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In this study, the pregnant female Sprague Dawley (SD) rats were used to evaluate the potential toxicological effect of strontium citrate, a dietary supplement, on embryo-fetal development. Strontium citrate at doses of 0 mg/kg, 680 mg/kg, 1360 mg/kg, and 2267 mg/kg was administrated orally by gavage to rats at day 6 to day 15 of pregnancy. Each group contained 20 pregnant rats. On the 20 th day of gestation, rats was anesthetized and dissected by cesarean section. The appearance, internal organs, gravid uterus weight, embryo implantation number, and implantation loss rate in maternal rats of each group did not reveal any lesions. In fetuses, there were no statistical differences in the fetus weight, sex ratio, embryo resorption number, stillbirth number, and fetal visceral examination in all testing groups compared to the control group. However, in 2267 mg/kg strontium citrate group, the fetuses showed the statistical differences in the anomalies of the bones and eyes compared to the control group. These findings indicate that high-dose strontium citrate possesses an adverse effect on embryonic and fetal development in SD rats. The no observed adverse effect level (NOAEL) of strontium citrate for prenatal development toxicity in SD rats may be regarded as 1360 mg/kg/day., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Teratology is the study of anatomical and physiological abnormalities, commonly known as birth defects. If an embryo is exposed to a harmful substance, or teratogen, during the critical period of development, an ensuing malformation may occur. These malformations and their associated mechanisms are studied and analyzed in laboratory animals in order to prevent them from occurring in humans. Rodents such as rats and mice have commonly been used in such studies because of their similarity to humans. In 1959, James G. Wilson designed, developed, and tested a protocol on how to observe and analyze structural malformations in rodent fetuses, which included: external examination, skeletal evaluation, soft tissue analysis, and data collection/analysis. For standardization purposes, i.e., to normalize findings from one lab to another, it is important that this protocol be followed with precision. Although many years have passed since Wilson initially created this protocol, it is still widely used to this day, and only minor changes have been made to his instructions such as the chemical reagents used in the experiments and methods of analysis of the experimental data. Such testing has resulted in major advances in the dissemination of teratology information, including the identification of an increasing number of teratogens and the understanding of the pathogenesis of birth defects. While mechanistically birth defect prevention will include the understanding of individual genomes and pharmacogenomics, overall, morphological assessment will still be required as an integral part of birth defects research. As the interaction between teratogenic and genetic factors is better understood, it is anticipated that the incidence of most types of defects will substantially be reduced.

Currently it is common practice to evaluate the developmental toxicity hazard of chemicals or pharmaceuticals by evaluation of fetuses after administration of the compound to pregnant animals. These studies are designed to provide possible compound-related fetal changes near term, which are usually classified into malformations or variations. Malformations, but not variations are expected to adversely affect the survival or health. Therefore, classification has striking different regulatory consequences. For categorization as variation reversibility is an important criterion, but it is usually not examined in a standard guideline study. Although this issue has already been recognized long time ago, data dealing with the postnatal reversibility of fetal alterations are still rare. In the current review, literature data, regulatory documents as well as in-house data were compiled. Beside skeletal alterations of skull, vertebral column, ribs, shoulder and pelvic girdle, and extremities, kidney and heart defects are discussed and assessed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

This study was undertaken to test the effects of sildenafil citrate (SC), a type 5 phosphodiesterase inhibitor, on valproic acid (VPA)-induced teratogenesis. On gestation day (GD) 8, ICR (CD-1) mice were treated by gastric intubation with SC at 0 (vehicle), 1.0, 2.5, 5.0 or 10mg/kg. One hour later, animals received a teratogenic dose of VPA (600mg/kg) or vehicle. Developmental endpoints were evaluated near the end of gestation. Twenty-eighth percent of fetuses exposed to VPA had neural tube defects (exencephaly). Pretreatment with SC at 2.5, 5.0 or 10mg/kg significantly reduced the rate of VPA-induced exencephaly to 15.9%, 13.7%, and 10.0%, respectively. Axial skeletal defects were observed in 75.8% of VPA-exposed fetuses. Pre-treatment with SC at 10mg/kg, but not at lower doses, significantly decreased the rate of skeletally affected fetuses to 61.6%. These results show that SC, which prolongs nitric oxide (NO) signaling action protects from VPA-induced teratogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In order to investigate teratogenic effects, especially on cartilage and bone formation, zebrafish embryos were exposed for 144h to the dithiocarbamate pesticide disulfiram (20-320μg/L) and acetic acid hydrazide (0.375-12g/L), a degradation product of isoniazid. After fixation and full-mount staining, disulfiram could be shown to induce strong cartilage malformations after exposure to ≥80μg/L, whereas acetic acid hydrazide caused cartilage alterations only from 1.5g/L. Undulating notochords occurred after exposure to disulfiram even at the lowest test concentration of 20μg/L, whereas at the two lowest concentrations of acetic acid hydrazide (0.375 and 0.75g/L) mainly fractures of the notochord were observed. Concentrations of acetic acid hydrazide≥1.5g/L resulted in undulated notochords similar to disulfiram. Cartilages and ossifications of the cranium, including the cleithrum, were individually analyzed assessing the severity of malformation and the degree of ossification in a semi-quantitative approach. Cartilages of the neurocranium such as the ethmoid plate proved to be more stable than cartilages of the pharyngeal skeleton such as Meckel's cartilage. Hence, ossification proved significantly more susceptible than cartilage. The alterations induced in the notochord as well as in the cranium might well be of ecological relevance, since notochord malformation is likely to result in impaired swimming and cranial malformation might compromise regular food uptake., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Methylmercury (MeHg) is an environmental contaminant that is found in many ecosystems. Many studies reported that MeHg toxicity is accompanied by increased lipid peroxidation that may lead to oxidative damage to DNA, RNA, and proteins. Vitamin E is considered as the most effective antioxidant preventing lipid peroxidation. The aim of this study was to evaluate the effects of MeHg exposure during pregnancy on the development of the appendicular skeleton in rat fetuses and whether vitamin E administration could reduce this toxicity. Positively mated adult female Sprague-Dawley rats were used and divided into the following experimental groups: control group, received only deionized water, and four MeHg treated groups received 1 mg of MeHg/kg/d, 2 mg of MeHg/kg/d, 1 mg of MeHg/kg/d plus 150 mg of vitamin E/kg/d, and 2 mg of MeHg/kg/d, plus 150 mg of vitamin E/kg/d starting from Day 0 of gestation. On Day 20 of gestation, the fetuses from the pregnant rats were extracted and the fetal growth parameters were evaluated. Skeletal evaluation of ossification of both fore- and hind-limbs, and coxal bones were undertaken. Results showed that treatment with MeHg caused adverse effects on fetal growth parameters and ossification of the bones. The coadministration of vitamin E with MeHg revealed an improvement in these parameters. These results suggest that vitamin E may ameliorate some aspects of MeHg developmental toxicity. The underlying and human health implications warrant further investigations., (Copyright © 2012 Wiley Periodicals, Inc.)

