The Teratogenic Activity of a Thalidomide Analogus Rats on a Low-Zinc Diet ANDRE J. JACKSON * AND HERBERT J. SCHUMACHER
EM12
in
3
University of Cincinnati, Department of Environmental Health, Cincinnati, Ohio 45229
ABSTRACT The relationship between the teratogenicity of EMl2, 2-(2,6dioxopiperiden-3‘-yl) phthalimidine, a stable analogue of thalidomide, and zinc status in the maternal animal was investigated using pregnant rats on a lowzinc diet (1 ppm zinc, days 0-14 gestation) as the experimental model. Previous studies with this compound in rats fed a commercial diet at oral doses up to 250 m g k g per day for three days and intravenous doses up to 10 mg/kg per day for three days failed to produce “typical” thalidomide malformations. However, when a dose of 150 m g k g was given intraperitoneally to rats on a low-zinc diet, typical thalidomide malformations occurred with a n incidence of 57.5%. Teratological studies using diets low in mations seen with intravenous dosing were metals have established the importance of es- not the “typical” thalidomide malformations sential micronutrients in embryonic develop- (Schumacher et al., ’68). The compound is an ment (Hurley e t al., ’66). Experimental results analogue of thalidomide and is metabolized have established that normal pregnancy in similarly to thalidomide by opening the gluthe rat could be maintained with a specially tarimide ring (Jackson, ’72). With the glutariprepared soy diet containing nine parts per mide ring open, several functional groups bemillion (ppm) of zinc (Hurley, ’68). When come available to serve as potential sites for maternal animals were placed on a zinc-defi- metal complexation. In order to investigate cient diet from days 0-21 of gestation, 90% of this possible interaction, the marginally zincthe surviving young exhibited gross con- deficient rat was used as the animal model. genital malformations (Hurley, ’68). However, The rat on a low zinc diet provides a model zinc deficiency from days 0-10 of gestation which is marginal with regard to an essential produced young of which only 22%were mal- nutrient, and thus any agent given during the formed (Hurley et al., ’71).In addition, malfor- period of organogenesis which is capable of demations have been produced by feeding rats a creasing the zinc level, or of altering its activdiet of 3% ethylenediamine-N,N,N,N-tetra-ity, should theoretically act as a teratogenic acetic acid from days 6-21 of gestation while or fetotoxic agent. maintaining the animals on a diet containing MATERIALS A N D METHODS 100 ppm zinc. These teratological effects were Virgin female Wistar rats weighing apprevented by simultaneous supplementation with 1,000 ppm of dietary zinc (Swenerton e t proximately 200 g were fed Purina Laboratory al., ’71). These results indicate that a chelat- Chow and housed in galvanized cages prior to ing agent can change the zinc homeostasis in being mated. Estrous cycles were determined the maternal animal resulting in congenital by vaginal smears, and the animals in proestrous cycle were caged overnight with normalformations in the offspring. mal stock-fed males. Matings were confirmed This paper reports on the embryotoxicity of by the presence of sperm in the vaginal smear. phthalimiEMl2, 2-(2,6-dioxopiperiden-3‘-yl) Received Mar. 6, ‘78. Accepted Nov. 6. ’78. dine (fig. I), in the rat. This compound has ’ This work was supported by the National Institute of Environbeen shown to be teratogenic in rabbits and mental Health Science8 training Grant No. ES00127-04. monkeys (Schumacher et al., ’72). Rats have Present address: Howard University, College of Pharmacy and Sciences. Washington, D.C. 20059. proven to be resistant to the teratogenic Pharmacal Present addresa: National Center for Toxicological Research. action of this compound upon oral and intra- Jefferson, Arkansas 72079. ‘Harlan Industries, Cumberland, Indiana 46229. peritoneal administration, and the malforTERATOLOGY (1979) 19: 341-344.
