Moderate alcohol consumption during pregnancy increases potency of two different drugs (the antifungal fluconazole and the antiepileptic valproate) in inducing craniofacial defects: prediction by the in vitro rat whole embryo culture.

The prenatal exposure to ethanol (Eth), fluconazole (FLUCO) and sodium valproate (VPA) is related to effects on development, producing characteristic syndromic pictures. Among embryotoxic effects described for the three molecules, the alteration on craniofacial morphogenesis is a common feature in humans and animal models, including rodent embryos developed in vitro. The aim of the present work is to evaluate the developmental effects of low Eth serum concentration (17 mM, corresponding to the legal limit to drive in UK, USA, Canada, and many other countries) in mixture with increasing realistic concentrations of the antifungal drug FLUCO (62.5-500 µM) or with increasing realistic concentrations of the antiepileptic drug VPA (31.25-250 µM). Groups exposed to Eth alone (17-127.5 mM), FLUCO alone (62.5-500 µM) or VPA alone (31.25-750 µM) were also included. The chosen alternative animal model was the post-implantation rat whole embryo culture (WEC). E9.5 embryos were exposed in vitro to the test molecules during the whole test period (48 h, corresponding to the developmental stages characteristics of any vertebrate, for human embryos post-fertilization days 23-31). Data were statistically analyzed and processed for modelling applying the benchmark dose (BMD) and relative potency factor (RPF) approaches. Concentration-related effects on facial outcomes were observed in all experimental groups, with a significant enhancement in the groups co-exposed with Eth in comparison to the single exposures. Data obtained by the present work suggest an additional alert for the assumption of even low levels of alcohol in pregnant women during FLUCO or VPA therapy.

Predictive assays for craniofacial malformations: evaluation in Xenopus laevis embryos exposed to triadimefon.

Craniofacial defects are one of the most frequent abnormalities at birth, but their experimental evaluation in animal models requires complex procedures. The aim of the present work is the comparison of different methodologies to identify dose- and stage-related craniofacial malformations in Xenopus laevis assay (R-FETAX, where the full cartilage evaluation, including flat mount technique, is the gold standard for skeletal defect detection). Different methods (external morphological evaluation of fresh samples, deglutition test, whole mount cartilage evaluation and Meckel-palatoquadrate angle measurements) were applied. Triadimefon (FON) was selected as the causative molecule as it is known to induce craniofacial defects in different animal models, including the amphibian X. laevis.FON exposure (0-31.25 µM) was scheduled to cover the whole 6-day test (from gastrula to free swimming tadpole stage) or each crucial developmental phases: gastrula, neurula, early morphogenesis, late morphogenesis, tadpole. Dose-dependent effects (fusions among craniofacial cartilages) were evident for groups exposed during the morphogenetic periods (neurula, early morphogenesis, late morphogenesis); gastrula was insensitive to the tested concentrations, tadpole group showed malformations only at 31.25 µM. The overall NOAEL was set at 3.9 µM. Results were evaluated applying benchmark dose (BMD) approach. The comparison of relative potencies from different methods showed deglutition as the only assay comparable with the gold standard (cartilage full evaluation).In conclusion, we suggest deglutition test as a reliable method for a rapid screening of craniofacial abnormalities in the alternative model X. laevis. This is a rapid, inexpensive and vital test allowing to preserve samples for the application of further morphological or molecular investigations.

Safety of Artemisinin Derivatives in the First Trimester of Pregnancy: A Controversial Story.

Artemisinin combination therapy (ACT) is recommended by the World Health Organization (WHO) as first line treatment for uncomplicated malaria both in adults and children. During pregnancy, ACT is considered safe only in the second and third trimester, since animal studies have demonstrated that artemisinin derivatives can cause foetal death and congenital malformation within a narrow time window in early embryogenesis. During this period, artemisinin derivatives induce defective embryonic erythropoiesis and vasculogenesis/angiogenesis in experimental models. However, clinical data on the safety profile of ACT in pregnant women have not shown an increased risk of miscarriage, stillbirth, or congenital malformation, nor low birth weight, associated with exposure to artemisinins in the first trimester. Although further studies are needed, the evidence collected up to now is prompting the WHO towards a change in the guidelines for the treatment of uncomplicated malaria, allowing the use of ACT also in the first trimester of pregnancy.