A possible teratogenicity of multi-wall carbon nanotube (MWCNT) was assessed using ICR mice. MWCNTs were suspended in 2% carboxymethyl cellulose and given intraperitoneally or intra-tracheally to pregnant ICR mice on day 9 of the gestation. All fetuses were removed from the uterus on day 18 of the gestation, and were examined for external and skeletal anomalies. In the intraperitoneal study, various types of malformation were observed in all MWCNT-treated groups (2, 3, 4 and 5 mg/kg body weight, intraperitoneal). In contrast, such malformations were observed in groups given 4 or 5 mg/kg body weight, but not in that treated with 3 mg/kg in the intratracheal study. In either study, the number of litters having fetuses with external malformation and that of litters having fetuses with skeletal malformations were both increased in proportion to the doses of MWCNT. The present results are the first to report that MWCNT possesses the teratogenicity at least under the present experimental conditions. Mechanism(s) to result such malformations is yet unclear and further experiment is necessary.

Anticonvulsant drugs taken by pregnant women to prevent seizures are among the most common causes of potential harm to the fetus. It has been suggested that carbamazepine was less teratogenic than the other drugs. Here, we report a case of fetal carbamazepine syndrome presenting with facial dysmorphism, congenital heart defect, skeletal abnormalities, renal agenesis, ambiguous genitalia, anal atresia, and right hemihypoplasia of the entire body. To the best of our knowledge this is the most severe case of fetal carbamazepine syndrome in the literature. This case can provide useful data about teratogenicity of carbamazepine therapy during the pregnancy.

Manganese (Mn) has been associated with embryo toxicity as it impairs differentiation of neural and skeletogenic cells in vertebrates. Nevertheless, information on the mechanisms operating at the cellular level remains scant. We took advantage of an amenable embryonic model to investigate the effects of Mn in biomineral formation. Sea urchin (Paracentrotus lividus) embryos were exposed to Mn from fertilization, harvested at different developmental stages, and analyzed for their content in calcium (Ca), expression of skeletogenic genes, localization of germ layer markers, and activation of the extracellular signal-regulated kinase (ERK). By optical and immunofluorescence microscopy, we found that Mn exposure produced embryos with no skeleton, by preventing the deposition of the triradiate calcitic spicules usually produced only by specialized mesoderm cells. On the contrary, ectoderm and endoderm differentiation was not impaired. Endogenous Ca content in whole embryos and its localization in Golgi regions of skeletogenic cells was strongly reduced, as measured by atomic absorption spectrometry and in vivo calcein labeling. Spicule-lacking embryos showed persistent ERK activation by immunocytochemistry and immunoblotting, contrary to the physiological oscillations observed in normal embryos. The expression of the skeletogenic genes, Pl-msp130 and Pl-sm30, was also differentially affected if compared with controls. Here, we showed for the first time the ability of Mn to interfere with Ca uptake and internalization into skeletogenic cells and demonstrate that Ca content regulates ERK activation/inactivation during sea urchin embryo morphogenesis. The use of Mn-exposed sea urchin embryos as a new model to study signaling pathways occurring during skeletogenesis will provide new insights into the mechanisms involved in Mn embryo toxicity and underlie the role of calcium in the biomineralization process in vertebrates.

This study was conducted on determining the effects of phenytoin on the skeletal system of the fetuses of 13 Wistar Albino rats. The female rats were divided into two groups after the vaginal smear test: the group 1 (control group) included 6 individuals, whereas the group 2 (phenytoin group) comprised 7 animals. A dose of 25mg/kg/day phenytoin was administered intraperitoneally to pregnant rats on the 8th-10th days of pregnancy and fetuses were obtained by C-section on the 20th day. A number of 82 fetuses were observed by double staining technique. Their lengths and weights were measured, revealing the statistically significant differences between the two groups (p<0.001). The lengths of the fetuses in the group 2 were determined as to be 14% shorter and the weights 13% lower compared to those in the group 1. Similarly, number of the fetuses obtained in one gestation decreased 9% in the group 2. Ossification of the skull bones in the fetuses of the group 2 was observed eminently to be deteriorated through using dissection microscope and inspection. Costal separation anomaly was observed in the 10 fetuses of the group 2. The separated-laterally located costal components were not attached to the costal arch. Shape malformations in the last two ribs and wide angularity, particularly in the last six ribs, were also determined. This study has documented that intraperitoneal usage of the pheytoin during pregnancy may cause to different skeletal malformations, even with lower doses, in rat fetuses., (Copyright © 2010 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

A toxicity study was planned to assess the teratogenic potential of cyfluthrin that is widely used as a household insecticide to control mosquitoes, flies and cockroaches. Pregnant Swiss albino mice of one group were orally administered two doses of the pesticide (16 mg/kg and 32 mg/kg body weight) daily during the organogenetic phase (days 5-14) of gestation. The second group received the same two doses daily during the maturation phase (days 14-18) of gestation. The animals receiving the higher dose exhibited burrowing behaviour, which is a characteristic symptom of pyrethroid poisoning. The autopsies were performed on the 18th day of gestation and routine teratological observations were made. No external malformations occurred in any of the fetus. The higher dose significantly reduced the number of live fetuses, litter size and increased the resorption of embryos when administered during organogenesis, while exposure to the pesticide during the maturation phase did not significantly affect the reproductive parameters. During both the phases, the higher dose reduced the maternal weight gain and the average weight of the fetuses. The fetal anomalies observed were reduced ossification of skull bones, widened cranial sutures, short or absent ribs, hydrocephaly of the ventricles, microphthalmia, anophthalmia, pulmonary edema and subcutaneous edema.

VPA axial malformations are related to embryonic somitic histone hyperacetylation. In cancer, histone hyperacetylation activates apoptosis. To verify if apoptosis is involved in somitic abnormalities, VPA-exposed embryos were evaluated for DNA fragmentation and for pro- (p53, acetylated p53, caspase 3) and anti-apoptotic (Sirt 1) protein expression. Pregnant mice were i.p. dosed on day 8 with VPA 400mg/kg or TSA (16 mg/kg). Embryos, collected 3, 5, 9 or 24h after treatment, were examined and processed for apoptosis or protein analysis. An event cascade has been observed at the level of somites and proposed as related to VPA-induced axial skeletal defects: increased p53 (3h), DNA fragmentation (9h), abnormalities (24h). TSA, used as alternative HDAC inhibitor, induced apoptosis and somitic abnormalities, strengthening our hypothesized link between HDAC inhibition and axial defects., (Copyright 2009 Elsevier Inc. All rights reserved.)

We developed and optimized a screening procedure, in which zebrafish embryos were explored as a model for the evaluation of the specific embryotoxic and teratogenic potential of chemicals. A selection of known positive (retinoic acid, valproic acid, caffeine, lithium chloride) and negative (glucose, saccharin) compounds for developmental toxicity were used to evaluate this method. We exposed embryos and evaluated embryotoxicity and morphological characteristics of the embryos at 24, 48, 72 and 144 h post fertilization. After evaluation of the induced effects, concentration-response curves were created for both embryotoxicity and teratogenic effects. Values for teratogenic indices (TI) were calculated as the ratio LC(50)/EC(50). The results obtained were compared to existing data from studies with laboratory animals and humans. We demonstrated that our classification of the compounds, based on TI values, allows to distinguish teratogens from non-teratogens and supports the application of zebrafish embryos as an alternative method for developmental toxicity studies to predict effects in mammals.

