Antiepileptic drugs (AEDs) during pregnancy and risk of congenital jaw and oral malformation.

In the United States, an estimate of 1.3 million women suffering from epilepsy are in their childbearing age. Potential teratogenicity of antiepileptic drugs (AEDs) is of concern to these women considering pregnancy because discontinuing pharmacotherapy during pregnancy may not be advised due to the risk of seizures that may be dangerous to the mother as well as the fetus. Using a Relational Online Analytical Processing (ROLAP) software licensed by Simultek, we searched for medications reported for congenital jaw and oral cavity malformation on the FDA Adverse Event Reporting System (AERS), a voluntary adverse event reporting program that contains over 55 million adverse event reports of medical products in the United.States. Our results indicate that various forms of valproic acid, and more importantly, newer generation antiepileptic agents including lamotrigine, topiramate, and gabapentin show signals for either congenital jaw or oral malformation. Although teratogenic potential of valproic acid has long been confirmed, information on teratogenicity of the newer generation antiepileptic drugs is relatively scarce and inconclusive. Early safety signals on the teratogenic potential of AEDs detected in this study are crude statistics that do not establish causation nor exclude confounding. The results require validation and further investigation via properly controlled epidemiological studies.

Reproductive and developmental toxicity of dioxin in fish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a global environmental contaminant and the prototypical ligand for investigating aryl hydrocarbon receptor (AHR)-mediated toxicity. Environmental exposure to TCDD results in developmental and reproductive toxicity in fish, birds and mammals. To resolve the ecotoxicological relevance and human health risks posed by exposure to dioxin-like AHR agonists, a vertebrate model is needed that allows for toxicity studies at various levels of biological organization, assesses adverse reproductive and developmental effects and establishes appropriate integrative correlations between different levels of effects. Here we describe the reproductive and developmental toxicity of TCDD in feral fish species and summarize how using the zebrafish model to investigate TCDD toxicity has enabled us to characterize the AHR signaling in fish and to better understand how dioxin-like chemicals induce toxicity. We propose that such studies can be used to predict the risks that AHR ligands pose to feral fish populations and provide a platform for integrating risk assessments for both ecologically relevant organisms and humans.

Molecular targets that link dioxin exposure to toxicity phenotypes.

Many toxicology studies have elucidated health effects associated with exposure to various chemicals, but few have identified the molecular targets that cause specific endpoints of toxicity. Our understanding of the toxicity of dioxins, a group of chemicals capable of causing toxicity at environmentally relevant levels of exposure, is no exception. Dioxins are unique compared to most chemicals that we are exposed to in the environment because they activate a high affinity receptor, aryl hydrocarbon receptor (AhR), that was identified more than three decades ago. In recent years, several lines of experimental evidence have provided clues for opening the "black box" that contains the molecular mechanisms of dioxin action. These clues have emerged by toxicologists beginning to identify the molecular targets that link AhR signaling to tissue-specific toxicity phenotypes. Endpoints of dioxin toxicity for which downstream molecular targets have begun to be elucidated are observed in developmental or tissue regeneration processes, and include impaired prostate development and hydronephrosis in mouse fetuses and pups, reduced midbrain blood flow and jaw malformation in zebrafish embryos, and impaired fin regeneration in larval and adult zebrafish. Significant progress in identifying molecular targets for dioxin-induced hepatotoxicity in adult mice also has occurred. Misregulation of AhR downstream pathways, such as conversion of arachidonic acid to prostanoids via cyclooxygenase-2, and altered Wnt/ß-catenin signaling downregulating Sox9, and signaling by receptors for inflammatory cytokines have been implicated in tissue-specific endpoints of dioxin toxicity. These findings may not only begin to clarify the molecular targets of dioxin action but shed light on new molecular events associated with development and disease.

Irreversible effects of retinoic acid pulse on Xenopus jaw morphogenesis: new insight into cranial neural crest specification.

Jaws are formed by cephalic neural crest (CNCCs) and mesodermal cells migrating to the first pharyngeal arch (PA1). A complex signaling network involving different PA1 components then establishes the jaw morphogenetic program. To gather insight on this developmental process, in this study, we analyze the teratogenic effects of brief (1-15 min) pulses of low doses of retinoic acid (RA: 0.25-2 µM) or RA agonists administered to early Xenopus laevis (X.l.) embryos. We show that these brief pulses of RA cause permanent craniofacial defects specifically when treatments are performed during a 6-hr window (developmental stages NF15-NF23) that covers the period of CNCCs maintenance, migration, and specification. Earlier or later treatments have no effect. Similar treatments performed at slightly different developmental stages within this temporal window give rise to different spectra of malformations. The RA-dependent teratogenic effects observed in Xenopus can be partially rescued by folinic acid. We provide evidence suggesting that in Xenopus, as in the mouse, RA causes craniofacial malformations by perturbing signaling to CNCCs. Differently from the mouse, where RA affects CNCCs only at the end of their migration, in Xenopus, RA has an effect on CNCCs during all the period ranging from their exit from the neural tube until their arrival in the PA1. Our findings provide a conceptual framework to understand the origin of individual facial features and the evolution of different craniofacial morphotypes.

