Facial formation in normal and cadmium-treated golden hamsters

Recent investigations have established the easily reproducible teratogenic effects of cadmium on the embryonic facial structures of the golden hamster (Ferm, 1967; Ferm & Carpenter, 1967, 1968; Ferm, 1969). Among the malformations produced were unilateral and bilateral cleft lips and palates and complex facial fissures.

In our attempts to understand the nature of the palatal clefts produced by cadmium, it became necessary to define clearly the normal events of palatal development in the golden hamster. Although detailed descriptions of palatal formation exist for the mouse (Walker & Fraser, 1956; Larsson, 1961) and the rat (Coleman, 1965), previous papers on the development of the golden hamster do not deal specifically enough with the events of palatal closure (Graves, 1945; Boyer, 1953).

In this study the events of palatal closure in golden hamster embryos are discussed as a basis of subsequent considerations of the interference by cadmium with the normal events of palatal development. The morphology of these cadmium-produced effects is also presented.

Ten pregnant golden hamsters (Cricetus auratus) were divided into control (5) and experimental (5) groups. The day following the evening of conception was designated day 1 of gestation. On the eighth day of gestation five experimental animals were injected intravenously with a solution of cadmium sulfate (3CdSO₄.8H₂O) in the amount of 2 mg per kg, made up in distilled water. The five control animals received no treatment. Animals from both groups were sacrificed on each of days 12, 13 and 14 of pregnancy. The numbers of control and experimental embryos obtained at each time interval were as follows: day 12, 3 controls and 3 experimentals; day 13, 2 controls and 7 experimentals; day 14, 1 control and 2 experimentals. The heads of the embryos from both normal and experimental groups were fixed in Bouin’s solution, processed according to standard histologic procedures, and embedded in paraffin wax. Serial sections of 7 p thickness were obtained in the frontal plane. Comparative sections were stained with either H. and E., Masson’s trichome, or M filler’s colloidal ferric oxide.

For clarity of presentation, the palate is divided into the following areas, proceeding antero-posteriorly : primary palate, anterior hard palate, mid-hard palate, posterior hard palate and soft palate. The following descriptive results were obtained.

In the region of the primary palate, the cartilaginous nasal septum and nasal capsule are well demarcated. The lateral palatine processes in this area are horizontal and have completely joined with the nasal septum (Fig. 1 A). In all other sections of the palate the palatine shelves are directed in a vertical direction alongside the tongue (Fig. 1C); the only exception to this being in the extreme posterior region where the tongue is actually part of the floor of the mouth and the palatal shelves are lying horizontally above it (Fig. 1 E). Although ‘preosteoblast’ (osteoid) areas (Larsson, 1961) can be seen throughout the palate, no sign of actual ossification is yet discernible.

Areas of congenital malformations are most evident in the anterior portions of the palate. In the areas of the primary palate (Fig. 1B) and anterior hard palate (Fig. 1D), the nose is extremely foreshortened. The vertical portion of the cartilaginous nasal septum is completely absent. A bilateral cleft exists in the area of the primary median palatal triangle and the nasal septum deviates to the side of the narrower cleft (Fig. IB). In the anterior hard palate the lateral palatine processes are quite small, if not almost redundant (Fig. ID). In the posterior region an abnormality which was found in several cadmium-treated animals was a thyroglossal cleft (Fig. 1F). Except for a decreased amount of cartilaginous nasal capsule in the mid-hard palate, the palate itself appears normal from the mid-hard palate posteriorly (Fig. ID, F).

The lateral palatine processes have already undergone transposition and are united with each other in the midline (Fig. 2C). Epithelial remnants are seen in all areas of the secondary palate except in the most posterior aspects of the soft palate and in the region of the primary median palatal triangle (Fig. 2A). Fusion has not yet occurred between the nasal septum and the secondary palate (Fig. 2 C). Bone formation is present in all sections of the hard palate to about the same degree, except in the mid-hard palate where ossification is further advanced (Fig. 2 A, C).

Unlike the previous day, malformations can be seen throughout the length of the palate. The nose is foreshortened and markedly deviates away from the cleft side (Fig. 2B, D). The cartilaginous nasal septum is bifurcated throughout its length (Fig. 2B, D). The lateral palatine process on the cleft side appears redundant throughout the length of the hard palate. The ability of the palatal shelves to undergo transposition is apparently not affected, as they are all horizontally located above the tongue (Fig. 2B, D, F). Bone formation is present but markedly retarded when compared to normal (Fig. 2B, D, F).

The secondary palate is now fully formed (Fig. 3 A, C, E). Epithelial remnants are no longer present in any areas of the palate indicating that fusion has been completed (Fig. 3C, E). The nasal septum has completely fused with the anterior portion of the hard palate and the vomerian bone beneath the cartilaginous nasal septum is well calcified (Fig. 3C). Bone formation, in general, has progressed much farther and has extended to the midline of the palate in the midhard palate (Fig. 3E) and posterior hard palate.

The animal exhibits a bilateral cleft which continues as a midline cleft partway into the hard palate. The nose once more is extremely blunted and has a bifurcation of the cartilaginous nasal septum. In all areas where a cleft exists, bone formation appears retarded. From the region of the mid-hard palate posteriorly, however, where palatal formation appears normal, ossification also appears normal (Fig. 3F). Despite the bilateral nature of the cleft the nasal septum still appears to deviate to one side. In the region of the mid-hard palate, it is of interest that, while the lateral palatine processes have undergone fusion and appear quite normal, the nose itself exhibits a bifurcated septum (Fig. 3F).