Single oral doses of artesunate, dihydroartemisinin, arteether and artemether administered to rats during a sensitive period of organogenesis caused embryo deaths and malformations (malformed long bones and ventricular septal defects). Extended oral dosing (12 days or more) of monkeys once daily with 12 mg/kg-d artesunate also caused embryo deaths. The initial embryotoxic effect in both species was to kill primitive erythroblasts which are present in the embryo for a few days of gestation in rats and several weeks in primates. The malformations that occurred in rats are attributed to a transient depletion of the primitive erythroblasts. In monkeys, when treatment at 12 mg/kg-d was shortened to 3 or 7 days, the embryos survived but likely suffered a transient loss of primitive erythroblasts. Limited clinical data including 123 first trimester pregnancies have not indicated any adverse effects on pregnancy. However, in rats and monkeys, the embryonic erythroblasts are much more sensitive to artemisinins than are erythroblasts in the adult bone marrow; the latter are indicated by decreases in reticulocyte count. Since decreases in reticulocyte count occur at therapeutic doses in humans, there is reason for concern that any treatment of pregnant women during the putative sensitive period (from approximately postconception Day 21 to approximately postconception week 9) that causes even minor decreases in adult reticulocyte count could also cause a marked depletion of embryonic erythroblasts which could lead to death or malformation of the embryo.

Background: Valproic acid (VPA) is used to treat epilepsy and bipolar disorders, as well as for migraine prophylaxis. However, its clinical use is limited by two life-threatening side effects: hepatotoxicity and teratogenicity. To develop a more potent and safer second-generation VPA drug, the urea derivatives of four VPA analogs (2-ethyl-3-methylpentanoyl urea, 2-ethylhexanoyl urea, 2-ethyl-4-methylpentanoyl urea, and 2-methylbutanoyl urea) were synthesized., Methods: Four CNS-active analogs of a VPA urea derivative testedthe anticonvulsant activity in the maximal electroshock seizure test (MES) and subcutaneous metrazol seizure threshold test (scMet). Teratogenic effects of these compounds were evaluated in NMRI mice susceptible to VPA-induced teratogenicity by comparison with VPA., Results: All four VPA analogs showed superior anticonvulsant activity over VPA. Compared with VPA, which induced neural tube defects (NTDs) in fetuses at 1.8 and 3.6 mmol/kg, the analog derivatives induced no NTDs at any concentration up to 4.8 mmol/kg (except for a single abnormality at 3.6 mmol/kg with 2-ethyl-3-methylpentanoyl urea). Skeletal examination also revealed that the acylurea derivatives induced vertebral and rib abnormalities in fetuses markedly less frequently than VPA. Our results confirmed that the analogue derivatives are significantly less teratogenic than VPA in NMRI mice., Conclusions: The CNS-active VPA analogs containing a urea moiety, which have better anticonvulsant potency and lack teratogenicity, are good potential candidates as second-generation VPA antiepileptic drugs., ((c) 2009 Wiley-Liss, Inc.)

Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not specific, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also affected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic effects remains to be elucidated. When considering the potential inhibitory effect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis.

Background: Oxidative stress is hypothesized to mediate embryotoxicity during organogenesis, yet the reactive oxygen species involved are not defined. The superoxide oxygen radical is converted to hydrogen peroxide, a less reactive species, by superoxide dismutases (SODs). If superoxide is important in mediating embryotoxicity, increased SOD expression should protect embryos against insult. Exposure to hydroxyurea during organogenesis causes brain defects, cleft palate, tail anomalies, and limb defects; administration of D-mannitol, a free radical scavenger, ameliorates hydroxyurea embryotoxicity, suggesting that oxidative stress is important. To elucidate the role of superoxide in mediating hydroxyurea embryotoxicity, we assessed the impact of human SOD1 expression in a murine model., Methods: hSOD1 hemizygous male mice, carrying the human SOD1 gene, were mated to wild-type or hSOD1 hemizygous females. Dams were treated on gestation day (GD) 9 with saline (control) or 400 (low) or 600 (high) mg/kg hydroxyurea (n = 8-13/group). Mice were euthanized on GD 18 and developmental toxicity was assessed., Results: Exposure to hydroxyurea caused a dose-dependent increase in fetal deaths that was not affected by hSOD1 expression; hydroxyurea decreased fetal weights in litters from wild-type but not hemizygous dams. Hydroxyurea increased the incidence of external and skeletal malformations; fetuses from hemizygous dams treated with high-dose hydroxyurea had fewer malformations compared to wild-type dams. There was no correlation between embryonic phenotype and genotype or SOD activity., Conclusion: Maternal hSOD1 expression protected fetuses against malformations induced by hydroxyurea, providing evidence that superoxide plays a role in mediating the response of organogenesis stage embryos to this teratogen.

Acetylsalicylic acid (ASA) at single doses of 125, 250, and 500mg/kg was administered to pregnant rats on Gestation Day (GD) 10, and skeletal changes in fetuses harvested on GD 20 and pups on post-natal (PN) Day 21 were evaluated. Changes in cartilage and ossified bones identified by Alizarin Red S single-staining were compared with Alizarin Red S and Alcian Blue double-staining. By the single-staining technique, skeletal abnormalities including fused rib, incomplete ossification of the cervical arch, absent/hemicentric body of thoracic or lumbar vertebra, deformation of lumbar arch, and absent sacral arch were demonstrated in at 250 and 500mg/kg ASA on GD 20. The double-staining technique facilitated identification of additional cartilaginous changes in the vertebrae, paws, and ribs: including discontinuous rib cartilage, fused carpus, and split cartilage of thoracic centrum at same doses. Discontinuous rib cartilage and fused carpus persisted in pups until PN Day 21 demonstrating that these changes were irreversible. With use of the double-staining technique, the incidence of abnormalities at 250mg/kg were dramatically increased, thus this technique was more sensitive for identifying fetal cartilaginous and ossified skeletal changes.

Toluene is a commonly abused organic solvent. Inhalant abusers are increasingly women in their prime childbearing years. Children born to mothers who abused solvents during pregnancy may exhibit characteristics of a "fetal solvent syndrome" which may include dysmorphic features. This study examined the teratological effects of an abuse pattern of binge toluene exposure during gestation on skeletal and soft tissue abnormalities, body weight, and body size in fetal rats. Pregnant Sprague-Dawley rats were exposed for 30 min, twice daily, from gestational day (GD) 8 through GD20 to either air (0 ppm), 8000 ppm, 12,000 ppm, or 16,000 ppm toluene. Two-thirds of each litter was prepared for skeletal examination using Alizarin Red S staining while the remaining third of each litter was fixed in Bouin's solution for Wilson's soft tissue evaluation. Exposure to toluene at all levels significantly reduced growth, including decreases in placental weight, fetal weight, and crown-rump length. In addition, numerous gross morphological anomalies were observed such as short or missing digits and missing limbs. Skeletal examination revealed that ossification of the extremities was significantly reduced as a result of toluene exposure at all levels. Specific skeletal defects included misshapen scapula, missing and supernumerary vertebrae and ribs, and fused digits. Soft tissue anomalies were also observed at all toluene levels and there was a dose-dependent increase in the number of anomalies which included cryptorchidism, displaced abdominal organs, gastromegaly, distended/hypoplastic bladder, and delayed cardiac development, among others. These results indicate that animals exposed prenatally to levels and patterns of toluene typical of inhalant abuse are at increased risk for skeletal and soft tissue abnormalities.

