- Email: [email protected]
Reduction in cadmium teratogenesis by prior cadmium exposure
ENVIRONMENTAL
RESEARCH
Reduction
18,
347- 350 (1979)
in Cadmium Cadmium
Teratogenesis Exposure
by Prior
VERGIL H. FERM AND WILLIAM M. LAYTON, Department
of AnatomylCytology,
Dartmouth Medical Hampshire 03755
School,
JR. Hanover,
New
Received August 16, 1978 The administration of cadmium 24-48 hr prior to an optimal teratogenic dose reduces both the number and severity of congenital malformations in hamster embryos. Intraperitoneal administration is more effective in indueing this protective effect than is the subcutaneous route. Oral administration of cadmium at the same pretreatment intervals does not seem to afford any protection. It is suggested that this protection is due to the induction of maternal synthesis of a metal-binding protein, possibly metallothionein. The significance of this protective induction as it relates to the possible role of cadmium in human malformations as well as teratogenic testing is discussed.
INTRODUCTION
Cadmium is a potent teratogen in a number of laboratory animals, affecting the development of a variety of organ systems (Ferm, 1971; Ferm and Layton, 1978). This teratogenic action of cadmium can be modified in a number of ways. The simultaneous administration of zinc will reduce, almost completely, the deleterious effect of cadmium on the hamster embryo. This protective effect of zinc is apparently limited to a brief time span of a few hours (Ferm, 1972). Selenium also has a similar protective effect against cadmium in the same animal model system (Holmberg and Ferm, 1969). It has also been known for some time that small amounts of cadmium can cause severe damage to the rodent testis and that zinc will protect against the testisdamaging effect of cadmium (Parizek, 1960; Webb, 1972). More recently it has been shown that small amounts of cadmium administered to male mice and rats prior to a testis-damaging dose will also significantly protect the testis from cadmium-induced testicular damage (Ito and Sawauchi, 1965). Semba et al. (1974) have reported preliminary findings on the reduction of cadmium-induced cranial abnormalities in mice following pretreatment with small doses of cadmium. The present study reports the striking reduction in number and type of malformations caused by cadmium in the hamster by prior treatment with cadmium utilizing different routes of administration. MATERIALS
AND METHODS
Pregnant hamsters were divided into five groups. The first group (I) received 2mg/kg cadmium sulfate in demineralized water intravenously on the morning of Day 8 of gestation. Groups II and III received a single subcutaneous injection of 1 mg/kg CdSO, on either Day 6 (Group II) or Day 7 (Group III) followed by an intravenous injection of 2 mg/kg on Day 8 as in the first group. Groups IV and V received a single intraperitoneal injection of 2 mg/kg CdSO, on either Day 6 (Group IV) or Day 7 (Group V) followed by an intravenous injection of CdSO, on 347 0013-9351/79/020347-04$02.00/O Copyright All rights
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
348
FERM
AND
LAYTON
Day 8 as in Group 1. The techniques for the injection of animals have been described previously (Ferm, 1967). In addition, 12 animals were given 2 mg/kg CdSO, by gastric intubation on Day 6 of gestation and then injected intravenously with 2 mg/kg CdSO, on Day 8 of gestation. The fetuses were recovered on Day 13 and examined for gross congenital defects. The number of resorption sites was also determined. RESULTS
The data are summarized in Tables 1 and 2. Administration of cadmium 24-48 hr prior to the critical teratogenic-sensitive period of Day 8 in this species reduces the teratogenic impact of cadmium. A preexposure period of 48 hr is more effective than a 24-hr period, and an intraperitoneal route slightly more effective than a subcutaneous route. Not only is the frequency of malformations reduced by prior exposure to cadmium but also it is apparent from Table 2 that the pattern of malformations is shifted toward a less severe type. Thus, no exencephalies, eye defects, or facial abnormalities were seen in those fetuses from mothers pretreated with cadmium. The feeding of 2 mg/kg CdSOI by gavage on Day 6 or 7 followed by an intravenous injection of 2 mg/kg CdSO, on Day 8 did not reduce the number of resorptions or malformations as compared to an intravenous injection of 2 mg/kg CdSO, on Day 8 alone. DISCUSSION
