The present authors carried out a parallel experiment of hypervitaminosis A using CF#1 mice, Sprague-Dawley rats and Japanese white rabbits. Special attention was paid to 1) the relationship between the time of administration and the frequency of fetal deaths as well as oculofacial syndrome in the three species (stage-response experiment) and 2) the relationship between dose administered and the incidence of oculofacial syndrome in the three species (dose-response experiment). Prior to the present study the equivalent ages in the rat and rabbit embryos for each successive mean age in the mouse embryos (the 8th-13th day of pregnancy) were determined by grossly comparing some morphogenetic patterns. In the stage-response experiment, mice were given a single, intraperitoneal injection of vitamin A palmitate at a dose of 600,000 IU/kg from the 8th to the 13th day of pregnancy, rats were given at a dose of 500,000 IU/kg from the 10th to the 15th day and rabbits were administered 300,000 IU/kg from the 8th to the 16th day. In the dose-response experiment, various dose levels of vitamin A (50,000-250.000 IU/kg) were administered on the 8th day of pregnancy in mice and rabbits and on the 10th day in rats. The results are summarized as follows: 1. The susceptibility expressed by the frequency of fetal deaths including embryonic losses was the highest in rabbits, next in mice and the lowest in rats. 2. Oculofacial syndrome was produced in all species treated on earlier stage, i.e. , 8th-10th day in mice, 10th-11th day in rats and 9th-10th day in rabbits. 3. The minimal teratogenic dose for the production of oculofacial syndrome as well as anomalous craniofacial bones was almost the same in the three species. (50,000-75,000 IU/kg). while the dose-response relationship of those anomalies was the greatest in the rat, next in the mouse and the smallest in the rabbit. 4. Though many other characteristic malformations such as exencephalia, spina bifida, cleft palate, abdominal hernia, anal atresia, tail abnormalities and malformations of vertebrae and ribs were also produced mainly in mice and rats according to the stage injected, no clear dose-response was found except a case of cleft palate. or. The minimal teratogenic dose and the dose-response relationship were depended upon the stage injected. Therefore, more reliable study on the equivalent age in the three species will be necessary for the determination of the species-difference in the teratogenic effect of hypervitaminosis A.
Pregnant mice (ddN and CF#1 strains) were divided into six groups according to gestational day from the 8th to 13th day of gestation. Each group of mice was administered a single dose of 15,000 I.U./mouse vitamin-A water miscible solution intraperitoneally, and the fetuses were examined for malformations of facial bones. The fetuses were observed in bone-stained cleared specimens, cartilage-stained ones and supplementally in H.E stained sections. The characteristic changes were features of the deformed mandibles and existence of abnormal cartilage in the upper jaw. In the groups treated on the 8th or 9th day of pregnancy, the deformity of the mandible was of hypoplastic nature and the developmental arrest was prominent in the proximal portion, especially in the ramus. While in the groups treated on the 10th or 11th day, the deformity of the mandible was evident in the middle-distal portions and associated with hyperplastic tendency, which manifested itself in a thickness of the mandibular body. The abnormal cartilages in the upper jaws were produced only by the treatments on the 8th or 9th day of pregnancy. They first appeared in the base of skull primordiums on the 14-15th embryonic day and actively proliferated with the development of chondrocranium. It suggested they were not heterotopic cartilages but dysplasias originated from the cartilaginous tissue of chondrocranium. The dysplastic cartilages remained as immature masses and showed no tendency of ossification in the fetal stage. The Meckel's cartilage was short and crooked but presented no evidence of dysplasia. The hypoplastic malformations of facial bones could be first detected as a developmental arrest of mandibular process shortly after the treatment of vitamin-A in the early embryonic stage. While the dysplastic cartilage appeared after the normal cartilage had begun to differentiate from the mesenchyme. The results indicnte that hypervitaminosis-A in the early embryonic stage acts as a potent inhibitory agent on the undifferentiated mesodermal tissue and also, as a late-effect, reveals an action to cause hyperplastic dysplasia in some cartilages in a certain stage of their development.
The experiment was designed to examine the nature of teratogenicity of myleran(busulfan) to the skeletal system of developing rat embryos. Myleran was administered orally using a stomach tube to pregnant Wistar rats during the organogenesis of their litters. Given doses were 2, 5, 10, 20, 40mg/kg/day both in a single and successive administrations. In the successive administration, the time range was 9-11th, 12-14th or 9-14th day of pregnancy. In the single treatment, a dose was administered to each graup on different days. The times were 9, 10, 11, 12, 13, 14th day of pregnancy. The fetuses were removed surgically from the mothers on the 20th day of pregnancy and their skeletons were examined in bone-stained cleared specimens and supplementally in cartilage-stained ones. Myleran revealed a potent teratogenicity on the mesodermal tissue of the rat embryos in the early stage of development and produced severe skeletal malformations. Their characteristic manifestations were found in the vertebra and the long bones of the extremities. The vertebral bones were the most susceptible to myleran, but the sensitive stage for the malformations was within a narrow time range restricted around the 9th day of pregnancy. When treated with a small dose of myleran on the 9th day of pregnancy, the malformations were localized in the thoracic vertebra. With increasing the dose in the same day treatment, the involved sites extended both to the head and tail directions from the thoracic region and finally the whole region of the vertebra was involved in a severe malformation. There was no head-to-tail gradient shift of vertebral malformations as the developmental stage of treatment advanced. It was noticed as agent-specific tendencies that the vertebral malformations were produced only by the treatments in the begining of somite-formation, and that the anomalies of segmentation of somites such as block-formation, absence or excess of vertebral bones were conspicuous among the malformations. The malformations of the long bones of the extremities were produced by the treatments on the 9th and 10th day of pregnancy. It indicates the malformations of the long bones are not due to damage of their primordiums after the limb-bud formation but are originated from developmental error of the undifferentiated mesenchymal tissue at the begining of limb-bud formation. The characteristic features of the malformations were developmental arrest of either one out of two parallel long bones in the forearms and the legs. In the forearms, either the radius or ulna was involved in an aplasia or hypoplasia, but both the radius and ulna were never involved in a severe malformation at the same time. In the legs, only the tibia was involved in an aplasia or hypo-plasia, while the fibula was almost normally developed.