Harmonization of terminology in developmental toxicology is a prerequisite to ensure a better risk assessment of chemicals. As part of an international effort of the International Programme on Chemical Safety (IPCS) to harmonize terminology in developmental toxicology, workshops have taken place in Berlin since 1995. This publication reports the main outcomes of the Fifth and Sixth Berlin Workshops held in 2005 and 2007, respectively. The objective of the Fifth workshop was to discuss a draft international proposal for updating the glossary of descriptive terms for fetal abnormalities put forward by Wise et al. [Wise LD, et al. Terminology of developmental abnormalities in common laboratory mammals (version 1). Teratology 1997;55:249-92]. The participants were asked to classify the new external, visceral and skeletal observations included within this new version 2 of Terminology of Developmental Abnormalities in common Laboratory Mammals according to the two-category scheme (malformation and variation) agreed at previous Berlin workshops. The discussions held during the Sixth Workshop were mainly focused on the causes of uncertainty and low agreement regarding classification of some fetal observations as malformations or variations. Lack of precision in descriptive terms and insufficient knowledge of the postnatal consequences of fetal observations had been identified as major causes of uncertainty and lower agreement among evaluators regarding the classification of "grey zone anomalies", i.e. abnormalities that do not fit readily into one of the two categories (malformation or variation). Imprecise anatomical terms, observation terms that are too broad, lack of information on severity and the use of different terms for the same change or different severities of the same change, were found to be the main reasons that descriptive terms are often not sufficiently precise to allow accurate classification of findings. It was agreed that provision of additional information, including sub-location within the affected structure, more detailed description of the nature of the change, in conjunction with presentation of photographs wherever possible, and a grading for severity would make descriptive terms more precise, thereby reducing misclassifications. A better knowledge of the adversity and postnatal consequences of fetal observations was considered as the key issue for achieving a substantial reduction in the number of misclassifications and grey zone anomalies. The urgent need for additional research along this line as a prerequisite for a better risk assessment was emphasized by the participants.

The potential interactivity of the antimicrobials miconazole and metronidazole in the induction of teratogenic effects was investigated in the present study. Drugs were injected at 60 mg/kg either individually or in combination to pregnant mice on gestation day (GD) 8, 9, or 10. Teratological assessments were carried out on GD 18. A potent teratogenic interaction resulted from miconazole-metronidazole co-administration, causing an increment in axial skeletal defects incidence. While, the individual exposure to miconazole or metronidazole produced axial skeletal defects at frequencies that did not exceed 5.6% in the various treatment groups, the percentage of fetuses with malformed skeleton reached 26% after co-exposure on GD 8 or GD 9. No significant synergism was noted when drugs were co-administered on GD 10. This study shows that a teratogenic interaction can result from miconazole-metronidazole concomitant exposure.

Objective: To investigate the teratogenic and anti-estrogenic effect of environment teratogen on rat skeleton development., Methods: The fetal rat models with congenital skeleton malformation were constructed with 2, 3, 7,8-tetrachlorodibenzo-p-dioxin (TCDD) on pregnant day 10. The transparent skeleton of fetal rat was made with alizarin bordeaux. The cell pathology of chondrocyte in fetal rat center of ossification was observed by light microscope. The estradiol in rat serum was measured by radioimmunity method., Results: Treated with 5-15 microg/kg TCDD, the single or multiple rat fetal skeletal development malformation had been induced. The teratogenic effect of TCDD on the osteogenesis was in dose-dependent manner. There were decreased center of ossification and cell degeneration of chondrocyte in fetal extremity limbs. The estradiol increased significantly in TCDD-treated rat, compared with normal rat (P < 0.05)., Conclusion: TCDD can induce congenital fetal skeletal malformation under interfering the biological activity of estrogen and the function of chondrocyte and the primary center of ossification.

In spite of the large use of salicylates, introduced into clinical practice more than 100 years ago, their anti-inflammatory and cancer preventive mechanisms are still under study. Teratogenic effects of salicylates have been reported in experimental animals since 1959 but the pathogenic pathways and the mechanisms of action were never described until now. The aim of this work is to verify if the inhibition of embryonic histone deacetylase (HDAC) enzymes and the consequent tissue hyperacetylation could be the mechanism responsible for axial skeletal defects described after the exposure of pregnant rodents to sodium salicylate (SAL). E8 pregnant CD-1 mice were intraperitoneally treated with SAL 0-150-300-450 mg/kg and sacrificed at 1, 3, 5 h after treatment or at term of gestation (E18). E8 embryos were processed for Western blotting and immunostaining analyses, while skeletons of E18 fetuses were double stained for bone and cartilage. A group of control E8 embryos were used to prepare embryonic nuclear extract for the HDAC enzyme assay. A significant SAL dose-related HDAC inhibition activity, compatible with a mixed-type partial inhibition mechanism, was detected. A clear dose-related hyperacetylation of histones was observed in embryos exposed in utero to SAL, with a peak at 3 h after treatment of dams. The most hyperacetylated organs were somites and the heart. Histone hyperacetylation is suggested to be the mechanism accounting for SAL-related axial skeletal and cardiovascular defects and is proposed as the mechanism responsible for other biological effects of salicylates.

Aim of the Study: Perovskia abrotanoides is a medicinal herb which traditionally has been used as a topical application to treat leishmaniasis in Iran. Due to similarities of the flowers of Perovskia abrotanoides and Echium species, it is added as an adulterant to Echium preparations in Iran. Echium flowers have been used in Iranian traditional medicine as an "anti-stress", tranquilizer, and energizer drink, fighting common cold and bronchitis particularly during pregnancy. In this study, the in vivo embryotoxicity of 0.125 and 0.25 g/kg of ethanolic and aqueous extracts of Perovskia abrotanoides during organogenesis in mice has been investigated., Materials and Methods: Maternal body weights were recorded and pregnant females were observed daily throughout gestation. After cesarean section, fetuses were weighed, lengthed and examined for gross external malformations. Others were fixed then stained with Alcian blue and Alizarin red to view any skeletal malformations., Results: Our results indicated that this herb causes minimal maternal toxicity, while resorption, stillborn, fetal malformations including polydactyly, spina bifida, aglossia, tarsal extensor, gastroschisis, and numerous skeletal abnormalities were detected particularly in the ethanolic extract groups. Specific skeletal abnormalities based on type of extract were also observed., Conclusions: Since Perovskia abrotanoides is widely used in traditional medicine intentionally or inadvertently, it is advisable its use during pregnancy should be avoided and strict regulations are imposed to prevent its addition to commercial preparations of other herbal products.