Teratogenic potential of nanoencapsulated vitamin A evaluated on an alternative model organism, the tunicate Ciona intestinalis.

Nano-encapsulation is a technology used to pack substances in order to enhance their stability and bioavailability, but this packing may interact with living systems, causing unexpected toxicity. Vitamin A (vit A) is a substance that has received attention, because in developed countries, the increasing availability of supplements is leading to its excessive intake. This study aims to compare teratogenic effects caused by exposure to the traditional formulation of vit A versus nano-encapsulated vit A. We used ascidian embryos as an alternative model. Ascidians are marine organisms closely related to vertebrates that share with them a body plan and developmental programme, including the morphogenetic role of retinoic acid (RA). Our data showed that the adverse effects of exposure to the same concentration of the two formulations were different, suggesting that the nano-encapsulation increased the bioavailability of the molecule, which could be better absorbed and metabolised to RA, the effective teratogenic substance.

Effects of mixtures of azole fungicides in postimplantation rat whole-embryo cultures.

The effect of mixtures of azole fungicides on development of postimplantation rat whole-embryos cultured in vitro has been tested. On the basis of bench mark dose (BMD) modeling of the in vitro effect in rat embryo, the potency of 7 azoles was determined and compared. Then, relative potency factors have been derived based on either the NOAEL or on the BMD curve. Alternatively, each compound was used as index compound (IC), and IC-equivalent concentrations have been calculated for each mixture. Expected effects of such IC-equivalent concentrations of the mixture were derived from the appropriate BMD curve. Test mixture includes the agrochemicals triadimefon and imazalil (MIX2) or triadimefon, imazalil, and the clinically used fluconazole (MIX3) at their previously determined no-effect concentration, corresponding to approximately a benchmark response of 5-10 %. Subsequently, we tested the effect of a mixture of the agrochemicals triadimefon, imazalil, triadimenol, cyproconazole, tebuconazole, and flusilazole (MIX6) at concentration levels derived from their established human acceptable daily intake. MIX6 was also added with fluconazole at concentration levels indicated as the minimum therapeutically effective plasmatic concentration (MIX7A) or ten times this level (MIX7B). Generally, the experimental response was higher than the estimated one, by a factor of 2-6. Our data suggest that it is in principle correct to assume that azoles act as teratogens via a common mode of action and therefore should be grouped together for risk assessment. The synergistic effect needs to be confirmed with more combinations of concentrations/compounds in vitro and with specific in vivo experiments.

Methionine pretreatment enhances the effects of valproate on axial development in a CD1 mouse model.

BACKGROUND: The present study was performed to evaluate the effect of methionine (Met) pretreatment on valproate (VPA) axial defects, induced in CD1 mice by a single intraperitoneal (i.p.) injection of 400 mg/kg VPA on E8 dams. This VPA dosage regimen has been in the past related to a specific pathogenic pathway cascade: (1) VPA in utero exposure, (2) H4 histone hyperacetylation (hAC) at the level of somites, (3) expression of pro-apoptotic factors in somite tissues, (4) apoptosis of somite cells, and (5) axial defects in embryos (abnormal or fused somites) and fetuses (fusions, duplications, respecifications of vertebrae, and/or ribs). METHODS: On the basis of literature suggestion, E8 CD1 mice were i.p. injected with 70 mg/kg Met 30 min before the i.p. injection with 400 mg/kg VPA. Some females were sacrificed, 1 or 3 hr after the VPA injection, embryos explanted, and used to evaluate the H4 histone hAC. The remaining females were sacrificed at term (E18) and fetuses processed for external and skeletal examination. RESULTS: The pretreatment with Met worsened the axial skeletal malformative picture in fetuses (we observed a larger number of affected segment per fetus in respect to the groups treated with VPA alone). In embryos, Met pretreatment increased the H4 hAC index and shifted the timing of the H4 hAC peak. CONCLUSIONS: Our data suggest that Met pretreatment enhances the effects of VPA in deregulating the epigenetic control of gene expression in somites, and by consequence, induces more extended dysmorphogenic effects along the axial axis.