341
342
ANDRE J. JACKSON AND HERBERT J. SCHUMACHER
Beginning with day 0 of gestation, the pregnant females were fed 10 g/day of the low-zinc diet for 14 days. The food jars were changed daily and water bottles were replaced every third day. On day 15 the animals were removed from the low-metal environment, put into galvanized cages and fed laboratory chow which contains an abundant supply of zinc. Thalidomide Maternal animals were injected intraperitoneally with 150 mg/kg of EM,,6 in 95% dimethyl sulfoxide (DMSO) and 5% water (prepared fresh daily in order to prevent any hydrolysis), on days 8, 9 and 10 of pregnancy. Control animals received a corresponding volume (i.e., 1 ml/kg) of 95% DMSO and 5% water or were injected with a corresponding volume of isotonic saline. The maternal animals were anesthetized on 2- (2,6-dioxopiperiden - ~ ' - ~ l phthalimidine ) day 20 of gestation, the fetuses removed and the followinn observations recorded: total number of implantation sites; number of dead EM12 or resorbed fetuses; weight and number of norFig. 1 Structural formulas of thalidomide and EM,,. mal or deformed live fetuses. One-third of the fetuses from each maternal rat were selected The day of finding sperm was considered day at random and fixed in Bouins solution and examined for internal malformations; the rezero of pregnancy. After pregnancy was confirmed by the pres- mainder were stained with alizarin red S and ence of sperm in the vaginal smear, the preg- examined under a dissecting microscope to nant females were housed in an isolated room check for any skeletal abnormalities. having a temperature of 24°C with 80%of the RESULTS air being recirculated. The room had a 12-hour The data for the marginally zinc-deficient dark and 12-hour light cycle. All stainless rats that were dosed intraperitoneally with steel items, including cages, were soaked in 1% ethylenediamine-N,N,N,N,-tetraacetic 150 m g k g of EM,, in 95% DMSO water are acid disodium salt, and glass items in 10% presented in table 1along with the values for nitric acid for six hours prior to use which ren- the control rats. Treatment of rats on a lowdered them zinc free. These items were then zinc diet with EMl2resulted in a n increase in thoroughly rinsed with deionized water before embryotoxicity as illustrated by the higher being used. All animals were housed in indi- percentage of resorptions - 67.6% for the vidual stainless steel cages on stainless steel treated animals compared with 1.8%for the racks and were provided with deionized water low-zinc control animals. Surviving animals ad libitum. There was no possibility of zinc had a 57.5% incidence of malformations compared with 12.9%for controls. The incidence of contamination from the drinking bottles. 5General Biochemicals, Chargrin Falls. Ohio 44022 - ComposiThe animals were fed an egg-white diet,5 is available from authors upon request. which had a zinc content of 0.8-1.0 ppm (con- tionDonated by Chemie Grunenthal, Stolberg/Rheinland, West firmed by atomic absorption spectroscopy). Germany.
-
TABLE 1
Embryopathic effects of E M , , in the offspring of low-zinc Wistar rats treated (i.p.1 with 150 m d k g of E M , , o n days 8-10 of pregnancy Dead or resorbed
Survivors malformed
Treatment No. of rats
Total implantations
No.
~
EM,? DMSO control
9
6
102 55
69 1
No.
%
67.6 1.8
% ~
~
19 7
~
_
_
_
57.5 12.9
_
_
TERATOGENIC ACTIVITY THALIDOMIDE ANALOGUE
343
Fig. 2 Alizarin red S-stained skeleton showing curly tail and retardation of ossification in sacral region. Fig. 3 Alizarin red S-stained skeleton exhibiting shortening of left arms as well as absence of the ulna from the arm.
TABLE 2
Summary of major EMlrinduced malformations in Wistar rats on a marginally zinc-deficient diet compared to controls
Total number of fetuses Total number of deformed fetuses Fetuses showing more than one malformation Total number of limb abnormalities Forelimbs Left only Total number of rib abnormalities Left only Right only Bilateral Total number of tail abnormalities
’ Significantly different from control at p
EM,2
Control
33 19
54
11 1
0 0
1 11 3 2 12
0 7 3 4
I
0
I
0
< 0.05, student’s t.teat
(Snedecor and Coehrsn. ‘67).