Ethanol is a known teratogen and has been implicated in the etiology of human fetal alcohol syndrome (FAS), which is characterized by distinct craniofacial abnormalities such as microcephaly, agnathia, and ocular aberrations. Attempts at quantifying the craniofacial anomalies arising from ethanol exposure have largely been limited to radiographic evaluation in postnatal rats. Such studies discount the role of the cranial soft tissue in the morphology of FAS. We present a study whose aim was to conduct measurements of the entire head including soft and hard tissue in full-term fetuses of mice by means of a digital analyzer, while at the same time comparing stained skeletal tissues in treated and untreated animals. Thirteen pregnant C57BL/6J mice were fed with 25% ethanol (vol/vol) on gestation days (E) 6, 7, and 8, whereas 10 pregnant mice received water only. Fetuses were retrieved from the animals just before delivery on E18, digitally photographed, measured, and assessed for abnormalities. Ethanol-exposed mice showed a number of abnormalities such as anophthalmia and agnathia, but these were not significantly increased over those from nontreated fetuses (P=.5). Birth weight (P=.5), crown-rump length (P=.8), and mandibular length (P=.9) were also not significantly reduced compared to control fetuses. However, defects in some cranial bones and degrees of ossification that trailed same-stage controls were observed in treated animals, at a nonstatistically significant level (P=.14). Acute maternal ingestion of alcohol in mice during pregnancy may not cause a significant increase in craniofacial or skeletal defects when evaluated at term. However, these effects may be latent, manifesting postnatally. The postnatal ability of mice for recovery from alcohol-induced birth defects deserves further investigation.

Skeletal variations are common in humans, and potentially are caused by genetic as well as environmental factors. We here review molecular principles in skeletal development to develop a knowledge base of possible alterations that could explain variations in skeletal element number, shape or size. Environmental agents that induce variations, such as teratogens, likely interact with the molecular pathways that regulate skeletal development., ((c) 2007 Wiley-Liss, Inc.)

Standard evaluations for characterizing selective developmental toxicity are traditionally undertaken in vivo. These studies incur significant cost in animal use, labor and compound, ultimately limiting the selection of compounds that can be evaluated in vivo. Such limitations hinder the ability to address questions regarding whether teratogenic outcome was caused by intended pharmacology or attributed to off-target effects associated with the structure of the small molecule. Ascertaining a better understanding of the published literature can enhance interpretation of existing in vivo datasets and hypotheses regarding critical windows of sensitivity and underlying mechanisms of teratogenicity. Thoughtful execution of investigative in vivo and in vitro studies can test and further define the underlying mechanism of teratogenicity. Skeletal variations and malformations are frequently encountered in in vivo studies and can be difficult to interpret in context of defining hazard assessment and mechanisms of abnormal development. This commentary reviews how investigative approaches can be integrated to better understand teratogenic mechanism as it pertains compounds that produce skeletal abnormalities. Approaches are discussed in context of how they could be used to study a compound that has been found to produce fused and wavy ribs in rat fetuses. An investigative approach is described that utilizes three strategies: 1) maximizing the data available from in vivo studies; 2) performing critical window studies in vivo; and 3) performing mechanism of action evaluations using gene expression studies and developmental model systems., ((c) 2007 Wiley-Liss, Inc.)

The developmental toxicity of the three main metabolites of N-methyl-2-pyrrolidone (NMP) was studied in Sprague-Dawley rats. Pregnant rats were given 5-hydroxy-N-methyl-2-pyrrolidone (5-HNMP; 0, 250, 500, 750 or 1000 mg kg(-1) day(-1)), N-methylsuccinimide (MSI; 0, 500, 750, 1000 or 1250 mg kg(-1) day(-1)), or 2-hydroxyN-methylsuccinimide (2-HMSI; 0, 250, 500, 1000 or 1500 mg kg(-1) day(-1)), by gavage, on gestational days (GD) 6-20. No evidence of maternal toxicity was observed in dams given 5-HNMP. Administration of 2-HMSI resulted in overt maternal toxicity at 500 mg kg(-1) day(-1) and higher doses, as indicated by a significant reduction in weight gain and food consumption at the beginning of treatment. There was no evidence of embryo/fetal toxicity in any of the groups treated with 5-HNMP or 2-HMSI. MSI produced marked developmental toxicity in the presence of maternal effects. Maternal body weight gain and food consumption were affected at 750 mg kg(-1) day(-1) MSI, and above. A significant increase in post-implantation loss occurred at 1250 mg kg(-1) day(-1) MSI, and the incidence of fetuses with external or with visceral malformations was significantly increased at 1000 and 1250 mg kg(-1) day(-1) MSI. Malformations mainly consisted of anasarca, cardiovascular defects and diaphragmatic hernia. Fetal weight was significantly reduced at 1000 and 1250 mg kg(-1) day(-1). The incidence of skeletal variations (predominantly cervical ribs, and delayed ossification of skull bones and sternebrae) was significantly elevated at 750 mg kg(-1) day(-1) and higher doses. However, MSI was much less potent than the parent compound. These results indicate that the embryotoxic and teratogenic effects of NMP are not attributable to these metabolites.

Leflunomide is an immunosuppressive agent that inhibits de novo synthesis of pyrimidine nucleotides and the activity of protein tyrosine kinase. This study examined the teratogenicity of Leflunomide in mice. Pregnant mice were treated orally with Leflunomide at a dose of 10, 30 or 70 mg/kg/day from day 6 to 15 of pregnancy. At 70 mg/kg, all embryos were resorbed and no live fetuses were detected. At 30 mg/kg, Leflunomide reduced fetal viability, and increased the incidence of multiple external, skeletal and visceral malformations. Characteristic external malformations were neural tube defects, cleft palate and tail deformities. Limb malformations were observed in a small number of fetuses. Skeletal examinations revealed malformations of cervical to sacral vertebrae, ribs and sternebrae. In the viscerae, the main anomalies were membranous ventricular septum defect and persistent truncus arteriosus. The results of this study indicate that Leflunomide administered at 30 mg/kg on days 6 to 15 of pregnancy can induce craniofacial malformations and deformities of the axial skeleton, heart and great vessels in mice.

The effects of indium on bone and cartilage development in rat fetuses were examined. Pregnant Sprague Dawley (SD) rats were treated with indium trichloride (0.1, 0.2, or 0.3 mg/kg) by single intravenous administration on Day 10 of gestation, and their fetuses were examined on Day 21. Half of each litter was prepared for skeletal examinations using a skeletal double-staining technique to allow evaluation of cartilage as well as bone. Dose-related increased incidences of external and skeletal fetal malformations occurred at doses of 0.2 mg/kg or more. The incidences of cartilage malformations in the vertebrae, ribs, and forepaw phalanges were significantly increased at 0.3 mg/kg. Malformations of the axial bone were accompanied by cartilage malformations. It was concluded from these results that indium produced cartilage malformations, that were considered to be the underlying cause for the majority of fetal skeletal malformations observed in rats in this study.

