Effects of Salicylamide and Protein Restriction on the Skeletal Development of the Rat Fetus ENID KNIGHT AND DAPHNE A. ROE Diuision of Nutritional Sciences, Cornell University, Ithaca, New York 14853
ABSTRACT Salicylamide has previously been shown to be teratogenic to the rat. Aims of the present study were to examine separate effects and interactions of salicylamide administration and protein restriction on development of the rat fetus. In experiments using Holtzman rats, the percentage of fetal resorptions was significantly increased by drug treatment and by protein restriction. Malformations were seen in fetuses from dams given the drug from the fifth to eleventh days of gestation; the incidence of these malformations being independent of the diet. Factors affecting appearance of ossification centers included drug dosage, placental and fetal weight and litter size. Protein restriction had no significant effect on the development of centers of ossification. It is concluded that salicylamide, as well as protein restriction, produces adverse effects on pregnancy outcome in the rat. In earlier studies, the non-narcotic, analgesic drug, salicylamide (SA) has been shown to be teratogenic to the rat (Knight, '77). When this drug is incorporated into the diet of pregnant rats and fed from the fifth or seventh days of gestation, malformed fetuses have been observed. The incidence of gross external defects has been shown to be dose dependent. Salicylamide has also been used as a model compound, to test the hypothesis that drugs which yield an arylsulfate as a major urinary metabolite may create a sulfate deficit in pregnant animals which adversely affects fetal sulfate utilization for skeletal development. Fetuses from rat dams receiving SA show decreased radiosulfate incorporation into skeletal as well as other tissues (Knight et al., '77). Drug-induced changes in radiosulfate uptake by fetal rats are modified by the protein level of the maternal diet as well as by drug dosage and gestational dates (McGarry et al., '73). An association has been proposed between development of defects and impaired sulfate utilization by the fetus. Therefore this study was undertaken to examine whether the adverse effects of this drug on outcome of pregnancy and fetal development or the prenatal development of the rat skeleton are influenced by protein restriction or variables pertaining to drug intake. TERATOLOGY(1978)18: 17-22.
MATERIALS AND METHODS
Sperm positive rats of the Holtzman strain were obtained on the fourth day of gestation and grouped according to drug and diet treatment. Diets were either protein adequate (25%casein) or protein restricted (8%casein) and other nutrients supplied were as recommended for rats during gestation (Knight et al., '78). Within each diet group the following subgroups were made: zero drug groups, hereafter considered as high protein or low protein controls, rats fed SA as a 2% dietary supplement from the fifth to eleventh days of gestation, and rats given SA from the twelfth to eighteenth days of gestation (table 1). Animals in the zero drug groups were pair fed to the level of intake of those in the corresponding drug groups. The weight gain of the dams was recorded. Dams were killed on the eighteenth day of gestation and fetuses and placentas extracted and counted. Resorption sites were examined and the viability of fetuses determined. Fetuses were examined for external malformations under a dissecting microscope. Thereafter the fetuses and placentas were weighed. Clearing and staining of the fetuses with Alizarin Red S was performed following the method described by Dawson ('26). Stained fetal specimens were examined for Received Apr. 7, '77
Accepted Mar. 3, '78.
17
18
ENID KNIGHT AND DAPHNE A. ROE TABLE 1
at groups and treatment design Group
Period of gestation when drug was administered (days1
Protein level subgroups (% casein)
I
5-18
8 25
I1
Pair-fed Days 5-1
8
111
8 25 25
Pair-fed Days 12. 8
8
8 5 25
Drug level (% SA)
0 0 0 2 0 2 0 2 0 2
skeletal abnormalities and centers of ossification were counted following the guidelines developed by Ward and Readhead (‘70). Intergroup differences in the outcome of gestation, the incidence of fetal skeletal defects and skeletal maturity were examined by analysis of variance. Three factor interactions were analyzed by the G-test (Sokal and Rahl, ’69). Analysis of covariance was used to examine data on fetuses from individual dams. RESULTS
Salicylamide did not produce a consistent effect on maternal weight or weight gain in all groups. Introduction of the drug into the diet on the fifth or twelfth day of gestation resulted in a transient decline in weight of the dams. Thereafter, the drug-treated dams gained weight, but the weight gain of the dams in the drug groups fed a 25%casein diet was less than those in the pair fed controls. Whether the drug was started early (day 5) or late (day 121, the drug did not significantly affect maternal weight gain in the protein restricted group (fig. 1). The drug did not influence fetal and placental weights uniformly. Average placental and fetal weights were significantly lower in drug than non-drug groups in rats receiving the drug from days 12-18 of gestation. Slightly lower weights of both placentas and fetuses in drug treated (5-11 day) versus control rats only reached an acceptable level of significance with respect to the placentas of the rats that received 25%casein diet. Protein restriction alone had no significant effect on placental or fetal weights (table 2). The overall percentage of resorptions was significantly increased by drug treatment (P