2,3,7,8-Tetrachlorodibenzo-p-dioxin upregulates FoxQ1b in zebrafish jaw primordium.

Vertebrate jaw development can be disrupted by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-a potent activator of the aryl hydrocarbon receptor (AHR) transcription factor required for transducing the toxic effects of TCDD. We used zebrafish (Danio rerio) embryos to investigate transcriptional responses to TCDD with the goal of discovering novel, jaw-specific genes affected by TCDD exposure. Our results uncovered a novel target of TCDD-activated Ahr belonging to the evolutionarily conserved family of forkhead box transcription factors. Quantitative real-time polymerase chain reaction analysis demonstrated that FoxQ1b was upregulated by TCDD 7- and 10-fold at 24 and 48 h postfertilization (hpf), respectively. The rate of TCDD-induced FoxQ1b expression was more rapid than that of Cyp1a, a known direct target of TCDD-activated Ahr. TCDD-mediated induction of FoxQ1b was suppressed in the presence of an Ahr antagonist, alpha-naphthoflavone, as well as following knockdown of Ahr2 expression using an Ahr2-specific morpholino antisense oligonucleotide. In situ hybridization analysis of FoxQ1b expression at 48 hpf demonstrated that FoxQ1b is specifically expressed in the jaw primordium where it discretely outlines a developing jaw structure known as Meckel's cartilage--a conserved structure in all jawed vertebrates that develops abnormally in the presence of TCDD. These results identify a novel target of TCDD-activated Ahr and suggest that FoxQ1b may play a role in craniofacial abnormalities induced by developmental exposure to TCDD.

Aryl hydrocarbon receptor-mediated down-regulation of sox9b causes jaw malformation in zebrafish embryos.

Exposure to environmental contaminants can disrupt normal development of the early vertebrate skeleton. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) impairs craniofacial skeletal development across many vertebrate species, and its effects are especially prominent in early life stages of fish. TCDD activates the aryl hydrocarbon receptor, a transcription factor that mediates most if not all TCDD responses. We investigated the transcriptional response in the developing zebrafish jaw after TCDD exposure using DNA microarrays. Zebrafish larvae were exposed to TCDD at 96 h after fertilization, and jaw cartilage tissue was harvested for microarray analysis at 1, 2, 4, and 12 h after exposure. Numerous chondrogenic transcripts were misregulated by TCDD in the jaw. Comparison of transcripts altered by TCDD in jaw with transcripts altered in embryonic heart showed that the transcriptional responses in the jaw and the heart were strikingly different. Sox9b, a critical chondrogenic transcription factor, was the most significantly reduced transcript in the jaw. We hypothesized that the TCDD reduction of sox9b expression plays an integral role in affecting the formation of the embryonic jaw. Morpholino knockdown of sox9b expression demonstrated that partial reduction of sox9b expression alone was sufficient to produce a TCDD-like jaw phenotype. Loss of a single copy of the sox9b gene in sox9b(+/-) heterozygotes increased sensitivity to jaw malformation by TCDD. Finally, embryos injected with sox9b mRNA and then exposed to TCDD blocked TCDD-induced jaw toxicity in approximately 14% of sox9b-injected embryos. These results suggest that reduced sox9b expression in TCDD-exposed zebrafish embryos contributes to jaw malformation.

PCB126 exposure disrupts zebrafish ventricular and branchial but not early neural crest development.

We have used zebrafish and 3,3',4,4',5-pentachlorobiphenyl (PCB126) to investigate the developmental toxicity of polychlorinated biphenyls (PCBs) that exert their effects through the aryl hydrocarbon receptor (AHR). We found that cardiac and neural crest (NC)-derived jaw and branchial cartilages are specifically targeted early in development. The suite of malformations, which ultimately leads to circulatory failure, includes a severely dysmorphic heart with a reduced bulbus arteriosus and abnormal atrioventricular and outflow valve formation. Early NC migration and patterning of the jaw and branchial cartilages was normal. However, the jaw and branchial cartilages failed to grow to normal size. In the heart, the ventricular myocardium showed a reduction in cell number and size. The heart and jaw/branchial phenotype could be rescued by pifithrin-alpha, a blocker of p53. However, the function of pifithrin-alpha in this model may act as a competitive inhibitor of PCB at the AHR and is likely independent of p53. Morpholinos against p53 did not rescue the phenotype, nor were zebrafish with a mutant p53-null allele resistant to PCB126 toxicity. Morpholino knockdown of cardiac troponin T, which blocks the onset of cardiac function, prevented the PCB126-induced cardiac dysmorphogenesis but not the jaw/branchial phenotype. The cardiovascular characteristics appear to be similar to hypoplastic left heart syndrome (HLHS) and introduce the potential of zebrafish as a model to study this environmentally induced cardiovascular malformation. HLHS is a severe congenital cardiovascular malformation that has previously been linked to industrial releases of dioxins and PCBs.