The developmental toxicity of toluene was evaluated via whole body inhalation exposure, in pregnant Sprague Dawley rats exposed to toluene (99.9% pure) from gestation day (GD) 6-15 inclusive, 6h/day, at concentrations of 0, 250, 750, 1500 and 3000ppm (0, 938, 2812, 5625 and 11250mg/m(3)). Doses were selected from a preliminary study performed over a range of concentrations from 0 to 5000ppm, in which maternal and fetal toxicity were observed at 2000ppm and above. This study has been cited in various regulatory documents and is presented here to allow greater accessibility to results and conclusions. Toluene induced clinical signs in pregnant dams (ataxia, hyper-responsivity, increased water intake, decreased food consumption) at 3000ppm, ataxia and hyper-responsivity at 1500ppm, and reduced maternal body weight gain at 1500 during the exposure period only and at 3000ppm from initiation of exposure to GD20. At Caesarean section on GD20, no adverse effects on implantation, number and viability of fetuses, or fetal sex distribution were observed. Litter weight and mean fetal weight was reduced at 3000ppm and mean fetal weight was reduced at 1500ppm. Instances of reduced or unossified skeletal elements occurred at the same dose levels. Mean fetal weight was also reduced at 250ppm but not at 750ppm. Extensive statistical analysis of fetal body weight data support the conclusion that there is no toxicologically significant dose-related effect on fetal body weight at or below 750ppm. Low incidences (

Dietary exposures to food pollutants such as mycotoxin(s) or pesticide(s) are most significant due to their adverse effects on the production and reproduction in animals and the human population. The present investigation was conducted to evaluate the teratogenic potential of citrinin (CIT) and endosulfan either alone or in combination in pregnant rats during gestational days 6-20. Endosulfan (1 mg kg(-1) body weight, by oral intubation) and CIT (10 mg kg(-1) feed, through diet) when administered either alone or in combination in pregnant rats caused significant teratogenic effects in the developing fetuses. There was no maternal mortality, however, reduced maternal weight gain and number of live fetuses and increased fetal resorptions were recorded in all the treated groups. The fetal body weights and crown to rump lengths were significantly decreased and the per cent gross, visceral and skeletal anomalies were significantly increased in the fetuses of dams of all the treated groups. The internal hydrocephalus, cerebellar hypoplasia, microphthalmia, contracted and notched kidneys, multilobulated liver, dilated renal pelvis, incomplete ossification of skull bones, rib anomalies and sacral and caudal vertebrae agenesis were the important fetal malformations. The occurrence of fetal gross, skeletal and visceral malformations was more severe in the combination group, suggesting an additive interaction of CIT and endosulfan in inducing developmental toxicity in Wistar rats.

Background: To test whether the differentiating embryo is susceptible to the teratogenic effects of the nitric oxide (NO) synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME)., Methods: ICR-(CD-1) mice received a single intraperitoneal injection of L-NAME at 90, 150, or 300 mg/kg on Gestation Day (GD) 8 or 9. Controls were treated with vehicle on GD 8 and 9. Teratological assessments were carried out near term (GD 18)., Results: Maternal treatment with a single dose of L-NAME at 150 or 300 mg/kg on either GD 8 or 9 produced axial skeletal defects in the ICR (CD-1) mouse fetuses. Other treatment-related effects included increased embryo lethality and fetal growth restriction., Conclusions: This study provides evidence that in utero exposure to L-NAME can affect organogenesis of the axial skeleton., ((c) 2007 Wiley-Liss, Inc.)

This study investigated the potential adverse effects of amitraz on the initiation and maintenance of pregnancy in Sprague-Dawley rats as well as its effects on embryo-fetal development after maternal exposure during the entire pregnancy period. Amitraz was administered to pregnant rats by gavage from days 1 to 19 of gestation at dose levels of 0, 3, 10, and 30 mg/kg/day. All dams underwent a caesarean section on day 20 of gestation and their fetuses were examined for any external, visceral, and skeletal abnormalities. At 30 mg/kg, maternal toxicity manifested as an increase in the incidence of abnormal clinical signs and a lower body weight gain and food intake. Developmental toxicity included an increase in the fetal death rate, a decrease in the litter size, and a reduction in the fetal body weight. In addition, there was an increase in the incidence of fetal external, visceral, and skeletal abnormalities. At 10 mg/kg, maternal toxicity observed included a decrease in the body weight gain and a decrease in food intake. In addition, minimal developmental toxicity, including a decrease in the fetal body weight, an increase in the visceral and skeletal aberrations, and a delay in fetal ossification. There were no signs of either maternal toxicity or developmental toxicity at 3 mg/kg. These results show that amitraz administered during the entire pregnancy period in rats is embryotoxic and teratogenic at the maternally toxic dose (i.e., 30 mg/kg/day) and is minimally embryotoxic at a minimally maternally toxic dose (i.e., 10 mg/kg/day). Under these experimental conditions, the no-observed-adverse-effect level of amitraz for both dams and embryo-fetal development is estimated to be 3 mg/kg/day.

Pregnant rats were given 1,3-di-o-tolylguanidine (DTG) by gavage at 0, 10, 20 or 40 mg/kg bw/day on days 6-19 of pregnancy and the pregnancy outcome was determined on day 20 of pregnancy. At 40 mg/kg bw/day, deaths were observed in four out of 24 females. The incidences of females showing mydriasis at 20 and 40 mg/kg bw/day and showing decreased locomotor activity at 40 mg/kg bw/day were significantly increased. Alopecia, bradypnea, prone position and tremor were also observed at mg/kg bw/day. The maternal body weight gain at 20 and 40 mg/kg bw/day and food consumption at 40 mg/kg bw/day were significantly reduced. A significantly decreased weight of the gravid uterus, increased incidence of postimplantation loss, decreased number of live fetuses, and lowered weights of fetuses and placentae were found at 40 mg/kg bw/day. The incidences of the total number of fetuses with external malformations at 40 mg/kg bw/day and with skeletal malformations at 20 and 40 mg/kg bw/day were significantly increased. Significantly higher incidences of fetuses with brachydactyly and short tail and defects of caudal vertebrae, phalanges and metacarpals were observed at 40 mg/kg bw/day. Delayed ossification was also noted at 40 mg/kg bw/day. The data indicate that DTG is teratogenic at maternal toxic doses and the NOAELs of DTG for maternal and developmental toxicity are 10 mg/kg bw/day in rats.

The current study was performed to assess the potential of 4,5-dihydroxy 1,3-benzene disulfonic acid di sodium salt (Tiron) and glutathione (GSH) either individually or in combination against aluminum (Al)-induced developmental toxicity in fetuses and sucklings of Wistar rats. Female rats were exposed to aluminum chloride at a dose of 345 mg/(kg day) oral from days 0 to 16 of gestation and 0 to 16 of post-partum (P.P.). Tiron and GSH were administered at a dose of 471 mg/(kg day) i.p. and 100 mg/(kg day) oral, respectively, on days 5, 7, 9, 11, 13, 15 and 17 of gestation and post-partum. Al caused reduction in number of corpora lutea, number of implantation sites, placental and fetal weight and stunted growth. Skeletal malformations were also observed in fetuses. Maternal toxicity was demonstrated by reduction in body weight gain. Induction of oxidative stress was also recorded in the brain of mother as well as in fetuses and sucklings after Al exposure. Significant decrease was recorded in reduced glutathione, glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), acetyl cholinesterase (AChE) and increase was observed in TBARS and glutathione-S-transferase (GST) in brain of pregnant mothers, fetuses and sucklings. Most of the above parameters responded positively with individual therapy with Tiron, but more pronounced beneficial effects on the above-described parameters were observed when Tiron was administered in combination with GSH. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) studies also showed significantly high concentration of Al in suckling's brain and maternal blood, brain, placenta and fetal brain. Treatment with Tiron individually or in combination with glutathione, reduced the accumulation of the Al in almost all the organs studied. It is concluded that chelating agents reduced the Al-induced toxicity and Tiron was more effective in reducing blood Al concentration than glutathione when given individually.