In the golden hamster, formation of the secondary palate occurs between days 12 and 14 of development. By day 12 the most anterior portions of the lateral palatine processes have already united with the primary median palatal triangle which is between them. As one proceeds posteriorly, the palatal shelves slowly take on a more and more vertical inclination as they come to hang alongside the developing tongue, except in the most posterior areas where the tongue is actually part of the floor of the mouth and the palatine shelves are horizontally directed above it.

Palatal transposition takes place on or about day 12 of gestation. This is in disagreement with Boyer (1953), who reported that it occurred on day 11. Unfortunately, we have been unable to obtain sections from a palate undergoing transposition. Until we are able to do so, it is not possible to say whether transposition takes place anteriorly-posteriorly as in the rat (Coleman, 1965), or posteriorly-anteriorly as in the mouse (Walker & Fraser, 1956). By day 13 the shelves have already undergone transposition. Indeed, the fusion process between the shelves is almost complete. Although epithelial remnants are present in the midline throughout the hard palate, there are none seen in the soft palate. This is therefore consistent with Wood’s hypothesis that the shelves in this area unite with each other by mergence rather than by fusion (Wood & Draus, 1962).

By day 13 palatal bone formation in the normal hamster embryo appears to have progressed farthest in the regions of the primary median palatal triangle and the mid-posterior hard palate. These could be centers of ossification from which bone formation proceeds both anteriorly and posteriorly.

When cadmium is administered to pregnant hamsters, marked effects are noted in the facial development of the offspring. Among those noted in this paper and in previous reports are unilateral and bilateral clefts of the palate, midline clefts through the nose, thyroglossal clefts, and anophthalmia (Ferm, 1967; Ferm & Carpenter, 1967, 1968; Ferm, 1969).

Cadmium appears to have marked deleterious effects on cartilage and bone formation. In some animals the vertical cartilaginous nasal capsule was entirely missing. In others it was greatly foreshortened and bifurcated. Bone formation tended to be retarded in all areas where clefts were present. It is of interest that in those areas where clefts were not present, bone formation appeared quite normal. It is difficult to explain why a generalized bodily function, such as ossification, should be so closely related to a local happening such as shelf fusion; unless both processes represent two different expressions of derangement of embryonic mesenchymal cell activity.

Several present observations, as well as those by previous authors, support this view. The inability of the processes to join is most likely due to a mesodermal deficiency as suggested by Stark (Stark, 1961). The foreshortening of the nasal process has already been mentioned. The bifurcation of the nasal septum probably represents a lack of union between the medial nasal processes. These processes are thought to merge with one another rather than fuse with each other (Patten, 1961). Therefore, since these processes are always in contact with each other, a midline cleft is most probably due to a mesodermal deficiency.

Similar reductions in tissue mass were also seen in the lateral palatine processes. Furthermore, there was no evidence that the shelves were not able to undergo transposition at the proper time as has been reported following cortisone administration (Walker & Fraser, 1956). Therefore, the wide clefts were once again most probably due to a deficiency in mesodermal tissue, rather than delayed shelf transposition.

Marin-Padilla has demonstrated the effect of the teratogens vitamin A and dimethylsulfoxide on the hamster embryo (Marin-Padilla & Ferm, 1965; Marin-Padilla, 1966 a, b). Both of these agents have a marked effect on the embryonic mesenchyme. In a similar fashion, the administration of cadmium sulfate may affect a vulnerable cephalic mesoderm and interfere with the role this mesoderm plays during the morphogenetic mechanism of palatal closure. The facial malformations thus produced could be a direct consequence of a cadmium-induced mesodermal disturbance. The existence and nature of the direct action of the cadmium on the embryonic mesenchyme have yet to be determined.

The placental transfer of radioactive Cd¹⁰⁹ has recently been demonstrated in this experimental system (Ferm, Hanlon & Urban, 1969). Further studies are needed to determine whether the mesodermal deficiency underlying the facial deformities resulting from this treatment might be due to causes other than direct embryonic damage. For example, these malformations may be due to interference with placental transfer of some essential metabolite (Walker & Fraser, 1956), or to the specific action of the teratogen on maternal enzymes and metabolism, with secondary effects on the differentiating embryonic tissue.

Chez le Hamster doré, la période critique de la formation du palais se place entre les 12éme et 14ème jours de la gestation. Pendant cette période, les crêtes se déplacent de la position verticale sur les côtés de la langue à la position horizontale au dessus d’elle et se fusionnent. La transposition du palais per se se place probablement au 12ème jour, ou vers le 12ème jour. Les données actuelles sont insuffisantes pour indiquer dans quelle direction cela se passe. Alors que des restes épithéliaux se retrouvent sur la ligne médiane du palais au jour 13, ils ont complètement disparu au jour 14, ce qui montre ainsi la complétion du processus de fusion. Des aires de preostéoblastes sont clairement visibles au jour 12 mais l’ossification réelle n’est perceptible qu’au jour 13.

Le cadmium exerce apparemment un effet délétère marqué sur le mésoderme de la tête de hamsters dorés, provoquant la production de nombreuses malformations, y compris des fentes labiales unilatérales ou bilatérales et des fentes palatines. Il est suggéré que les fentes trouvés dans les hamsters traités au cadmium sont dues à une déficience du mésoderme plutôt qu’à un retard de la transposition des crêtes. Des anomalies sévères de la formation du cartilage et de retards de l’ossification ont également été décrites.

Le mécanisme de l’action du cadmium, qu’elle soit directe sur le tissu embryonnaire en différenciation, ou qu’elle agisse indirectement par l’intermédiaire des tissus maternels, reste à être élucidé.

We wish to acknowledge the technical assistance of Miss Lorraine Stevens and Miss Anna Morse.

This investigation was supported by USPHS grants DE 02849 (J. M.), HD 02616 (V. F.), and GM 10210 (V. F.).