Immunostaining patterns reveal potential morphogenetic role of Toll-like receptors 4 and 7 in the development of mouse respiratory system, liver and pancreas.

Toll-like receptors (TLRs) are the mammalian ortholog of Drosophila melanogaster protein Toll, originally identified for its involvement in embryonic development. In mammals, TLRs are mainly known for their ability to recognize pathogen- or damage-associated molecular patterns and, consequently, to initiate the immune response. However, it is becoming clear that TLRs can play a role also in mammal embryo development. We have previously described TLR4 and TLR7 expression in developing mouse peripheral nervous system and gastrointestinal tract. In the present study, we extended the investigation of TLR4 and TLR7 to the respiratory system and to the two main accessory organs of the digestive system, the liver and pancreas. TLR4 and TLR7 immunostaining was performed on mouse conceptuses collected at different stages, from E12 to E18. TLR4 and TLR7 immunoreactivity was evident in the embryo pancreas and liver at E12, while, in the respiratory apparatus, appeared at E14 and E17, respectively. Although further studies are required to elucidate the specific role of these TLRs in embryo development, the differential spatiotemporal TLR4 and TLR7 appearance may suggest that TLR expression in developing embryos is highly regulated for a possible their direct involvement in the formation of the organs and in the acquisition of immune-related features in preparation for the birth.

The problem of maternal toxicity in developmental toxicity studies.

Guidelines for developmental toxicity studies require that the highest dose(s) should induce some signs of maternal toxicity. However, the interpretation of the results is often difficult when developmentally toxic effects are recorded only at maternotoxic doses, as it is impossible to ascertain whether the developmental effects are maternally mediated or not. In order to avoid this source of misinterpretation we suggest to use in developmental toxicity tests for environmental chemicals the maximum dose unable to produce maternal toxic effects extrapolated by previous short term toxicity studies.

Modified Xenopus laevis approach (R-FETAX) as an alternative test for the evaluation of foetal valproate spectrum disorder.

In compliance to animal welfare 3Rs principle there is a great demand for refined tests alternative to classical mammal teratogenicity tests. We propose a refined alternative amphibian method (R-FETAX) to evaluate chemical induced embryotoxicity. The human foetal valproate spectrum disorder (FVSD) characteristics are morphological defects (including cranio-facial, neural tube defects) and behavioural alterations due to valproate (VPA) exposure in pregnancy. Vertebrate assays to evaluate FVSD include classical and alternative mammal (implying adult sacrifice), and non-mammal developmental models (zebrafish, amphibians, chick). Among these latter only zebrafish assays report in the same test both morphological and behavioural examinations. Compared to zebrafish, the amphibian Xenopus laevis excels having a more comparable organ development and morphology to mammalian systems. We used X. laevis embryos exposed during developmental specific windows to VPA therapeutic concentrations. Different VPA effects were observed depending on the exposure window: concentration-related embryo-lethal and teratogenic effects (neural tube, facial, tail defects) were observed in groups exposed at the organogenetic phylotypic stages. Neurobehavioral deficits were described using a functional swimming test at the highest VPA concentration exposure during the phylotypic stages and at any concentration during neurocognitive competent stages. Malformations were compared to those obtained in a mammalian assay (the rat post-implantation whole embryo culture method, WEC), that we used in the past to evaluate VPA teratogenicity. R-FETAX and WEC data were modelled and their relative sensitivity was calculated. We suggest the amphibian R-FETAX as a refined windowed alternative test for the evaluation of chemicals inducing both morphological and behavioural anomalies, including VPA.

