Defining a critical period in calvarial development for Hedgehog pathway antagonist-induced frontal bone dysplasia in mice.

The Hedgehog (Hh) signalling pathway is essential for cellular proliferation and differentiation during embryonic development. Gain and loss of function of Hh signalling are known to result in an array of craniofacial malformations. To determine the critical period for Hh pathway antagonist-induced frontal bone hypoplasia, we examined patterns of dysmorphology caused by Hh signalling inhibition. Pregnant mice received a single oral administration of Hh signalling inhibitor GDC-0449 at 100 mg•kg-1 or 150 mg•kg-1 body weight at preselected time points between embryonic days (E)8.5 and 12.5. The optimal teratogenic concentration of GDC-0449 was determined to be 150 mg•kg-1. Exposure between E9.5 and E10.5 induced frontal bone dysplasia, micrognathia and limb defects, with administration at E10.5 producing the most pronounced effects. This model showed decreased ossification of the frontal bone with downregulation of Hh signalling. The osteoid thickness of the frontal bone was significantly reduced. The amount of neural crest-derived frontal bone primordium was reduced after GDC-0449 exposure owing to a decreased rate of cell proliferation and increased cell death.

[Hedgehog pathway antagonist-induced oromandibular limb hypogenesis in mouse].

Objective: To analysis teratogenic effect of GDC-0449 to fetus and set up the animal model of GDC-0449 induced oromandibular limb hypogenesis in mouse for further research of its pathogenesis. Methods: Twenty-seven pregnant Institute of Cancer Research (ICR) mice were randomly divided into: control group, embryonic day 8.5 (E8.5) exposed groups, E9.5 exposed groups, E10.5 exposed groups, E11.5 exposed groups, E12.5 exposed groups, E13.5 exposed groups, E14.5 exposed groups and E15.5 exposed groups. Each group had 3 mice. Exposed groups were treated with the Hedgehog pathway antagonist GDC-0449 at a single dose 150 mg/kg by oral gavage from E8.5 to E15.5. At E16.5, embryonic phenotypes were analyzed in detail by stereo microscope and histology. After establish an optimal dysmorphogenic concentration, 6 pregnant ICR mice were randomly divided into control group and the optimal group, embryonic phenotypes were analyzed by whole-mount skeletal staining and micro-computed tomography at E18.5. Results: The mice were exposed to GDC-0449 on E11.5 and E12.5 had a high incidence of cleft palate. GDC-0449 exposed between E9.5 and E10.5 caused craniofacial and limb dysmorphology, including micrognathia, microglossia, ectrodactylia, partial anodontia and cleft palate. Most interestingly, these are extremely similar to oromandibular limb hypogenesis syndrome. Conclusions: The results of this study indicate that GDC-0449 can be used to induce micrognathia, microglossia, ectrodactylia, partial anodontia and cleft palate. This work established a novel mouse model for oromandibular limb hypogenesis.

Congenital malformations and other reproductive losses in goats due to poisoning by Poincianella pyramidalis (Tul.) L.P. Queiroz (=Caesalpinia pyramidalis Tul.).

In the semiarid region of Brazil, in areas with vegetation composed mainly of Poincianella pyramidalis, several cases of congenital malformation and reproductive losses were observed in goats and sheep from 2012 to 2014. To determine the teratogenic effect of P. pyramidalis, two groups of eight goats each were used. Goats from Group 1 received fresh P. pyramidalis, harvested daily, as the only roughage during the whole breeding and pregnancy period. Goats in Group 2 (control) received Cynodon dactylon (tifton) hay free choice. Ultrasound examination for pregnancy diagnosis was performed every 28 days. Four goats from Group 1 were pregnant on day 28 but not on day 56, suggesting embryonic death or abortion. Another goat from Group 1 died at day 70 of pregnancy, and the fetuses exhibited micrognathia. The other three goats bore six kids, three of which showed bone malformations in the limbs, spine, ribs, sternum, and head, including arthrogryposis, scoliosis and micrognathia. One kid also showed hypoplasia of the left pulmonary lobes. In the control group, all goats bore a total of 13 kids and none of them exhibited malformations. These results demonstrated that P. pyramidalis causes congenital malformations and other reproductive losses in goats.

Specific congenital malformations after exposure to cyclophosphamide, epirubicin and 5-fluorouracil during the first trimester of pregnancy.