malformations for the saline-treated controls were similar to those observed for the DMSO controls. Table 2 summarizes the types of malforma-
tions observed in the low-zinc rats following treatment with 150 m g k g of EMlzintraperitoneally. Observed rib malformations included missing centers, lack of ossification of lumbar centra, split centra in the lumbar region, floating ribs, and missing thoracic vertebrae. Tail abnormalities included missing and fused vertebrae and curly tails (fig. 2). In this series of experiments one fetus from an EM,,treated rat had a shortened left forelimb and an absent ulna of that limb (fig. 3). The only soft-tissue malformations observed were bilateral hydronephrosis and hydroureter which were present in 16%of the EMl,-treated fetal survivors. DMSO-treated control animals exhibited a 12.9% malformation rate in survivors. The malformations were characterized by extra ribs on the seventh cervical vertebrae or absence of one pair of thoracic ribs. In no case
344
ANDRE J. JACKSON AND HERBERT J. SCHUMACHER
was there more than one malformation in a control animal. Furthermore, these malformations were seen in the offspring of only two maternal animals. The malformations observed are summarized in table 2. DISCUSSION
et al. (’711,in animals on a zinc-deficient diet. Therefore, i t appears that the dietary-zinc level in this study was sufficient to maintain a normal pregnancy in the rat, and that the effects of using DMSO as a solvent were minimal. Accordingly, the authors speculate that the low-zinc levels used in this study altered the normal-zinc equilibrium in such a manner that particular areas of actively proliferating tissues within the conceptus became more sensitive to embryotoxic or teratogenic damage than others. Possibly, metal-complexing agents, in this case EMlZ,further alter this balance resulting in a teratological, or embryotoxic effect, in the conceptus. The embryotoxic effects occur because of the inability of the maternal animal to mobilize zinc from body stores in amounts sufficient to supply the needs of the developing offspring.
Thalidomide and its derivative, EMl2,have been shown to be teratogenic in rabbits. However, a high incidence of the typical thalidomide malformations Le., tail, rib, limb, and multiple skeletal defects in a single fetus) have not been observed in rats with either of these compounds a t oral doses as high as 250 mgk. Also, EMlZ administered by intravenous injection (10 mg/kg) and orally (150 mg/kg) to rats on a Purina Lab Chow diet produced a low incidence of malformations (Schumacher et al., ’72). Furthermore, following intraperitoneal administration to rats on regular diet, ACKNOWLEDGMENTS the typical thalidomide malformations were The authors would like to acknowledge the not observed. The malformations seen were mainly hydronephrosis and pulmonary steno- technical assistance of Mrs. Cindy Bellini in sis. In fact, t h e malformations observed were collecting the data for preparation of this not the “typical EM,, malformations” as de- manuscript. LITERATURE CITED scribed in this paper or produced by thalidoHurley, L. S. 1968 Approaches to the study of nutrition mide in other species. in mammalian development. Fed. Proc., 27: No. 1, This study has shown that the rat, which is 193-198. refractory to thalidomide or EMIZ,is more Hurley, L. S., J. Gowan and H. Swenerton 1971 Teratogenic susceptible to the teratogenic effects of EMl2, effects of short-term and transitory zinc deficiency in rats. Teratology, 4: 199-204. when fed a low-zinc diet. In addition, the malformations observed were similar to those Hurley, L. S., and H. Swenerton 1966 Congenital malformations resulting from zinc deficiency in rats. Proc. SOC. caused by E M l 2in rabbits (Schumacher et al., for Biol. and Exp. Med., 123: 692-696. ’72) with the major effects being seen in the Jackson, A. J. 1972 Unpublished thesis results, University of Cincinnati. vertebral column, ribs, and tail. The major difference between the two species, in response Schumacher, H. J., D. A. Blake, J. M. Gurian and J. R. Gillehe 1968 A comparison of the teratogenic activity of to EM,2, is the incidence of limb malforthalidomide in rabbits and rats. J. Pharmacol. Exp. Ther., mations which was considerably lower for the Z60: No. 1, 189-200. low-zinc rats compared with rabbits (Schu- Schumacher, H. J., J. Terapane, R. L. Jordan and J. G. Wilson 1972 The teratogenic activity of a thalidomide macher et al., ’72) and which may be related to analogue, EM,,, in rabbits, rats, and monkeys. Teratolspecies differences in response to the comogy, 5: 233-240. pound. Snedecor, G. W., and W. G. Cochran 1967 Statistical Methods. Iowa State University Press, Ames, Iowa, pp. The control r a t s on t h e low-zinc diet 104-106. (Saline- or DMSO-treated) showed markedly Swenerton, J., L. S. Hurley 1971 Teratogenic effects of a low incidence of malformations and were of chelating agent and their prevention by zinc. Science, different types than those observed by Hurley 1973: 62-63.