Meglumine antimoniate (MA), a pentavalent antimonial (SbV) drug, has been used for the treatment of leishmaniases for over half a century but there is almost no data on its safety and kinetics during pregnancy. This study was undertaken to investigate the developmental toxicity of MA as well as the transplacental transfer of antimony (Sb) in rats. Wistar rats (approximately 20 per group) were treated subcutaneously (s.c.) with MA (0, 75, 150, 300 mg SbV/(kg BW day)) during pregnancy (days 1-20). An untreated control group was evaluated as well. Caesarean sections were performed on day 21 and implantations, living and dead fetuses, and resorptions were recorded. Fetuses were weighed and fixed in Bouin's solution and one-third of each litter was examined for soft-tissue anomalies. The remaining fetuses were cleared and stained with Alizarin red S for skeleton evaluation. No adverse effect of MA on the mothers was noted at any dose level. No embryotoxicity was observed at the lowest dose but, at the highest dose, MA increased embryolethality, reduced fetal weight and augmented the occurrence of some soft-tissue and skeleton variations. Therefore, the no-observed-adverse-effect level for MA-induced embryotoxicity was 75 mg SbV/(kg BW day). In a separate group of rats treated with 300 mg SbV/(kg BW day) s.c. during whole pregnancy, blood (0.2 mL) was taken from the tail vein 1, 2, 4, 6, 12 and 24 h after treatment on day 1 and thereafter every other day immediately before drug injection. Blood was also taken from fetuses removed on day 21, 24h after the last injection of MA. Blood levels of Sb were determined by ICP-MS and results showed that most of administered Sb was eliminated rapidly (in less than 6 h), but nadir blood concentrations increased gradually during treatment from 1 to 2 microg/g, 24h after the first dose, up to approximately 38 microg/g after the 20th dose. Levels of Sb in fetal blood were as high as 10-15 microg/g, i.e. approximately 30% their mothers' nadir levels near term. These findings indicated that repeated administration of MA during gestation led to an accumulation of Sb in mothers and fetuses.

Objectives: Valproic acid (VPA) is a widely used anticonvulsant medication with well-known teratogenic effects in both humans and in experimental animal model systems. The most commonly observed malformations induced by VPA in experimental animals include neural and skeletal defects. In this study the potential alterations in somitic tissue gene expression relative to the development of observed axial skeletal defects were examined., Methods: SWV mice were treated at 8.5 days post coitum (d.p.c.) with 1.36 mmol/kg or 2.72 mmol/kg VPA by i.p. injection. At 18.5 d.p.c., animals were killed and stained for morphological and skeletal examination. Cervical malformations consisting of vertebral fusions and cervical ribs were consistently observed. Phenotypic analysis confirmed the presence of dose-dependent axial skeletal malformations induced by in-utero VPA-exposure. Using antisense RNA amplification and cDNA microarrays, we examined the expression of approximately 5700 genes in the first six postotic somites of control and treated embryos at 6, 12, 18 and 24 h after the 8.5 d.p.c. VPA treatment., Results: Analysis indicated that several ontological groups (e.g. histone deacetylase complex, guanosine triphosphatases, cell proliferation and cytoskeletal) have significantly enriched gene expression changes in response to the teratogenic insult. The RNA from 6 h post-treatment was also subjected to a microarray cross-platform validation, and genes identified on both platforms are presented., Conclusion: These data were then used to deduce candidate cellular pathways that may be responsible for the VPA-induced teratogenic skeletal phenotypes.

Diphenyl diselenide is an organoselenium compound with potential therapeutic use. The present study evaluates the effects of single maternal subcutaneous injection of 50 and 100mg/kg diphenyl diselenide [(PhSe)2] at gestational days (GD) 6, 10 or 17 in Wistar rats. The highest dose of (PhSe2 was also administered at GD 7-12. External and internal fetal soft-tissue examination was performed at GD 20. No mortality was observed in fetuses or dams at any (PhSe)2 treatment group. Neither did exposure to (PhSe)2 cause significant changes to fetal body weight, organ weight, or fetal size when administered at GD 6-8, 10-12 or 17. Exposure to 100mg/kg (PhSe)2 at GD 9 produced significant changes in fetal biometry (crown-rump (CR) length) and body weight. No significant increase in the proportion of fetuses with external visible abnormalities was observed in groups exposed to (PhSe)2. Skeletal anomalies were observed in fetuses in the GD 9-11 treatment groups and included incomplete ossification of cranial bones, misshapen and incomplete ossification of sternebrae, reduced sternebrae number, wavy and extra ribs, incomplete ossification of fore and hindpaw bones and incomplete ossification of sacral and caudal bones. We conclude that maternal administration of (PhSe)2 during GD 7-12 led to increased incidences of these skeletal variations or anomalies, but did not cause externally visible malformations in rat fetuses.

Topiramate is a new generation, antiepileptic drug used for the treatment of persistent partial crises. To date no specific teratogenic effects have been reported in humans, but they have appeared in experimental animals. We present the case of a neonate whose mother suffered from partial epilepsy, which was treated with topiramate throughout pregnancy at doses of 300 mg per day. When the child was born agenesis of the right thumb, hypoplasia of the left thumb, and syndactylia of the second and third toes of the foot with agnesis of some phalanges, and hypoplasia of the right orbicular muscle in the mouth were observed. No etiologic cause was found. We discuss whether there could have been a causal relationship with topiramate monotherapy.

In a database cohort study, the risk of adverse birth outcome in Danish women who redeemed a fluoroquinolone prescription during pregnancy was examined. Among 87 women who redeemed a fluoroquinolone prescription at any time during the pregnancy, the prevalence ratio of preterm birth was 1.4 (95% confidence interval (CI), 0.6-3.2), one woman had a stillbirth, and no children died during the perinatal period. Among 130 women who redeemed a prescription during the first trimester or 30 days before conception, the prevalence ratio of congenital malformation was 0.7 (95% CI, 0.3-2.0) and the prevalence ratio of bone malformations was 2.2 (95% CI, 0.7-6.7). Our study raises the concern that prenatal fluoroquinolone exposure may be associated with an increased risk of bone malformations.

Background: The individual effects of boric acid (BA) and hyperthermia on the development of the axial skeleton have been reported previously. Both cause an increased incidence of axial skeletal defects including a decrease in the total number of ribs and vertebrae. Because of the similarity in the effects of the two agents, we examined their interaction when given in combination to pregnant rats on gestational day (GD) 10., Methods: Dams were treated on GD 10 with BA (0, 250, or 500 mg/kg) and hyperthermia (37, 41, or 42 degrees C) and allowed to deliver their pups. Doses of BA were based on results from a dose-finding study. Litters were evaluated on postnatal days (PND) 1 and 3 for number, gender, and weight of pups. On PND3, pups were examined externally and viscerally, and double-stained for skeletal evaluation., Results: A dose-dependent, statistically significant increase in fetal skeletal defects was seen on PND 3 with BA or hyperthermia alone with even greater effects when given in combination. Defects included rib and vertebral fusions, split vertebral centra in the thoracic and lumbar areas, and a decrease in the total number of ribs and vertebrae., Conclusions: The increased incidence of skeletal defects resulting from combined exposure to hyperthermia and BA was additive for segmentation defects and synergistic for the reduction in numbers of vertebrae.