Retrospective cumulative dietary risk assessment of craniofacial alterations by residues of pesticides.

EFSA established cumulative assessment groups and conducted retrospective cumulative risk assessments for two types of craniofacial alterations (alterations due to abnormal skeletal development, head soft tissue alterations and brain neural tube defects) for 14 European populations of women in childbearing age. Cumulative acute exposure calculations were performed by probabilistic modelling using monitoring data collected by Member States in 2017, 2018 and 2019. A rigorous uncertainty analysis was performed using expert knowledge elicitation. Considering all sources of uncertainty, their dependencies and differences between populations, it was concluded with varying degrees of certainty that the MOET resulting from cumulative exposure is above 100 for the two types of craniofacial alterations. The threshold for regulatory consideration established by risk managers is therefore not exceeded. Considering the severity of the effects under consideration, it was also assessed whether the MOET is above 500. This was the case with varying levels of certainty for the head soft tissue alterations and brain neural tube defects. However, for the alterations due to abnormal skeletal development, it was found about as likely as not that the MOET is above 500 in most populations. For two populations, it was even found more likely that the MOET is below 500. These results were discussed in the light of the conservatism of the methodological approach.

Early genetic control of craniofacial development is affected by the in vitro exposure of rat embryos to the fungicide triadimefon.

BACKGROUND: Previous published experiments reported that in vitro exposure of postimplantation rat embryos to the triazole fungicide triadimefon (FON) resulted in specific abnormalities at the branchial apparatus and that the sensitive period is restricted to the first 24 hr of culture and is associated with the abnormal expression of TGF family genes (some of a large panel of genes regulated by retinoic acid (RA) and involved in branchial arch morphogenesis). The aim of this study is the determination of the sensitive window to FON-induced abnormalities during in vitro development and the evaluation of the expression of some genes controlled by RA and involved in early branchial arch morphogenesis (Gsc, Msx1, Msx2, Dlx1, Dlx2, Shh, Patched (the main Shh receptor)). METHODS: Rat embryos were exposed in vitro to the FON under condition known to be able to induce 100% of abnormal embryos (250 µM) at different stages and examined after 48 hr of culture. The sensitive window for FON-induced abnormalities was during the hours E9 h8.00 PM-E10 h8.00 AM. To evaluate the expression of selected genes, embryos exposed during the sensitive stages were processed to perform quantitative PCR after 18 and 24 hr of culture. RESULTS: FON was able to affect the expression of some genes in a stage-specific manner: earlier embryos were characterized by the downregulation of Msx2 and Gsc, later embryos showed the downregulation of Gsc, Shh, and Patched. The obtained data suggest that FON-induced abnormalities are mediated, at least in part, through the imbalance of the expression of RA-related signals.

Expression analysis of some genes regulated by retinoic acid in controls and triadimefon-exposed embryos: is the amphibian Xenopus laevis a suitable model for gene-based comparative teratology?

BACKGROUND: The use of nonmammal models in teratological studies is a matter of debate and seems to be justified if the embryotoxic mechanism involves conserved processes. Published data on mammals and Xenopus laevis suggest that azoles are teratogenic by altering the endogenous concentration of retinoic acid (RA). The expression of some genes (Shh, Ptch-1, Gsc, and Msx2) controlled by retinoic acid is downregulated in rat embryos exposed at the phylotypic stage to the triazole triadimefon (FON). In order to propose X. laevis as a model for gene-based comparative teratology, this work evaluates the expression of Shh, Ptch-1, Gsc, and Msx2 in FON-exposed X. laevis embryos. METHODS: Embryos, exposed to a high concentration level (500 µM) of FON from stage 13 till 17, were examined at stages 17, 27, and 47. Stage 17 and 27 embryos were processed to perform quantitative RT-PCR. RESULTS: The developmental rate was never affected by FON at any considered stage. FON-exposed stage 47 larvae showed the typical craniofacial malformations. A significant downregulation of Gsc was observed in FON-exposed stage 17 embryos. Shh, Ptch-1, Msx2 showed a high fluctuation of expression both in control and in FON-exposed samples both at stages 17 and 27. CONCLUSION: The downregulation of Gsc mimics the effects of FON on rat embryos, showing for this gene a common effect of FON in the two vertebrate classes. The high fluctuation observed in the gene expression of the other genes, however, suggests that X. laevis at this stage has limited utility for gene-based comparative teratology.