Both time and route of pretreatment seem to affect the results, and these would appear to be in predictable patterns. A 48-hr preexposure to cadmium is more effective than a 24-hr preexposure period and the intraperitoneal route is more effective than the subcutaneous route. This can probably best be explained by the fact that longer time intervals and the intraperitoneal route of administration permit more cadmium to reach the protein synthesis sites where cadmium is bound more rapidly. The oral route of administration in these experiments is not effective in protecting the embryo probably because cadmium is not absorbed fast enough or in enough quantities to induce protective protein synthesis in the 4%hr interval. Cadmium exposure is known to result in the induction of specific metal-binding proteins in a variety of tissues (Winge and Rajagopalan, 1972). Synthesis of such a protein (metallothionein) in rats may occur in as little as 5-24 hr following a subcutaneous injection of cadmium. The induction of such a metal-binding protein probably accounts for the protection afforded by subtoxic amounts of cadmium prior to a testis-damaging dose. Such an explanation may also account for the protective effect of prior exposure to cadmium against a teratogenic amount of cadmium as demonstrated in these experiments and in those reported by Semba et al. (1974) in mice. This phenomenon of protective induction is rather unique in experimental teratology. If the same situation holds in man, the prior and chronic exposure to low concentrations of cadmium in the human may well be the explanation for the lack of clear identification of cadmium as a human teratogen, and in that sense such exposure may not necessarily be detrimental. A suspicion of teratogenicity has not been raised as yet in the extensive investigations of Itai-itai disease in Japan although certain mutagenic possibilities of cadmium have been summarized (Ferm and Layton, 1978).
REDUCTION
IN
CD
TERATOGENESIS
TABLE METHODS
AND RESULTS OF CADMIUM
BY
349
CD
1
ADMINISTRATION
TO FIVE GROUPS
OF PREGNANT
HAMSTERS
No. of litters with one or more malformed fetuses / total No. of litters (%)
Total No. of implantation sites
Total No. of resorptions (%)
No. of fetuses with one or more malformations (%)
Cd (2)b iv Day 8
19127 (70)
364
78 (21)
107 (37)
Cd (1) SC Day 6 Cd (2) iv Day 8
4/11 (36)
145
5 (3)
16 (11)
Cd (1) SCDay 7 Cd (2) iv Day 8
4113 (31)
177
18 (10)
50 (31)
Cd (2) ip Day 6 Cd (2) iv Day 8
l/9 (II)
126
19 (15)
1 (1)
Cd (2) ip Day 7 Cd (2) iv Day 8
6117 (35)
208
38 (18)
13 (7)
Group a I II
III
IV
V
a iv, intravenous; ip, intraperitoneal; SC, subcutaneous. b Values in parentheses indicate amount of CdSO, administered in mg/kg.
TABLE
2
TYPES OF MALFORMATIONS”
Group I II III IV V
Exencephaly 1 0 0 0 0
Encephalocele 71 6 0 0 3
Eye defects*
Face and lip
71 1 0 0 0
42 5 0 0 0
Forelimb 10 1 2 0 0
Hindlimb 18 3 2 1 0
Ribs
Tail
58 8 15 0 3
36 2 2 1 4
’ See Table 1 for treatment regimen and number of animals in each group. Multiple malformations in a fetus are recorded separately. b Includes microphthalmia and anophthalmia.
Nonpregnant mice treated with small doses of lead and injected 48 hr later with a larger dose of lead showed a marked decrease in mortality (Yoshikawa, 1968). Sanai et al. (1972) showed that pretreatment of rats with small doses of lead caused a change in the distribution and excretion of lead following a subsequent injection of that metal, possibly accounting for this protective mechanism. This phenomenon of induction as demonstrated by these results should em-
FERM AND LAYTON
350
phasize the fact least one study embryogenesis. some instances,
that all drug testing for teratogenic properties should include at of a single dose of a test compound during the critical stages of Multiple doses of a compound throughout early gestation may, in mask a significant teratogenic threat. ACKNOWLEDGMENT
This work was supported by USPHS Grant ES-00697.