EM12
in
3
University of Cincinnati, Department of Environmental Health, Cincinnati, Ohio 45229
ABSTRACT The relationship between the teratogenicity of EMl2, 2-(2,6dioxopiperiden-3‘-yl) phthalimidine, a stable analogue of thalidomide, and zinc status in the maternal animal was investigated using pregnant rats on a lowzinc diet (1 ppm zinc, days 0-14 gestation) as the experimental model. Previous studies with this compound in rats fed a commercial diet at oral doses up to 250 m g k g per day for three days and intravenous doses up to 10 mg/kg per day for three days failed to produce “typical” thalidomide malformations. However, when a dose of 150 m g k g was given intraperitoneally to rats on a low-zinc diet, typical thalidomide malformations occurred with a n incidence of 57.5%. Teratological studies using diets low in mations seen with intravenous dosing were metals have established the importance of es- not the “typical” thalidomide malformations sential micronutrients in embryonic develop- (Schumacher et al., ’68). The compound is an ment (Hurley e t al., ’66). Experimental results analogue of thalidomide and is metabolized have established that normal pregnancy in similarly to thalidomide by opening the gluthe rat could be maintained with a specially tarimide ring (Jackson, ’72). With the glutariprepared soy diet containing nine parts per mide ring open, several functional groups bemillion (ppm) of zinc (Hurley, ’68). When come available to serve as potential sites for maternal animals were placed on a zinc-defi- metal complexation. In order to investigate cient diet from days 0-21 of gestation, 90% of this possible interaction, the marginally zincthe surviving young exhibited gross con- deficient rat was used as the animal model. genital malformations (Hurley, ’68). However, The rat on a low zinc diet provides a model zinc deficiency from days 0-10 of gestation which is marginal with regard to an essential produced young of which only 22%were mal- nutrient, and thus any agent given during the formed (Hurley et al., ’71).In addition, malfor- period of organogenesis which is capable of demations have been produced by feeding rats a creasing the zinc level, or of altering its activdiet of 3% ethylenediamine-N,N,N,N-tetra-ity, should theoretically act as a teratogenic acetic acid from days 6-21 of gestation while or fetotoxic agent. maintaining the animals on a diet containing MATERIALS A N D METHODS 100 ppm zinc. These teratological effects were Virgin female Wistar rats weighing apprevented by simultaneous supplementation with 1,000 ppm of dietary zinc (Swenerton e t proximately 200 g were fed Purina Laboratory al., ’71). These results indicate that a chelat- Chow and housed in galvanized cages prior to ing agent can change the zinc homeostasis in being mated. Estrous cycles were determined the maternal animal resulting in congenital by vaginal smears, and the animals in proestrous cycle were caged overnight with normalformations in the offspring. mal stock-fed males. Matings were confirmed This paper reports on the embryotoxicity of by the presence of sperm in the vaginal smear. phthalimiEMl2, 2-(2,6-dioxopiperiden-3‘-yl) Received Mar. 6, ‘78. Accepted Nov. 6. ’78. dine (fig. I), in the rat. This compound has ’ This work was supported by the National Institute of Environbeen shown to be teratogenic in rabbits and mental Health Science8 training Grant No. ES00127-04. monkeys (Schumacher et al., ’72). Rats have Present address: Howard University, College of Pharmacy and Sciences. Washington, D.C. 20059. proven to be resistant to the teratogenic Pharmacal Present addresa: National Center for Toxicological Research. action of this compound upon oral and intra- Jefferson, Arkansas 72079. ‘Harlan Industries, Cumberland, Indiana 46229. peritoneal administration, and the malforTERATOLOGY (1979) 19: 341-344.