The treatment of pregnant women with chemotherapeutic drugs leads to congenital malformations in 10-20% of newborn children. We present a case of an ongoing 19-week-long pregnancy which was diagnosed in a 39-year-old woman who was being treated with CEF (cyclophosphamide, epirubicin, 5-fluorouracil) chemotherapy for an infiltrating ductal carcinoma of the breast. After termination of the pregnancy, subsequent examination of the fetus revealed micrognathia and bilateral malformations of the hands and feet. The peak exposure of the fetus to the chemotherapeutic agents was in the 5th to 6th week of the pregnancy. Both the nature of the malformations and the timing of the administration of chemotherapy are similar to another case reported previously. We conclude that chemotherapy treatments with CEF in the 5th to 6th week of pregnancy specifically generate hand and foot abnormalities and micrognathia, which is consistent with an inhibition of proliferation, leading to cell death at this embryonic stage.

Effects of light ethanol consumption during pregnancy: increased frequency of minor anomalies in the newborn and altered contractility of umbilical cord artery.

This study explores the effects of light maternal ethanol consumption during pregnancy on the appearance of minor malformations in neonates as well as on the contractile properties of their umbilical cord arteries (UCAs). Clinical external findings of newborns of women declaring light ethanol consumption during any period of their pregnancies [ethanol-exposed group (E group), n=79] were compared with those of nonexposed mothers [nonexposed to ethanol group (NE group), n=100]. Women who smoked or had any associated pathology were excluded. E group mothers consumed, on average, 200-250 mL ethanol/trimester (upper limit 700 mL/trimester). Sixty-six percent of the neonates in the E group presented at least one minor malformation (retromicrognathia and minor anomalies of the auricular/preauricular area were the more common), whereas only 16% of the NE group did (p=0.0000). The percentage of children exhibiting Apgar scores <7 was significantly greater in the E group (11% versus 2%, p=0.0119). UCAs from the E group developed significantly less contractile force (p<0.05) than those of the NE group when exposed to 1 microM serotonin (5-HT) or to a high K+ depolarizing solution. This difference persisted after inhibition of endothelial release of nitric oxide (NO) and prostacyclin. In conclusion, even light drinking should be considered a risk during pregnancy.

Effects of irradiation and methyl-triazene on craniofacial development in mouse embryos: a semiautomated morphometric analysis.

OBJECTIVE: The purpose of the present study was a 2D-semiautomated morphometric analysis of craniofacial growth in nuclear magnetic resonance imaged (NMRI) mouse embryos. METHODS: The NMRI mouse embryos were exposed in utero to either a single dose of 2 Gy X-irradiation on day 9 of gestation (113 embryos) or to 1.5 mg methyl-triazene administered orally to their pregnant mothers on gestational day 10.5 (124 embryos). An additional group of 108 embryos was used as controls. Digitized pictures of embryos from gestational days 14 to 17 were taken in lateral right view using a video system. Landmarks were located and digitized for computerized analysis of growth changes in relation to developmental stages of the face. RESULTS: The results revealed that the snout of control embryos lengthens during the developmental period considered. The snout of embryos previously submitted to methyl-triazene displayed micrognathia, and all treated fetuses exhibited macroscopic signs of microcephaly with a reduced mandible. The snouts of irradiated embryos appeared shortened at the 14-day stage and continued to shorten as development proceeded. A shortening of the midface was detected macroscopically in 83% of the cases. CONCLUSION: The results of this morphometric analysis enabled us to trace the developmental progression of the induced dysmorphosis and to assess the differences compared with normal development.

[Observation of micrognathic development in mouse fetus induced by sulfadimethoxine].

The purpose of this study was to investigate the process of micrognathic development in mouse fetus induced by Sulfadimethoxine (SDM). SDM of 3,000 mg/kg was administered orally to pregnant ICR mice on days 8, 9, 10, 11, 12, and 13 of gestation. The fetuses were removed on day 15 to analyze the incidence of micrognathia. The incidence of micrognathia was the highest in the treated group on day 10. The fetuses, SDM-treated on day 10, were sacrificed on days 11, 12, 13, 14, 15, and 18 and prepared for histological and morphological analyses. Histomorphological analyses were performed by lateral and horizontal graphic reconstruction using serial frontal sections. It was also performed by clearing and alcian blue-alizarin red double staining. In the treated group, bilateral sigmoid buckling and shortening of Meckel's cartilage became evident after day 14, however, the nasal septal and capsule cartilage were less affected. It was concluded that remarkable hypoplastic and malformed Meckel's cartilage, caused by the administration of SDM, induced mandibular dysmorphogenesis.