< 0.05). Protein restriction also resulted in a significant increase in the percentage of resorptions (P < 0.001). Interaction of drug administration and protein restriction increased the percentage of resorptions (P < 0.001). Within drug-treated dams that showed resorption sites, the percentage of resorptions was correlated with the total placental weight (r = 0.67, P < 0.01), average placental weight (r = 0.72, P < 0.011, average fetal weight (r = 0.67, P < 0.05) and litter size (r = - 0.88, P < 0.001). There was no significant correlation between the percent of resorptions and total placental weight, average placental weight, fetal weight or litter size in the dams on zero drug intake. Malformations were associated with fetal drug exposure. External malformations were found in the fetuses of animals receiving SA from the fifth to eleventh days of gestation, and in single fetuses in the groups receiving SA from days 12-18. Malformations included micromelia of the hind legs, phocomelia, s y n dactyly and s ort or absent tails. The percentage of fetuses with external malformations in the drug group was correlated directly with the percentage of resorptions (r = 0.60, P < 0.001) and inversely to the litter weight (r = 0.37, P < 0.011. In individual dams, the percentage of external malformations was correlated with the number of dead fetuses (r = 0.47, P < 0.05). A diet-drug interaction effect was demonstrated by the G-test such that the percent of malformed fetuses was lower in the drug groups on the protein restricted diet (P < 0.001) (table 3). In the high protein and low protein control rats there were no malformed fetuses. Skeletal malformations included missing and abnormal ribs, fused ileum, lordosis and absence of lumbar arches (fig. 2). More skeletal malformations were found in the fetuses from rats receiving the drug from the fifth to eleventh days of gestation. A very significant lower incidence of malformations was found in the fetuses of dams fed the drug from the twelfth to eighteenth days of gestation. The incidence of skeletal malformations was independent of the diet (table 4). Within the fetuses of the drug treated groups, the percent of skeletal malformations was inversely related to the number of centers of ossification present (r = -0.48, P < 0.01). Multiple variables pertaining to fetal development and protein restriction including fetal weight (P < 0.001) and litter size (P
ABSTRACT Salicylamide has previously been shown to be teratogenic to the rat. Aims of the present study were to examine separate effects and interactions of salicylamide administration and protein restriction on development of the rat fetus. In experiments using Holtzman rats, the percentage of fetal resorptions was significantly increased by drug treatment and by protein restriction. Malformations were seen in fetuses from dams given the drug from the fifth to eleventh days of gestation; the incidence of these malformations being independent of the diet. Factors affecting appearance of ossification centers included drug dosage, placental and fetal weight and litter size. Protein restriction had no significant effect on the development of centers of ossification. It is concluded that salicylamide, as well as protein restriction, produces adverse effects on pregnancy outcome in the rat. In earlier studies, the non-narcotic, analgesic drug, salicylamide (SA) has been shown to be teratogenic to the rat (Knight, '77). When this drug is incorporated into the diet of pregnant rats and fed from the fifth or seventh days of gestation, malformed fetuses have been observed. The incidence of gross external defects has been shown to be dose dependent. Salicylamide has also been used as a model compound, to test the hypothesis that drugs which yield an arylsulfate as a major urinary metabolite may create a sulfate deficit in pregnant animals which adversely affects fetal sulfate utilization for skeletal development. Fetuses from rat dams receiving SA show decreased radiosulfate incorporation into skeletal as well as other tissues (Knight et al., '77). Drug-induced changes in radiosulfate uptake by fetal rats are modified by the protein level of the maternal diet as well as by drug dosage and gestational dates (McGarry et al., '73). An association has been proposed between development of defects and impaired sulfate utilization by the fetus. Therefore this study was undertaken to examine whether the adverse effects of this drug on outcome of pregnancy and fetal development or the prenatal development of the rat skeleton are influenced by protein restriction or variables pertaining to drug intake. TERATOLOGY(1978)18: 17-22.