Impairment of lower jaw growth in developing zebrafish exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and reduced hedgehog expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to cause a multitude of detrimental effects to developing zebrafish (Danio rerio). Previously, we demonstrated that jaw growth was impaired by TCDD exposure, but the exact mechanism underlying these malformations remained unknown. In the present study, we investigated the involvement of hedgehog genes and their downstream signaling in TCDD-mediated jaw malformation. We demonstrate that the developing lower jaw expresses sonic hedgehog a (shha), sonic hedgehog b (shhb) and their receptors, patched1 (ptc1) and patched2 (ptc2), as well as the downstream transcription factors, gli1 and gli2a. Loss of Hh signaling in mutants (sonic you) and larvae treated with a Hh inhibitor (cyclopamine), resulted in similar effects as those caused by TCDD. Moreover, TCDD exposure caused downregulation of shha and shhb in a manner dependent on aryl hydrocarbon receptor 2 (ahr2). Although this suggested an involvement of Hh signaling in TCDD-mediated impairment of jaw growth, we did not observe downregulation of ptc1 and ptc2, receptors dependent on Hh signaling. Furthermore, while the overall occurrence of apoptosis in the developing jaw was minimal, it was significantly increased in larvae treated with cyclopamine. In contrast, both TCDD and cyclopamine markedly reduced immunoreactivity against phosphorylated histone 3, a cell proliferation marker in the developing jaw. Taken together, our data suggest that Ahr2-mediated downregulation of Hh signaling, leading to a failure of cell proliferation, contributes to TCDD induced inhibition of lower jaw growth. TCDD may impair jaw growth through other pathway(s) in addition to Hh signaling.

Retinoic acid isomers produce malformations in postembryonic development of the Japanese flounder, Paralichthys olivaceus.

We previously reported that characteristic deformities were induced by retinoic acid (RA) treatment of the Japanese flounder, Paralichthys olivaceus, at 6-9 days post-hatching (dph). To evaluate the toxic potency of nuclear retinoid receptors in induction of deformities by RA, we here investigated the effects of retinoic acid isomers on postembryonic development of this species. Larvae were exposed to either 25 nM of all-trans RA (atRA), 9-cis RA (9cRA) or 13-cis RA (13cRA) at 6-9 dph. All RA isomers induced deformities in the lower jaw, caudal fin and vertebrae. In the lower jaw, growth retardation of the dentary was evident. In the vertebrae, the major abnormalities were hypertrophy of the centrum, central fusion, and an increase in the number of abdominal vertebrae. Caudal fin deformities included deformity of caudal bone complex and absence of the entire caudal fin. The absence of the hypural primordium at 12 dph was the first sign of abnormality in caudal fin development, and resulted in complete blocking of the caudal fin development. Among the RA isomers, atRA induced the most severe deformity in all skeletons examined. Retinoic acid receptor (RAR) expression was activated by atRA and 9cRA, and pitx2 expression was inhibited in the lower jaw by atRA and 9cRA. Vitamin D receptor (VDR) expression was specifically inhibited by atRA treatment, suggesting that RA inhibits the lower jaw growth by suppressing the expression of these genes. These results suggest that RA exerted toxic effects on the skeletal systems, mainly through the RAR pathway.

Cyclopia-astomia-agnathia-holoprosencephaly association: a case report.

A female infant is described with cyclopia-astomia-agnathia-holoprosencephaly association. The authors discuss whether the use of salicylates in early pregnancy is implicated.