341
342
ANDRE J. JACKSON AND HERBERT J. SCHUMACHER
Beginning with day 0 of gestation, the pregnant females were fed 10 g/day of the low-zinc diet for 14 days. The food jars were changed daily and water bottles were replaced every third day. On day 15 the animals were removed from the low-metal environment, put into galvanized cages and fed laboratory chow which contains an abundant supply of zinc. Thalidomide Maternal animals were injected intraperitoneally with 150 mg/kg of EM,,6 in 95% dimethyl sulfoxide (DMSO) and 5% water (prepared fresh daily in order to prevent any hydrolysis), on days 8, 9 and 10 of pregnancy. Control animals received a corresponding volume (i.e., 1 ml/kg) of 95% DMSO and 5% water or were injected with a corresponding volume of isotonic saline. The maternal animals were anesthetized on 2- (2,6-dioxopiperiden - ~ ' - ~ l phthalimidine ) day 20 of gestation, the fetuses removed and the followinn observations recorded: total number of implantation sites; number of dead EM12 or resorbed fetuses; weight and number of norFig. 1 Structural formulas of thalidomide and EM,,. mal or deformed live fetuses. One-third of the fetuses from each maternal rat were selected The day of finding sperm was considered day at random and fixed in Bouins solution and examined for internal malformations; the rezero of pregnancy. After pregnancy was confirmed by the pres- mainder were stained with alizarin red S and ence of sperm in the vaginal smear, the preg- examined under a dissecting microscope to nant females were housed in an isolated room check for any skeletal abnormalities. having a temperature of 24°C with 80%of the RESULTS air being recirculated. The room had a 12-hour The data for the marginally zinc-deficient dark and 12-hour light cycle. All stainless rats that were dosed intraperitoneally with steel items, including cages, were soaked in 1% ethylenediamine-N,N,N,N,-tetraacetic 150 m g k g of EM,, in 95% DMSO water are acid disodium salt, and glass items in 10% presented in table 1along with the values for nitric acid for six hours prior to use which ren- the control rats. Treatment of rats on a lowdered them zinc free. These items were then zinc diet with EMl2resulted in a n increase in thoroughly rinsed with deionized water before embryotoxicity as illustrated by the higher being used. All animals were housed in indi- percentage of resorptions - 67.6% for the vidual stainless steel cages on stainless steel treated animals compared with 1.8%for the racks and were provided with deionized water low-zinc control animals. Surviving animals ad libitum. There was no possibility of zinc had a 57.5% incidence of malformations compared with 12.9%for controls. The incidence of contamination from the drinking bottles. 5General Biochemicals, Chargrin Falls. Ohio 44022 - ComposiThe animals were fed an egg-white diet,5 is available from authors upon request. which had a zinc content of 0.8-1.0 ppm (con- tionDonated by Chemie Grunenthal, Stolberg/Rheinland, West firmed by atomic absorption spectroscopy). Germany.
-
TABLE 1
Embryopathic effects of E M , , in the offspring of low-zinc Wistar rats treated (i.p.1 with 150 m d k g of E M , , o n days 8-10 of pregnancy Dead or resorbed
Survivors malformed
Treatment No. of rats
Total implantations
No.
~
EM,? DMSO control
9
6
102 55
69 1
No.
%
67.6 1.8
% ~
~
19 7
~
_
_
_
57.5 12.9
_
_
TERATOGENIC ACTIVITY THALIDOMIDE ANALOGUE
343
Fig. 2 Alizarin red S-stained skeleton showing curly tail and retardation of ossification in sacral region. Fig. 3 Alizarin red S-stained skeleton exhibiting shortening of left arms as well as absence of the ulna from the arm.
TABLE 2
Summary of major EMlrinduced malformations in Wistar rats on a marginally zinc-deficient diet compared to controls
Total number of fetuses Total number of deformed fetuses Fetuses showing more than one malformation Total number of limb abnormalities Forelimbs Left only Total number of rib abnormalities Left only Right only Bilateral Total number of tail abnormalities
’ Significantly different from control at p
EM,2
Control
33 19
54
11 1
0 0
1 11 3 2 12
0 7 3 4
I
0
I
0
< 0.05, student’s t.teat
(Snedecor and Coehrsn. ‘67).
