Prenatal exposure to the antiepileptic drug valproic acid (VPA) has been associated with the formation of spina bifida aperta, meningocele, and meningomyelocele in the human. Until now, a direct relationship between VPA application and spina bifida has not been experimentally demonstrated. VPA was known only to induce exencephaly in mice, a defect of the anterior neural tube. Maximal sensitivity toward production of this defect was on day 8 of gestation (plug day = day 0). The closure of the posterior neuropore occurs later in the development of mice than the closure of the anterior neuropore. To investigate whether there is a direct relationship between VPA application during pregnancy and induction of spina bifida in mice, we administered various doses of the drug on day 9 of gestation, at three time intervals (at 0, 6, and 12 hr). This administration of VPA produced spina bifida aperta and spina bifida occulta in mice. High doses of VPA (3 x 450 and 3 x 500 mg/kg) induced a low rate of spina bifida aperta in the lumbosacral region. High incidences of spina bifida occulta, a less serious form of spina bifida, were induced with lower doses. This malformation was demonstrated in double-stained fetal skeletons by measurements of the distance between the cartilaginous ends of each vertebral arch. The occurrence of this defect and its localization was dose-dependent. The lumbar region was affected by all doses investigated (3 x 300, 3 x 350, 3 x 400, 3 x 450, and 3 x 500 mg/kg). The sacral/coccygeal region was affected additionally, but with higher doses (3 x 400, 3 x 450, and 3 x 500 mg/kg). A comparison of the results obtained with day 16 and 17 control fetuses showed that the pattern of gaps present in the lumbar and sacral region of the spinal cord in treated groups was drug-specific and not related to a developmental delay. Our results indicate that multiple administrations of VPA on day 9 of gestation in mice result in a low incidence of spina bifida aperta and a high incidence of spina bifida occulta, and provides a relevant model for the study of human spina bifida defects.

The antiepileptic drug valproic acid (VPA) has been implicated as a human teratogen causing spina bifida aperta. Recently, we developed a mouse model inducing spina bifida aperta with VPA. To elucidate the pathogenesis of VPA-induced spina bifida aperta we now investigated the anatomy and histology of this defect in the mouse. The morphology of spina bifida aperta induced by all-trans-retinoic acid (RA) was used for comparison. Various doses of VPA and RA were administered at different times to determine the periods of sensitivity for inducing spina bifida aperta with these drugs. Each administration regimen consisted of three doses applied at intervals of 6 hr. RA induced spina bifida aperta during an earlier developmental period (day 8 of gestation) than VPA (day 9 of gestation). The most effective regimens for induction of spina bifida aperta in mice were injections of 3 x 500 mg VPA-Na/kg body weight (b.w.) intraperitoneally on day 9 of gestation at 0, 6, and 12 hr; RA (12.5 mg/kg b.w.) was given orally on day 8 of gestation at 12 and 18 hr, day 9 at 0 hr. VPA did not induce spina bifida aperta on day 8 of gestation and RA did not induce this effect on day 9 of gestation. Histological studies of day 18 fetuses carrying spina bifida aperta were performed. The spina bifida aperta induced by VPA shows a disorganized and necrotic spinal cord. In the vertebral canal were observed cell debris, blood cells, capillaries, macrophages, and rests of meninges. These results indicate that the spinal cord is almost destroyed at the affected section. In contrast, the spina bifida aperta induced by RA demonstrates a spinal cord organized in the gray and white matter, the dorsal and ventral horn. But the neural canal does not exist, only a layer of ependymal cells lies on the surface of the spinal cord. Our results indicate that the morphology of spina bifida aperta induced by VPA differed distinctly from that induced by RA in the mouse fetus. Moreover VPA produced a spina bifida aperta with a specific morphology. Also the period of sensitivity for induction of this lesion differed and occurred earlier for RA than for VPA. VPA and RA may possibly induce spina bifida aperta via different mechanisms in the mouse.

The response of pregnant marmosets (Callithrix jacchus) to the thalidomide derivative EM 12 was evaluated. EM 12 was selected for these studies because it is more active than thalidomide and is much more stable for hydrolysis. Skeletal gross structural abnormalities were observed when EM 12 was given to marmosets for 3-7 days during the period between days 49 and 60 post ovulation. Using the treatment schedule finally adapted in our laboratory, i.e. treatment during days 51-57 post ovulation, doses of 5 (or 10) mg EM 12/kg body wt induced the typical limb abnormalities known from man with an 80-100% certainty. In some animals we could observe the typical pattern of abnormalities even with doses as low as 1 mg EM 12/kg body wt. Abnormalities of the skeleton induced during this sensitive period are described. None of these (except some bifurcations of ribs) were seen in any of the ten litters (23 fetuses) serving as controls during the period of the study.