2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: altered regional blood flow and impaired lower jaw development.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on regional red blood cell (RBC) perfusion rate, as an index of blood flow, and lower jaw development were investigated quantitatively in zebrafish embryos (Danio rerio) during early development. As revealed by observation of live embryos and alcian-blue staining, TCDD retarded lower jaw development in a concentration-dependent manner with only a minor inhibitory effect on total body length. Both inhibitory effects were significant as early as 60 h postfertilization (hpf), at which time the area of goosecoid (gsc) mRNA expression was clearly reduced in the lower jaw. To examine effects of TCDD on RBC perfusion rate, time-lapse recording was performed using a digital video camera attached to a light microscope. TCDD did not show marked effects on RBC perfusion rate until 72 hpf, when vessel-specific effects emerged. TCDD severely inhibited RBC perfusion rate in intersegmental arteries of the trunk, but only modestly and slightly inhibited RBC perfusion rate in certain vessels of the head such as the central arteries and optic vein. Conversely, at both 72 and 84 hpf, TCDD significantly increased RBC perfusion rate in the hypobranchial artery branching to the lower jaw primordia, and then reduced it at 96 hpf. RBC perfusion rate in all vessels examined in TCDD-exposed embryos was inhibited at 96 hpf. The zebrafish aryl hydrocarbon receptor 2 (zfAhR2) mRNA was strongly expressed in the lower jaw primordia at 48 hpf, and expression of this transcript was augmented by TCDD treatment. Thus, TCDD exposure of the zebrafish embryo has a disruptive effect on local circulation and lower jaw cartilage growth. Initially, TCDD may act directly on the lower jaw primordia to impair lower jaw development. Reductions in hypobranchial RBC perfusion rate occurred well after the initial retardation in lower jaw development had become apparent, and may contribute further to the effect.

Exencephaly and axial skeletal malformations induced by maternal administration of sodium valproate in the MF1 mouse.

Clinical and epidemiological studies indicate that maternal use of valproic acid (VPA) during pregnancy causes an increased risk for spina bifida in the fetus. A proportion of infants exposed to VPA in utero exhibit a characteristic pattern of facial malformations. Despite the developmental interdependence of the neural plate and paraxial mesoderm during normal morphogenesis, the possible involvement of the axial skeleton in VPA-induced NTD has not been clearly documented. So the objective of this investigation was to determine the nature and extent of involvement of the axial skeleton in VPA-induced exencephaly in the mouse. A single dose of 600 mg/kg of sodium valproate was administered (IP) to MF1 mouse on day 8 of gestation. This treatment resulted in significant increase in resorption, reduction in mean fetal weight, and exencephaly (25%) of live fetuses. Several craniofacial malformations and subcutaneous haematomas were associated with exencephaly. Alizarin red-stained skeletal preparations revealed maxillary-, mandibular hypoplasia, absence of skull vault, hypoplasia and/or agenesis of basicranial bones, and obtuse angulation of the craniovertebral junction. Hemivertebrae, longitudinal fusion of the vertebral arches and bodies, accessory ribs (cervical and lumbar), fusion of thoracic ribs, and several patterns of sternal variations were observed. Nonexencephalic VPA-treated embryos exhibited mandibular, maxillary hypoplasia, arched and cleft palates, cleft lip, kinky tail, and vertebral and sternal anomalies. Treated embryos at early stages of development revealed delay in elevation and fusion of neural folds, distended IVth ventricle, kinky spinal cord, incomplete separation of somites and growth retardation. When viewed in light of the published work on VPA action on embryonic systems, these observations suggest that abnormalities associated with VPA-induced exencephaly may be due to either a direct action of VPA on the precursors of these organs or secondary to its action on neural tube. A significantly high incidence of NTD and their consistent association with defective development of the axial skeleton suggest that this is an excellent experimental model for investigating the pathogenetic mechanism(s) of VPA induced NTD.

Formhydroxamic acid-induced malformations of the temporo-mandibular joint.

Formhydroxamic acid was given to pregnant rats in order to produce anomalies of the temporo-mandibular joint. Especially successful in our hands was the dosage of 500-550 mg./kg. given intraperitoneally on day 13. The resulting malformations were very much akin to those seen in human patients.

[On the embryo-fetal alcohol syndrome (author's transl)]. / Uber das embryo-fetale Alkoholsyndrom

The symptomatology in 24 patients with the embryo-fetal alcohol syndrome in this study corresponded essentially to the clinical picture described by Lemoine et al., and Jones et al., although we were not able to confirm the maxillary hypoplasia and microphthalmia mentioned in the latter. In addition, we observed in 2 girls a virilization of the genitalia which corresponded to female pseudohermaphroditism. Auxologically a nearly regular pattern of the parameters measured was found. The intra-uterine hypotrophy continues after birth. In particular, the head circumference, reflecting the brain growth, remains low. The cerebral damage leads to oligophrenia with a typical pattern of psychic and motor behaviour. Moderate cases tend to improve. Concerning the pathogenesis, the clinical observations in connexion with recent animal experiments permit the following conclusions. Neither the malnutrition nor the liver damage of the mothers are necessary presuppositions. Ethanol itself appears to play the main teratogenetical rôle. Acetaldehyde, which is the primary metabolite of ethanol, is cytotoxic too. However, the liver of the embryo and the fetus, in consequence of its deficient alcohol dehydrogenase content, is not able to metabolize ethanol.