fetus are very complex interactions. The results of these experiments represent the summed total of these various possible effects. Some of these factors have been further investigated by us and are reported in a subsequent publication (McClain and Siekierka, 1975). In conclusion, the results of this study with respect to the embryo and fetus are in agreement with the generally observed protective effects of chelating agents in heavy metal toxicity.
442
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AND
SIEKIERKA
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