Arotinoid ethyl ester (RO 13-6298) is a new and very potent retinoid that exerts a profound influence on epithelial and mesenchymal differentiation in doses 500 times lower than those of compounds of the first and second retinoid generation. In the present study the teratogenicity of arotinoid ethyl ester was investigated in NMRI mice employing different treatment schedules. Recording of abnormalities was performed on day 18 (day 0 = day of conception) according to Wilson and with cleared skeletal preparations. Intraperitoneal application of the drug at a dosage of 10 micrograms/kg/day for three consecutive days (days 9-11 or 12-14) caused severe malformations, particularly in the skeletal system and the cavernous organs. Skeletal elements were reduced in number, shortened, or abnormally shaped. Ossification was diminished. Atresia of anus and urethra were frequent. Single application of 200 micrograms/kg between days 8 and 14 also caused multiple and severe malformations. However, no stage-specific pattern of abnormalities was detectable. Some skeletal malformations indicated more or less vulnerable stages that were in concordance with special developmental steps. Others, however, seemed to be equally susceptible over a longer period, eg, rays 1 and 5 of the hand or foot and the development of the mandibular joints. The pattern of abnormalities caused by these very low doses of RO 13-6298 is comparable to that obtained with other retinoids and is achieved within the same relative dose-response range. Preconceptional treatment of the animals did not induce any malformations.

The teratogenic potency of the thalidomide (Thd) derivative phthalimidophthalimide (Phtpht) was assessed in the common marmoset (Callithrix jacchus), by oral administration of the relatively high daily dose of 50 mg Phtpht/kg body wt, during the susceptible period (days 48-61 of pregnancy). Since in this species daily doses of only 100 micrograms/kg body wt of the Thd derivative EM12 already induce typical gross structural abnormalities in nearly 100% of the fetuses, investigations with a small number of these New World monkeys allow a rough estimation of the teratogenic potency of Thd-type substances. Macroscopic inspection and skeletal evaluation of ten fetuses gave no indication of dysmorphogenesis following treatment with Phtpht. We conclude that Phtpht has little, if any, Thd-type teratogenic potency in this non-human primate.

The antiepileptic drug valproic acid (VPA) has been implicated as a human teratogen causing spina bifida aperta. Recently we developed a mouse model inducing spina bifida aperta and occulta with VPA. In a search for novel antiepileptic agents the VPA metabolite E-2-n-propyl-2- pentenoic acid (2-en-VPA) had been developed. In the mouse, 2-en-VPA exhibits anticonvulsive potency similar to VPA but very low teratogenic potency (induction of exencephaly). We have now compared VPA and its metabolite 2-en-VPA in regard to induction of spina bifida in our mouse model. 2-en-VPA was administered 3 times during the period of gestation most sensitive for the induction of spina bifida aperta with VPA: on day 9 of gestation at 0, 6 and 12 h. The following doses were injected (in mmol 2-en-VPA-Na/kg): (a) 3 x 2.1, (b) 3 x 2.7 (the equimolar dose of VPA is the threshold dose for induction of spina bifida aperta) and (c) 3 x 3.0 (the equimolar VPA dose produced spina bifida aperta). 2-en-VPA did not induce spina bifida aperta in the mouse in any of these groups. We then investigated the induction of spina bifida occulta in the three dose groups. Spina bifida occulta is a less serious form of spina bifida and may provide a sensitive method to estimate the potency of a compound to induce more severe forms of spina bifida. This malformation was demonstrated in alcian-blue- and alizarin-red-stained fetal skeletons by measurements of the distance between the cartilaginous ends of each vertebral arch. Spina bifida occulta was not induced by 2-en-VPA; equimolar doses of VPA induced spina bifida occulta to a high percentage. Our results indicate that the multiple and high doses of 2-en-VPA on day 9 of gestation did not induce spina bifida aperta or spina bifida occulta in the mouse. The low teratogenic potency of 2-en-VPA in comparison with VPA was thus demonstrated with the hallmark defect induced by antiepileptic therapy with VPA in the human.