[Histological study of micrognathic development in mouse fetus induced by 3,3-dimethyl-1-phenyltriazene].

The purpose of this study was to investigate the influence of 3,3-dimethyl-1-phenyltriazene (DMPT) on development of craniofacial structures in mouse fetus. (1) Pregnant C57BL/6 mice were treated with 30 mg/kg DMPT injection on days 10.5 approximately 13.5 of gestation. The fetuses were analyzed on day 15.0. The incidence of micrognathia was formed 100% in the treated group on days 10.5 and 11.0, and then decreased remarkably after day 11.5. This result suggested that the critical period of micrognathic development is days 10.5 approximately 11.0. (2) The fetuses, injected 30 mg/kg DMPT on day 10.5, were sacrificed on days 11.0 approximately 18.0. Their heads were used for the following histomorphological analyses: 1. clearing and Alcian blue-alizarin red double staining and 2. lateral graphic reconstruction using serial frontal sections. Dysmorphology of the anterior portion of Meckel's and nasal septal cartilage became evident after day 13.0, however, nasal capsule cartilage was hardly affected. It was concluded that hypoplastic Meckel's cartilage, caused by the administration of DMPT, induced mandibular dysmorphogenesis.

Pathogenesis of bromodeoxyuridine-induced cleft palate in mice.

This study was designed to examine the pathogenesis of bromodeoxyuridine-induced (BrdU) clefts of the secondary palate in mice. Intraperitoneal injections of BrdU (500 mg/kg body weight) were given on days 11 and 12 to some pregnant mice and on days 12 and 13, and days 11, 12 and 13 to others. Evaluation of craniofacial relations and palate development in BrdU-treated mice revealed inhibition of vertical development of the palatal shelves, mandibular hypoplasia which led to failure of downward displacement of the tongue and the creation of an obstacle to reorientation of the palatal shelves. The results of this study demonstrate a strong correlation between induction of cleft palate and the presence of structural alterations in the mandible, and the mechanism of BrdU-induced cleft palate resembles the defect in the Pierre Robin anomaly.

Teratogenicity of 3,3-dimethyl-1-phenyltriazene in the rat: gross malformations including micrognathism.

3,3-Dimethyl-1-phenyltriazene (DMPT), an alkylating agent, has been reported to be teratogenic in chickens, mice, and rats. One of the most commonly affected structures is the mandible; however, a complete description and incidence rates of all malformations produced have not been published. Rats were treated on day 12 of gestation (day 0 = sperm in vaginal smear) with a single intraperitoneal injection of 30 mg DMPT/kg. Fetuses were examined on each subsequent day of gestation for external and skeletal abnormalities. Standard soft tissue examinations were performed on day-20 fetuses. A high percentage (greater than or equal to 80%) of treated litters contained numerous fetuses with micrognathism, cleft palates, syndactyly, adactyly, and misshapen digits and limbs. Lordosis, cerebellar and cerebral hypoplasia, decreased fetal size, and generalized delayed ossification were also observed. Dams exposed to DMPT had decreased food consumption and weight gains, although clinicopathologic and histopathologic evaluations failed to indicate other evidence of maternal toxicity. DMPT caused numerous permanent structural alterations that were not attributed to maternal toxicity.

Morphogenesis of isotretinoin-induced microcephaly and micrognathia studied by scanning electron microscopy.

Isotretinoin ingestion during the first trimester of human pregnancy can induce malformations of the skull, ears, face, central nervous system, eyes, palate, lungs, circulatory system, limbs, and digits. A single oral dose of isotretinoin on day 8 of gestation in hamsters induces a similar syndrome of congenital malformation. The present study concerned scanning electron microscopic (SEM) observation of embryonic and fetal hamster craniofacial structures at 4, 8, 12, 24, 48, and 72 hr after administration of an oral dose of 50 mg/kg isotretinoin or an equivalent volume of the vehicle. The variability in development among control embryos recovered 4 hr after treatment precluded objective assessment of pathologic change by SEM at very early time points. Craniofacial damage was obvious within 8-12 hr of isotretinoin treatment, and it included hypoplasia of the maxillary and mandibular processes of the first branchial arch, a rudimentary second arch, and apparent collapse of the forebrain. Equivalent fusion between the lateral nasal process and the maxillary process and between the medial nasal process and the maxillary process in treated and control embryos accounts for the very low incidence of cleft lip observed in fetuses. The terminal microstomia was not associated with excessive merging or overgrowth of the first arch components. Hypoplasia of the first arch can account for retinoid-induced macrostomia and microstomia.