REFERENCES Ferm, V. H. (1967). The use of the golden hamster in experimental teratology. Lab. Anim. Cure 17, 452-462. Ferm, V. H. (1971). Developmental malformations induced by cadmium. A study of timed injections during embryogenesis. Biol. Neonatorum Z. 19(l). lOl- 107. Ferm, V. H. (1972). Teratogenic effect of metals on mammalian embryos. In “Advances in Teratology” (D. H. M. Woollam, Ed.), Vol. V, Chap. 2, pp. 51-75. Logos Press, London. Ferm, V. H., and Layton, W. M., Jr. (1978). Teratogenic and mutagenic effects of cadmium. In “Biogeochemistry of Cadmium” (J. 0. Nriagu, Ed.), Ann Arbor Science Pub., Ann Arbor, Mich. Holmberg, R. E., Jr., and Ferm, V. H. (1969). Interrelationships of selenium, cadmium, and arsenic in mammalian teratogenesis. Arch. Environ. Health 18, 873-877. Ito, T., and Sawauchi, K. (1965). Inhibitory effects of cadmium-induced testicular damage by pretreatment with smaller cadmium dose. Okajimas Folia Anat. Japan. 42, 107- 117. Parizek, J. (l%O). Sterilization of the male by cadmium salts. J. Reprod. Ferr. 1, 294-298. Sanai, G. H., Hasegawa, T., and Yoshikawa, H. (1972). Pretreatment of rats with lead in experimental acute lead poisoning. J. Occup. Med. 14, 310-305. Semba, R., Ohta, K., and Yamamura, H. (1974). Low-dose preadministration of cadmium prevents cadmium-induced excencephalia. Teratology 10,96A. Webb, M. (1972). Biochemical effects of Cdl+ injury in the rat and mouse testis. J. Reprod. Ferr. 30, 83-98. Winge, D. R., and Rajagopalan, K. V. (1972). Purification and some properties of Cd-binding protein from rat liver. Arch. Biochem. Biophys. 153, 755-762. Yoshikawa, H. (1968). Tolerance to lethal doses of metals in mice pretreated with their low doses. Ind. Health
6, 88-89.
RESEARCH
Reduction
18,
347- 350 (1979)
in Cadmium Cadmium
Teratogenesis Exposure
by Prior
VERGIL H. FERM AND WILLIAM M. LAYTON, Department
of AnatomylCytology,
Dartmouth Medical Hampshire 03755
School,
JR. Hanover,
New
Received August 16, 1978 The administration of cadmium 24-48 hr prior to an optimal teratogenic dose reduces both the number and severity of congenital malformations in hamster embryos. Intraperitoneal administration is more effective in indueing this protective effect than is the subcutaneous route. Oral administration of cadmium at the same pretreatment intervals does not seem to afford any protection. It is suggested that this protection is due to the induction of maternal synthesis of a metal-binding protein, possibly metallothionein. The significance of this protective induction as it relates to the possible role of cadmium in human malformations as well as teratogenic testing is discussed.
INTRODUCTION
Cadmium is a potent teratogen in a number of laboratory animals, affecting the development of a variety of organ systems (Ferm, 1971; Ferm and Layton, 1978). This teratogenic action of cadmium can be modified in a number of ways. The simultaneous administration of zinc will reduce, almost completely, the deleterious effect of cadmium on the hamster embryo. This protective effect of zinc is apparently limited to a brief time span of a few hours (Ferm, 1972). Selenium also has a similar protective effect against cadmium in the same animal model system (Holmberg and Ferm, 1969). It has also been known for some time that small amounts of cadmium can cause severe damage to the rodent testis and that zinc will protect against the testisdamaging effect of cadmium (Parizek, 1960; Webb, 1972). More recently it has been shown that small amounts of cadmium administered to male mice and rats prior to a testis-damaging dose will also significantly protect the testis from cadmium-induced testicular damage (Ito and Sawauchi, 1965). Semba et al. (1974) have reported preliminary findings on the reduction of cadmium-induced cranial abnormalities in mice following pretreatment with small doses of cadmium. The present study reports the striking reduction in number and type of malformations caused by cadmium in the hamster by prior treatment with cadmium utilizing different routes of administration. MATERIALS
AND METHODS
Pregnant hamsters were divided into five groups. The first group (I) received 2mg/kg cadmium sulfate in demineralized water intravenously on the morning of Day 8 of gestation. Groups II and III received a single subcutaneous injection of 1 mg/kg CdSO, on either Day 6 (Group II) or Day 7 (Group III) followed by an intravenous injection of 2 mg/kg on Day 8 as in the first group. Groups IV and V received a single intraperitoneal injection of 2 mg/kg CdSO, on either Day 6 (Group IV) or Day 7 (Group V) followed by an intravenous injection of CdSO, on 347 0013-9351/79/020347-04$02.00/O Copyright All rights
@ 1979 by Academic Press, Inc. of reproduction in any form reserved.
