ABSTRACT
Cortisone has been demonstrated by several investigators to be a teratogenic agent when administered to pregnant mice (Baxter & Fraser, 1950; Fraser & Feinstat, 1951; Ingalls & Curley, 1957; Walker & Fraser, 1957; Gordon et al., 1961). The degree of occurrence was found to vary with strain (Walker & Fraser, 1957; Fraser et al., 1954), maternal age, weight and litter size (Kalter, 1956; 1957).
A number of investigators have demonstrated the relation of adenosine triphosphate to adrenal function (Notario & Casirola, 1957; Albeaux-Fernet et al., 1958). Grossfeld (1957), using tissue culture techniques, demonstrated that adenosine tiiphosphate could reverse the growth inhibitory action of hydrocortisone. In our work (Gordon et al., in print) we found that adenosine triphosphate administered to mice in low concentrations, about 50 μg., concomitantly with cortisone reduced the teratogenic effects of this hormone. The most significant protection appeared to be against congenital hydrocephaly and spinal deformities. However, from our studies one could not determine whether the protective effect was related to the administration of adenosine triphosphate or its decomposition products. The present study was designed to resolve this question.
MATERIALS AND METHODS
CFW female mice, 7 weeks old (about 23–25 g.) were mated with CFW male mice, 8 weeks old (about 25–27 g.). These were obtained from Carworth Farms. The mating procedure consisted of a preliminary premating of the primiparious mice for 48 hr., that is, four females were separated from one male by a wire screen partition. At the end of this period the five mice were placed together in a half-gallon fish bowl overnight. The following morning the males were separated from the females and the females were examined for vaginal plugs. Prior to mating, the animals were maintained on the diet used at Carworth Farms, the Wayne Lab-Blox high protein mouse diet. Subsequent to mating, they were transferred to a high fat diet, the Old Guilford mouse breeder ration. The high fat diet we reported in our earlier paper (Gordon et al., 1961), aided in reducing the number of resorptions produced by cortisone administration.
The pregnant animals received four consecutive injections beginning the 11th day and ending the 14th day of gestation. The mice were sacrificed by dislocation of the cervical vertebra on the 18th day of gestation, zero day being the day the vaginal plug was discovered. The cortisone used was a cortisone acetate suspension. Sodium pyrophosphate, adenosine-5-phosphoric acid (AMP), adenosine-5′-disphosphate monosodium (ADP) and adenosine-5′-tri-phosphate disodium (ATP) were prepared in M/15 phosphate buffer (pH 7·4) for injections. Although adenosine tetraphosphate (Atetra P) may not be considered as a decomposition product of ATP, its close relationship structurally to ATP made it an interesting compound to include in this study. It was prepared in M/15 phosphate buffer (pH 7·4) for injections. M/15 phosphate buffer (pH 7·4) was used as a control. All compounds were prepared fresh daily for injection.
The pregnant animals were divided into six experimental groups and six corresponding control groups. The decomposition products and Atetra P were administered at a concentration equivalent to 50 μg. of ATP per injection, since the protective effects were observed at that level in our earlier work. Each animal in Group I received 2·5 mg. cortisone intramuscularly per treatment and the corresponding control group 0-1 ml. phosphate buffer (pH 7·4) intramuscularly. Group II received 35·8 μg. sodium pyrophosphate intraperitoneally and 2·5 mg. cortisone intramuscularly per treatment. The control group received the same quantity of pyrophosphate intraperitoneally and 0·1 ml. phosphate buffer intramuscularly. Group III received 29·2 μg. AMP intraperitoneally plus 2·5 mg. cortisone intramuscularly per treatment. The control group received the same quantity of AMP intraperitoneally and 0·1 ml. phosphate buffer intramuscularly. Group IV received 37·4 μg. ADP intraperitoneally and 2·5 mg. cortisone intramuscularly per treatment, while the control group received that quantity of ADP intraperitoneally and 0·1 ml. phosphate buffer intramuscularly. Group V received 50 μg. of ATP intraperitoneally and 2·5 mg. cortisone intramuscularly per treatment. The control group received 50 μg. ATP intraperitoneally and 0·1 ml. phosphate buffer intramuscularly. Group VI received 53 μg. adenosine tetraphosphate intraperitoneally and 2·5 mg. cortisone intramuscularly per treatment. The control group consisted of 53 /xg. Atetra P intraperitoneally and 0·1 ml. buffer intramuscularly. The animals were sacrificed, as described, on the 18th day of gestation. The fetuses were washed, dried, weighed and examined for gross deformities. The number of resorptions and deaths were noted in each mother.
RESULTS
Litter size
There was no apparent effect of ATP or its decomposition products on litter size in the experimental groups (cortisone treated groups) and the control groups (buffer treated groups) (Table 1). However, as observed in our earlier studies, a decreased litter size was apparent in the experimental groups compared to the control series.
Resorptions and death
Administration of ATP, its decomposition product and Atetra P in conjunction with cortisone, as well as cortisone alone, produced a pronounced increase in resorption and death over what was observed in the control groups (p = 0·05) (Table 2). The buffer control group, as well as those receiving the decomposition products without cortisone, all demonstrated some resorptions during pregnancy. However, the ATP controls showed no signs of resorptions. No significant difference in the frequency of resorptions or deaths were noted between the experimental groups. An insignificant number of deaths were observed in the control groups.
Birth defects
Our findings indicated that the administration of ATP, and not its decomposition products, is responsible for reducing the teratogenic effects of cortisone, particularly hydrocephaly and spinal deformity.
Except for a small number of hydrocephalic fetuses, which were observed in the ADP and Atetra P control groups, none of the four deformities observed in the study were noted in the control groups.