malformations for the saline-treated controls were similar to those observed for the DMSO controls. Table 2 summarizes the types of malforma-
tions observed in the low-zinc rats following treatment with 150 m g k g of EMlzintraperitoneally. Observed rib malformations included missing centers, lack of ossification of lumbar centra, split centra in the lumbar region, floating ribs, and missing thoracic vertebrae. Tail abnormalities included missing and fused vertebrae and curly tails (fig. 2). In this series of experiments one fetus from an EM,,treated rat had a shortened left forelimb and an absent ulna of that limb (fig. 3). The only soft-tissue malformations observed were bilateral hydronephrosis and hydroureter which were present in 16%of the EMl,-treated fetal survivors. DMSO-treated control animals exhibited a 12.9% malformation rate in survivors. The malformations were characterized by extra ribs on the seventh cervical vertebrae or absence of one pair of thoracic ribs. In no case
344
ANDRE J. JACKSON AND HERBERT J. SCHUMACHER
was there more than one malformation in a control animal. Furthermore, these malformations were seen in the offspring of only two maternal animals. The malformations observed are summarized in table 2. DISCUSSION
et al. (’711,in animals on a zinc-deficient diet. Therefore, i t appears that the dietary-zinc level in this study was sufficient to maintain a normal pregnancy in the rat, and that the effects of using DMSO as a solvent were minimal. Accordingly, the authors speculate that the low-zinc levels used in this study altered the normal-zinc equilibrium in such a manner that particular areas of actively proliferating tissues within the conceptus became more sensitive to embryotoxic or teratogenic damage than others. Possibly, metal-complexing agents, in this case EMlZ,further alter this balance resulting in a teratological, or embryotoxic effect, in the conceptus. The embryotoxic effects occur because of the inability of the maternal animal to mobilize zinc from body stores in amounts sufficient to supply the needs of the developing offspring.
Thalidomide and its derivative, EMl2,have been shown to be teratogenic in rabbits. However, a high incidence of the typical thalidomide malformations Le., tail, rib, limb, and multiple skeletal defects in a single fetus) have not been observed in rats with either of these compounds a t oral doses as high as 250 mgk. Also, EMlZ administered by intravenous injection (10 mg/kg) and orally (150 mg/kg) to rats on a Purina Lab Chow diet produced a low incidence of malformations (Schumacher et al., ’72). Furthermore, following intraperitoneal administration to rats on regular diet, ACKNOWLEDGMENTS the typical thalidomide malformations were The authors would like to acknowledge the not observed. The malformations seen were mainly hydronephrosis and pulmonary steno- technical assistance of Mrs. Cindy Bellini in sis. In fact, t h e malformations observed were collecting the data for preparation of this not the “typical EM,, malformations” as de- manuscript. LITERATURE CITED scribed in this paper or produced by thalidoHurley, L. S. 1968 Approaches to the study of nutrition mide in other species. in mammalian development. Fed. Proc., 27: No. 1, This study has shown that the rat, which is 193-198. refractory to thalidomide or EMIZ,is more Hurley, L. S., J. Gowan and H. Swenerton 1971 Teratogenic susceptible to the teratogenic effects of EMl2, effects of short-term and transitory zinc deficiency in rats. Teratology, 4: 199-204. when fed a low-zinc diet. In addition, the malformations observed were similar to those Hurley, L. S., and H. Swenerton 1966 Congenital malformations resulting from zinc deficiency in rats. Proc. SOC. caused by E M l 2in rabbits (Schumacher et al., for Biol. and Exp. Med., 123: 692-696. ’72) with the major effects being seen in the Jackson, A. J. 1972 Unpublished thesis results, University of Cincinnati. vertebral column, ribs, and tail. The major difference between the two species, in response Schumacher, H. J., D. A. Blake, J. M. Gurian and J. R. Gillehe 1968 A comparison of the teratogenic activity of to EM,2, is the incidence of limb malforthalidomide in rabbits and rats. J. Pharmacol. Exp. Ther., mations which was considerably lower for the Z60: No. 1, 189-200. low-zinc rats compared with rabbits (Schu- Schumacher, H. J., J. Terapane, R. L. Jordan and J. G. Wilson 1972 The teratogenic activity of a thalidomide macher et al., ’72) and which may be related to analogue, EM,,, in rabbits, rats, and monkeys. Teratolspecies differences in response to the comogy, 5: 233-240. pound. Snedecor, G. W., and W. G. Cochran 1967 Statistical Methods. Iowa State University Press, Ames, Iowa, pp. The control r a t s on t h e low-zinc diet 104-106. (Saline- or DMSO-treated) showed markedly Swenerton, J., L. S. Hurley 1971 Teratogenic effects of a low incidence of malformations and were of chelating agent and their prevention by zinc. Science, different types than those observed by Hurley 1973: 62-63.