MATERIALS AND METHODS
Sperm positive rats of the Holtzman strain were obtained on the fourth day of gestation and grouped according to drug and diet treatment. Diets were either protein adequate (25%casein) or protein restricted (8%casein) and other nutrients supplied were as recommended for rats during gestation (Knight et al., '78). Within each diet group the following subgroups were made: zero drug groups, hereafter considered as high protein or low protein controls, rats fed SA as a 2% dietary supplement from the fifth to eleventh days of gestation, and rats given SA from the twelfth to eighteenth days of gestation (table 1). Animals in the zero drug groups were pair fed to the level of intake of those in the corresponding drug groups. The weight gain of the dams was recorded. Dams were killed on the eighteenth day of gestation and fetuses and placentas extracted and counted. Resorption sites were examined and the viability of fetuses determined. Fetuses were examined for external malformations under a dissecting microscope. Thereafter the fetuses and placentas were weighed. Clearing and staining of the fetuses with Alizarin Red S was performed following the method described by Dawson ('26). Stained fetal specimens were examined for Received Apr. 7, '77
Accepted Mar. 3, '78.
17
18
ENID KNIGHT AND DAPHNE A. ROE TABLE 1
at groups and treatment design Group
Period of gestation when drug was administered (days1
Protein level subgroups (% casein)
I
5-18
8 25
I1
Pair-fed Days 5-1
8
111
8 25 25
Pair-fed Days 12. 8
8
8 5 25
Drug level (% SA)
0 0 0 2 0 2 0 2 0 2
skeletal abnormalities and centers of ossification were counted following the guidelines developed by Ward and Readhead (‘70). Intergroup differences in the outcome of gestation, the incidence of fetal skeletal defects and skeletal maturity were examined by analysis of variance. Three factor interactions were analyzed by the G-test (Sokal and Rahl, ’69). Analysis of covariance was used to examine data on fetuses from individual dams. RESULTS
Salicylamide did not produce a consistent effect on maternal weight or weight gain in all groups. Introduction of the drug into the diet on the fifth or twelfth day of gestation resulted in a transient decline in weight of the dams. Thereafter, the drug-treated dams gained weight, but the weight gain of the dams in the drug groups fed a 25%casein diet was less than those in the pair fed controls. Whether the drug was started early (day 5) or late (day 121, the drug did not significantly affect maternal weight gain in the protein restricted group (fig. 1). The drug did not influence fetal and placental weights uniformly. Average placental and fetal weights were significantly lower in drug than non-drug groups in rats receiving the drug from days 12-18 of gestation. Slightly lower weights of both placentas and fetuses in drug treated (5-11 day) versus control rats only reached an acceptable level of significance with respect to the placentas of the rats that received 25%casein diet. Protein restriction alone had no significant effect on placental or fetal weights (table 2). The overall percentage of resorptions was significantly increased by drug treatment (P
< 0.05). Protein restriction also resulted in a significant increase in the percentage of resorptions (P < 0.001). Interaction of drug administration and protein restriction increased the percentage of resorptions (P < 0.001). Within drug-treated dams that showed resorption sites, the percentage of resorptions was correlated with the total placental weight (r = 0.67, P < 0.01), average placental weight (r = 0.72, P < 0.011, average fetal weight (r = 0.67, P < 0.05) and litter size (r = - 0.88, P < 0.001). There was no significant correlation between the percent of resorptions and total placental weight, average placental weight, fetal weight or litter size in the dams on zero drug intake. Malformations were associated with fetal drug exposure. External malformations were found in the fetuses of animals receiving SA from the fifth to eleventh days of gestation, and in single fetuses in the groups receiving SA from days 12-18. Malformations included micromelia of the hind legs, phocomelia, s y n dactyly and s ort or absent tails. The percentage of fetuses with external malformations in the drug group was correlated directly with the percentage of resorptions (r = 0.60, P < 0.001) and inversely to the litter weight (r = 0.37, P < 0.011. In individual dams, the percentage of external malformations was correlated with the number of dead fetuses (r = 0.47, P < 0.05). A diet-drug interaction effect was demonstrated by the G-test such that the percent of malformed fetuses was lower in the drug groups on the protein restricted diet (P < 0.001) (table 3). In the high protein and low protein control rats there were no malformed fetuses. Skeletal malformations included missing and abnormal ribs, fused ileum, lordosis and absence of lumbar arches (fig. 2). More skeletal malformations were found in the fetuses from rats receiving the drug from the fifth to eleventh days of gestation. A very significant lower incidence of malformations was found in the fetuses of dams fed the drug from the twelfth to eighteenth days of gestation. The incidence of skeletal malformations was independent of the diet (table 4). Within the fetuses of the drug treated groups, the percent of skeletal malformations was inversely related to the number of centers of ossification present (r = -0.48, P < 0.01). Multiple variables pertaining to fetal development and protein restriction including fetal weight (P < 0.001) and litter size (P