Teratogenic effect of trifluoperazine in rats and mice.

Trifluoperazine was administered to pregnant mice and rats by gastric intubation during the period of organogenesis. Dams were treated at doses of 0.5, 5, 50 (mice and rats) and 100 (only rats) mg/kg/day. The drug affected the pregnant weight increment in a dose-related manner in rats but only the 50 mg/kg/day dose level affected the weight gain of mice. The foetal weight was not markedly affected in either species. The drug induced cleft palate and micrognathia in rats but did not produce a teratogenic effect in mice. Trifluoperazine caused abortions in mice and there were significant increases in the number of resorptions with the top dose levels in both species.

Effect of intraamniotic administration of vitamin A on rat fetuses.

The present study examines the effect of intraamniotic (i/a) administration of vitamin A to rat fetuses. Vitamin A aquasol in concentrations of 50 I.U., 100 I.U., and 300 I.U., was mixed in 5 microliter of saline. On day 13 of gestation, each of the tree experimental subgroups were injected i/a with varying doses of vitamin A. The four control subgroups consisted of: (1) i/a injection of 5 microliter of saline only, (2) amniotic puncture only, (3) laparotomy or (4) i/a injection of sorethytan oleate. The fetuses were recovered on day 18 of gestation, and were weighed and examined for gross malformations. The viable fetuses from the experimental groups showed various malformations such as cleft palate, micrognathia and limb defects. The percentage of resorbed fetuses was significantly high. The control fetuses showed a moderate number of resorptions; however the percentage of malformations was small. The results show that the i/a method can be used effectively to study the direct teratogenic effect of vitamin A on the fetuses.

Effects of prenatal administration of hadacidin, a cancer chemotherapeutic agent, on the development of hamster fetuses.

A single intraperitoneal injection of 1-2-5 g/kg body weight of hadacidin was teratogenic when administered to pregnant hamsters between days 8 and 11 of gestation. Both the frequency of malformation and resorption were related to the dose and time of hadacidin administration. The drug produced both gross and microscopic malformation in the fetus and impaired its general growth. The malformation involved craniofacial structures, central nervous system, respiratory, digestive and urinary systems, limbs and tail. Morphologically different types of cleft lip and cleft palate were related to the dose and time of hadacidin treatment. An association was observed between cleft lip, cleft palate and micrognathia on one hand, and between cleft palate and micrognathia on the other. Significance of association between the lip, the palate and the mandibular malformation was discussed and the hypothesis that human cases of Pierre Robin syndrome may result from an environmental assault was supported. It was suggested that the fetal weight and the microscopic analysis should be included in the criteria for the teratological drug safety evaluation procedures.

Congenital malformations. Cleft palate, congenital heart disease, absent tibiae, and polydactyly.

A girl had cleft palate, micrognathia, Wormian bones, congenital heart disease, dislocated hips, absent tibiae, bowed fibulae, preaxial polydactyly of the feet, and abnormal dermal patterns at birth. She was born after a pregnancy complicated by exposure to multiple medications. This combination of malformations may represent a distinct entity unrelated to the medication or may be a complication of the intrauterine drug exposure.

Action of the phenothiazine derivative methophenazine on prenatal development in rats.

Single doses of 100-400 mg/kg or multiple doses of 10 or 50 mg/kg of the phenothiazine derivative methophenzaine were given per osto Wistar rats at various times on the 7th-14th days fo gestation and the fetuses examined near term. Results indicated that methophenazine was mainly embryolethal when administered on the 8th-11th days, and was teratogenic at later times, producing types of malformations that depended on the day of treatment, the most susceptible period being the 13th and 14th days of gestation. Teratogenicity occurred only when the dosages were highly toxic to the pregnant rats. Ribovlavin given ip on the 14th day significantly reduced the embryolethal but not the teratogenic action of methophenazine.