Background: Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans-retinoic acid (RA) was associated with altered expression of TGFalpha, EGF receptor, and binding of EGF. This study uses knockout (KO) mice to examine the roles of EGF and TGFalpha in teratogenic responses of embryos exposed to RA., Methods: Pregnant wild-type (WT) mice of mixed genetic background, EGF KO, C57BL/6J, and TGFalpha KO mice were given a single oral dose of RA (100 mg/kg, 10 ml/kg) or corn oil on GD 10 at 12 PM, GD 11 at 12 PM or 4 PM, or GD 12 at 8 AM or 12 PM (plug day = GD 0). GD 18 fetuses were examined for external, visceral, and skeletal effects., Results: After exposure to RA on GD 12, the incidence of CP in EGF KO was significantly reduced relative to WT. In TGFalpha KO fetuses, RA exposure on GD 10 increased the incidence of CP versus C57BL/6J. The incidence of skeletal defects in the limbs, vertebrae, sternebrae, and ribs were also affected by lack of expression of EGF or TGFalpha with region-specific amelioration or exacerbation of the effects of RA. In TGFalpha KO fetuses, incidences of forelimb long bone and digit defects increased relative to C57BL/6J. In EGF KO fetuses, relative to WT, the incidence of hindlimb oligodactyly was increased. In EGF KO, but not WT, RA produced short, bent radius, humerus, and ulna. Both TGFalpha and EGF KO mice had increased incidences of dilation of the renal pelvis and this was reduced by RA., Conclusions: RA exposure produced skeletal and visceral defects in all genotypes; however, EGF or TGFalpha KO influenced the incidence and severity of defects. This study supports a role for EGF and TGFalpha in the response to RA., (Published 2005 Wiley-Liss, Inc.)

Background: Triadimefon is an antifungal derived from triazole. In in vitro whole-rodent embryo cultures, triazole-derivatives showed specific teratogenic effects at the branchial apparatus. The aim of the present work was to test in vivo triadimefon (FON), in order to verify a relationship between triazole exposure, embryonic abnormalities, and/or fetal malformations., Methods: Pregnant CD-1 mice were treated with 0-300 mg/kg FON by gavage on day 8 post coitum (p.c.) at 10:00 AM, and sacrificed on day 8 p.c. at 1:00 PM, on day 9 p.c. at 10:00 AM, on day 10 p.c. at 10:00 AM, and at term of gestation (day 18 p.c.). At midgestation, the embryos were processed for specific immunostainings to visualize the hindbrain segmentation (day 8 p.c.) and the neural crest cell migration (days 8 and 9 p.c.). Fetuses explanted at term were all processed for skeletal examination after double-staining of osseous and cartilaginous tissues., Results: At midgestation, the immunostaining of rhombomeres 3 and 5 showed a light scattering of the immunostained areas; the neural crest cell migration was unaffected, but their localization at the branchial arch level was abnormal. At term, several severe malformations were observed at the craniofacial and at the axial skeletal level. Ectopic cartilage was observed at the upper jaw., Conclusions: Triadimefon is teratogenic. The observed craniofacial malformations could be explained by an alteration of the rhombomeric organization and neural crest migration to the branchial arches; the axial abnormalities could be explained by the abnormal segmental identity specification.

In 1998, a mine tailings dyke in southwestern Spain broke, flooding the Agrio-Guadiamar river system with acid tailings up to the borders of one of the largest breeding colonies of white storks in the western Palearctic, Dehesa de Abajo. Over the following years, a high proportion of nestlings developed leg defects, prompting this study. Ten fledglings with leg deformities from the spill area were compared with 11 normal storks of the same year class from another region far from the spill. However, metals were analyzed as a continuum rather than by site, as reference birds also contained high levels of metals. Gross pathology of the legs was supported by histopathology, which showed that bone remodeling activity was greater in the deformed storks, which also had more irregular subperiosteal bone, and tended to have higher residual islets of cartilage in their metaphyses, which, in turn were related to metal contaminant residues. Both Ca and P in bone were affected independently by metals. Deformed birds had lower serum bone alkaline phosphatase. Bone malformations, measured by leg asymmetry, was only partially explained by bone metals, indicating that a combination of factors was involved with the abnormal development in these young storks.

Ochratoxin A (OA) and Aflatoxin B1 (AFB1), the food borne mycotoxins are produced by several fungal species of the genera Aspergillus and Penicillium. To determine the teratogenic effects, these mycotoxins were administered orally either individually or in combination to the pregnant Wistar rats on days 6-15 of gestation. OA and AFB1 were dissolved in corn oil and different doses of OA (0.125, 0.25, 0.50, and 0.75 mg/kg), AFB1 (0.125, 0.25, 0.50, and 1.00 mg/kg), and a combination of OA+AFB1 (0.125+0.125; 0.25+0.50; 0.50+0.25 mg/kg) were given by gastric intubation to rats. During dosing period, the body weight and body weight gains significantly decreased at a higher dosage, in both individual and combined treatments. In all the combination treatments, the percent implants resorbed, fetal body weights, and crown-rump lengths were comparable to those of controls and with the individual mycotoxin treatment. The number of dead fetuses was significantly increased in the high OA combination (OA+AFB1 0.50+0.25) group as compared with the other two combinations. OA and AFB1 alone and in combination caused various gross, skeletal, and visceral anomalies. The occurrence was considerably less pronounced in fetuses of AFB1 and combination groups as compared with those of OA group fetuses. The exencephaly, incomplete closure of skull, wavy and fused ribs, agenesis of the ischium bone, and enlarged renal pelvis, recorded in OA treatment and ear abnormality and incomplete ossification of skull bones observed in AFB1 when given individually, were not seen in combination groups. However, new manifestations, such as gastroschisis and syndactyly were observed and the incidence of cardiac defects was increased in fetuses due to the combined treatment. The results of the present study indicated that there is some interaction between these mycotoxins that resulted in reduced teratogenic activity of OA in the presence of AFB1. Apparently, new manifestations observed in combination treatment points to the potential threat of teratogenicity in terms of public health hazards., (Copyright 2004 Wiley-Liss, Inc.)

Background: Valproic acid (VPA) is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its teratogenic mechanisms are unknown. We have attempted to describe a fundamental role of the Polycomb group (Pc-G) in VPA-induced transformations of the axial skeleton., Methods: Pregnant NMRI mice were given a single subcutaneous injection of vehicle or VPA (800 mg/kg) on gestation day (GD) 8. The expression of genes encoding Polycomb and trithorax groups was measured by quantitative real-time RT-PCR using total RNA isolated from the embryos exposed to vehicle or VPA for 1, 3, and 6 hr. In addition, the use of two less teratogenic antiepileptic chemicals valpromide (VPD) and valnoctamide (VCD) provide reliable evidence to support the relationship between VPA teratogenicity and the Polycomb group., Results: At a teratogenic level, VPA inhibits the expression of the Polycomb group genes, including Eed, Ezh2, Zfp144, Bmi1, Cbx2, Rnf2, and YY1 in the mouse embryos. In contrast, neither VPD nor VCD have significant effects on the expression of those genes affected by VPA. The trithorax group (trx-G) gene MLL, which is known to be required to maintain homeobox gene expression such as the Polycomb gene, is not affected by a teratogenic dose of VPA., Conclusions: We propose that, during embryonic development, VPA may affect the gene silencing pathway mediated by the Polycomb group complex. The epigenetic mechanism of VPA teratogenicity on anteroposterior patterning is suspected.