A significant increase in the frequency of fetuses with hydrocephaly and spinal deformities were observed in the group receiving AMP and cortisone compared to the groups receiving cortisone, Atetra P and cortisone, and ATP and cortisone (p = 0·05) (Table 3). However, a decided decrease of fetuses with hydrocephaly were noted in the fetuses whose mothers received ATP concomitantly with cortisone compared to the other experimental groups (p = 0·01). It is interesting to note that no spinal deformities were observed in the group receiving ATP with cortisone.
Although congenital absence of eyes were observed in a very small number of fetuses and no statistical significance can be attached to this deformity, the following is suggestive. The largest percentage of fetuses with congenital absence of eyes occurred in the experimental group receiving AMP. This anomaly was not noted in any of the fetuses of the experimental groups receiving ADP, ATP, or Atetra P and cortisone. Only a small number of fetuses with this anomaly appeared in the experimental group treated with cortisone alone.
Although there appears to be some protection with ATP against cortisone induced cleft palate, we were unable to obtain this with any degree of consistency.
Fetal weight
The groups receiving cortisone alone, or cortisone with ATP, Atetra P or any of the decomposition products, demonstrated an obvious decrease in weight compared to the control groups (p = 0·05) (Table 4). Within the experimental groups the fetuses bearing cleft palates tended to have a lower body weight than those with fused palates (p = 0·05).
Only three fetuses in the buffer control group were found to have cleft palate. Their average weight did not differ from the fetuses with fused palates of the same group.
DISCUSSION
In our earlier studies (Gordon et al., 1961) we found that administration of ATP in low concentrations (50 μg.) appeared to reduce the teratogenic action of cortisone in mice. However, high levels (3 mg.) enhanced its teratogenic effect. It was important to determine whether these effects resulted from the administration of ATP or its hydrolytic products. In view of the close association of adenosine tetraphosphate it was decided to include it in our investigation. From our findings in this study, it appears that the administration of ATP, and not its decomposition products or Atetra P, is responsible for reducing the teratogenic effect of cortisone. Further, administration of AMP appears to be largely responsible for enhancing the teratogenic action of cortisone.
Of interest are our consistent findings that the administration of ATP in low concentrations appears to reduce such cortisone induced congenital neurological deformities as hydrocephaly and spinal deformities. The potential of ATP administration in the problem of congenital neurological deformities, regardless of the inducing agent, presents an intriguing question.
Higher than normal values of adenosine triphosphate (Bodis et al., 1955) and cortisone (Robinson et al., 1955) are found during pregnancy. Recently evidence was presented which suggested that the occurrence of birth defects may be related to a defunct carbohydrate metabolism (Runner, 1959). The relation of adenosine triphosphate to carbohydrate metabolism is well established. In view of our findings in animals, it would be interesting to consider the possible role of ATP in human teratology.
SUMMARY
The present study was designed to determine whether the effect of ATP in reducing cortisone induced congenital defects, specifically hydrocephaly and spinal deformities in mice, was due wholly or in part to its decomposition products. The decomposition products were represented by sodium pyrophosphate, adenosine monophosphate and adenosine diphosphate. In view of its relationship to ATP adenosine tetraphosphate was also studied. The investigation was made using CFW mice from Carworth Farms.
There was no apparent effect of any of the compounds studied on litter size in both the experimental group (cortisone treated groups) and the control groups (buffer treated groups). However, the litter size in the experimental groups were lower than the control groups.
The experimental groups demonstrated a pronounced increase in resorptions and death compared to the control groups.
A significant increase in the frequency of fetuses with hydrocephaly and spinal deformities were observed in the group receiving AMP and cortisone compared to the groups receiving cortisone, Atetra P and cortisone and ATP and cortisone (p = 0·05). In the ATP-cortisone group, however, there was a decided decrease in these deformities as well as no cases of congenital absence of eyes.
The fetuses in the experimental groups had a lower body weight than the control groups and the fetuses with cleft palates had a lower body weight than those with fused palates.
RÉSUMÉ
L’action de ïadénosine-triphosphate et de ses produits de décomposition sur l’action tératogène de la cortisone
Ces recherches ont eu pour but de déterminer si l’action de l’ATP dans la diminution des malformations congénitales induites par la cortisone, en particulier l’hydrocéphalie et les malformations spinales chez les souris, était due en tout ou partie à ses produits de décomposition. Ceux-ci étaient représentés par le pyrophosphate de sodium, l’adénosine-monophosphate et l’adénosine-diphosphate. Etant donné ses rapports avec l’ATP, on a aussi étudié l’adénosine-tétraphosphate. On a utilisé des souris CFW des Carworth Farms.
Aucun des composés étudiés n’a eu d’action apparente sur la taille des portées, à la fois chez les groupes traités à la cortisone et les groupes témoins traités au tampon. Néanmoins, la taille des portées des groupes expérimentaux était inférieure à celle des témoins.
Les groupes expérimentaux ont présenté un accroissement marqué des résorptions et des morts, par comparaison avec les groupes témoins.
On a observé un accroissement significatif de la fréquence des foetus présentant de l’hydrocéphafie et des malformations spinales, chez les groupes recevant de l’AMP et de la cortisone, par comparaison avec les groupes recevant de la cortisone, de l’ATétraP et de la cortisone, et de l’ATP et de la cortisone (p = 0,05). Néanmoins, le groupe traité à l’ATP-cortisone a présenté moins de malformations, et aucun cas d’absence congénitale des yeux.
Les foetus des groupes expérimentaux avaient un poids corporel inférieur à celui des groupes témoins, et les foetus à palais fissuré avaient un poids corporel plus faible que ceux dont le palais était fermé.
ACKNOWLEDGEMENTS
This study was supported by the John A. Hartford Foundation Inc.