348
FERM
AND
LAYTON
Day 8 as in Group 1. The techniques for the injection of animals have been described previously (Ferm, 1967). In addition, 12 animals were given 2 mg/kg CdSO, by gastric intubation on Day 6 of gestation and then injected intravenously with 2 mg/kg CdSO, on Day 8 of gestation. The fetuses were recovered on Day 13 and examined for gross congenital defects. The number of resorption sites was also determined. RESULTS
The data are summarized in Tables 1 and 2. Administration of cadmium 24-48 hr prior to the critical teratogenic-sensitive period of Day 8 in this species reduces the teratogenic impact of cadmium. A preexposure period of 48 hr is more effective than a 24-hr period, and an intraperitoneal route slightly more effective than a subcutaneous route. Not only is the frequency of malformations reduced by prior exposure to cadmium but also it is apparent from Table 2 that the pattern of malformations is shifted toward a less severe type. Thus, no exencephalies, eye defects, or facial abnormalities were seen in those fetuses from mothers pretreated with cadmium. The feeding of 2 mg/kg CdSOI by gavage on Day 6 or 7 followed by an intravenous injection of 2 mg/kg CdSO, on Day 8 did not reduce the number of resorptions or malformations as compared to an intravenous injection of 2 mg/kg CdSO, on Day 8 alone. DISCUSSION
Both time and route of pretreatment seem to affect the results, and these would appear to be in predictable patterns. A 48-hr preexposure to cadmium is more effective than a 24-hr preexposure period and the intraperitoneal route is more effective than the subcutaneous route. This can probably best be explained by the fact that longer time intervals and the intraperitoneal route of administration permit more cadmium to reach the protein synthesis sites where cadmium is bound more rapidly. The oral route of administration in these experiments is not effective in protecting the embryo probably because cadmium is not absorbed fast enough or in enough quantities to induce protective protein synthesis in the 4%hr interval. Cadmium exposure is known to result in the induction of specific metal-binding proteins in a variety of tissues (Winge and Rajagopalan, 1972). Synthesis of such a protein (metallothionein) in rats may occur in as little as 5-24 hr following a subcutaneous injection of cadmium. The induction of such a metal-binding protein probably accounts for the protection afforded by subtoxic amounts of cadmium prior to a testis-damaging dose. Such an explanation may also account for the protective effect of prior exposure to cadmium against a teratogenic amount of cadmium as demonstrated in these experiments and in those reported by Semba et al. (1974) in mice. This phenomenon of protective induction is rather unique in experimental teratology. If the same situation holds in man, the prior and chronic exposure to low concentrations of cadmium in the human may well be the explanation for the lack of clear identification of cadmium as a human teratogen, and in that sense such exposure may not necessarily be detrimental. A suspicion of teratogenicity has not been raised as yet in the extensive investigations of Itai-itai disease in Japan although certain mutagenic possibilities of cadmium have been summarized (Ferm and Layton, 1978).
REDUCTION
IN
CD
TERATOGENESIS
TABLE METHODS
AND RESULTS OF CADMIUM
BY
349
CD
1
ADMINISTRATION
TO FIVE GROUPS
OF PREGNANT
HAMSTERS
No. of litters with one or more malformed fetuses / total No. of litters (%)
Total No. of implantation sites
Total No. of resorptions (%)
No. of fetuses with one or more malformations (%)
Cd (2)b iv Day 8
19127 (70)
364
78 (21)
107 (37)
Cd (1) SC Day 6 Cd (2) iv Day 8
4/11 (36)
145
5 (3)
16 (11)
Cd (1) SCDay 7 Cd (2) iv Day 8
4113 (31)
177
18 (10)
50 (31)
Cd (2) ip Day 6 Cd (2) iv Day 8
l/9 (II)
126
19 (15)
1 (1)
Cd (2) ip Day 7 Cd (2) iv Day 8
6117 (35)
208
38 (18)
13 (7)
Group a I II
III
IV
V
a iv, intravenous; ip, intraperitoneal; SC, subcutaneous. b Values in parentheses indicate amount of CdSO, administered in mg/kg.
TABLE
2
TYPES OF MALFORMATIONS”
Group I II III IV V
Exencephaly 1 0 0 0 0
Encephalocele 71 6 0 0 3
Eye defects*
Face and lip
71 1 0 0 0
42 5 0 0 0
Forelimb 10 1 2 0 0
Hindlimb 18 3 2 1 0
Ribs
Tail
58 8 15 0 3
36 2 2 1 4
’ See Table 1 for treatment regimen and number of animals in each group. Multiple malformations in a fetus are recorded separately. b Includes microphthalmia and anophthalmia.
Nonpregnant mice treated with small doses of lead and injected 48 hr later with a larger dose of lead showed a marked decrease in mortality (Yoshikawa, 1968). Sanai et al. (1972) showed that pretreatment of rats with small doses of lead caused a change in the distribution and excretion of lead following a subsequent injection of that metal, possibly accounting for this protective mechanism. This phenomenon of induction as demonstrated by these results should em-
FERM AND LAYTON
350
phasize the fact least one study embryogenesis. some instances,
that all drug testing for teratogenic properties should include at of a single dose of a test compound during the critical stages of Multiple doses of a compound throughout early gestation may, in mask a significant teratogenic threat. ACKNOWLEDGMENT
This work was supported by USPHS Grant ES-00697.
REFERENCES Ferm, V. H. (1967). The use of the golden hamster in experimental teratology. Lab. Anim. Cure 17, 452-462. Ferm, V. H. (1971). Developmental malformations induced by cadmium. A study of timed injections during embryogenesis. Biol. Neonatorum Z. 19(l). lOl- 107. Ferm, V. H. (1972). Teratogenic effect of metals on mammalian embryos. In “Advances in Teratology” (D. H. M. Woollam, Ed.), Vol. V, Chap. 2, pp. 51-75. Logos Press, London. Ferm, V. H., and Layton, W. M., Jr. (1978). Teratogenic and mutagenic effects of cadmium. In “Biogeochemistry of Cadmium” (J. 0. Nriagu, Ed.), Ann Arbor Science Pub., Ann Arbor, Mich. Holmberg, R. E., Jr., and Ferm, V. H. (1969). Interrelationships of selenium, cadmium, and arsenic in mammalian teratogenesis. Arch. Environ. Health 18, 873-877. Ito, T., and Sawauchi, K. (1965). Inhibitory effects of cadmium-induced testicular damage by pretreatment with smaller cadmium dose. Okajimas Folia Anat. Japan. 42, 107- 117. Parizek, J. (l%O). Sterilization of the male by cadmium salts. J. Reprod. Ferr. 1, 294-298. Sanai, G. H., Hasegawa, T., and Yoshikawa, H. (1972). Pretreatment of rats with lead in experimental acute lead poisoning. J. Occup. Med. 14, 310-305. Semba, R., Ohta, K., and Yamamura, H. (1974). Low-dose preadministration of cadmium prevents cadmium-induced excencephalia. Teratology 10,96A. Webb, M. (1972). Biochemical effects of Cdl+ injury in the rat and mouse testis. J. Reprod. Ferr. 30, 83-98. Winge, D. R., and Rajagopalan, K. V. (1972). Purification and some properties of Cd-binding protein from rat liver. Arch. Biochem. Biophys. 153, 755-762. Yoshikawa, H. (1968). Tolerance to lethal doses of metals in mice pretreated with their low doses. Ind. Health
6, 88-89.