An adverse outcome pathway on the disruption of retinoic acid metabolism leading to developmental craniofacial defects.

Adverse outcome pathway (AOP) is a conceptual framework that links a molecular initiating event (MIE) via intermediate key events (KEs) with adverse effects (adverse outcomes, AO) relevant for risk assessment, through defined KE relationships (KERs). The aim of the present work is to describe a linear AOP, supported by experimental data, for skeletal craniofacial defects as the AO. This AO was selected in view of its relative high incidence in humans and the suspected relation to chemical exposure. We focused on inhibition of CYP26, a retinoic acid (RA) metabolizing enzyme, as MIE, based on robust previously published data. Conazoles were selected as representative stressors. Intermediate KEs are RA disbalance, aberrant HOX gene expression, disrupted specification, migration, and differentiation of neural crest cells, and branchial arch dysmorphology. We described the biological basis of the postulated events and conducted weight of evidence (WoE) assessments. The biological plausibility and the overall empirical evidence were assessed as high and moderate, respectively, the latter taking into consideration the moderate evidence for concordance of dose-response and temporal relationships. Finally, the essentiality assessment of the KEs, considered as high, supported the robustness of the presented AOP. This AOP, which appears of relevance to humans, thus contributes to mechanistic underpinning of selected test methods, thereby supporting their application in integrated new approach test methodologies and strategies and application in a regulatory context.

Effect of nano-encapsulation of ß-carotene on Xenopus laevis embryos development (FETAX).

Vitamin A plays a vital role during embryo development as most precursor of embryonic retinoic acid, a key morphogen during embryogenesis. Carotenoids, including ß-carotene, are important vegetal source of Vitamin A and in contrast to teratogenic potential of animal-derived retinoids, ß-carotene is usually considered freed from embryotoxic effects and supplements in pregnancy with ß-carotene are suggested. The aim of the present work is to evaluate the effect of bulk and nano-encapsulated ß-carotene on embryo development, by using the animal model Frog Embryo Teratogenesis Assay: Xenopus- FETAX. Xenopus laevis embryos were exposed from late gastrula till pharyngula (the phylotypic stage for vertebrates) to the concentrations of BULK ß-carotene 150-3000 ng/mL and NANO ß-carotene 0.75-30 ng/mL. At pharyngula stage, some embryos were processed for whole mount neural crest cell immunostaining, while others embryos were allowed to develop till tadpole for morphological and histological evaluation of neural crest cells-derived structures. In this model, the nano-encapsulated ß-carotene induced specific malformations at craniofacial and eye level, while the bulk formulation only induced developmental delays. Finally, the applied alternative animal model resulted a rapid and sensitive screening method able to re-evaluate the teratogenic profile of nano-encapsulated micronutrients.

Expression of Toll-like receptors 4 and 7 in murine peripheral nervous system development.

BACKGROUND: Toll-Like Receptors (TLRs) play a critical role in the innate and adaptive immune system. They are the mammalian orthologs of Drosophila melanogaster protein Toll, which has been proved to have an early morphogenetic role in invertebrate embryogenesis that in the adult switches to an immune function. AIM: The aim of this study was to evaluate the expression of TLR4 and TLR7 during dorsal root ganglia (DRG), paravertebral ganglia (PVG), and enteric nervous system (ENS) murine development. METHODS: Mouse embryos from different stages (i.e. E12 to E18) were processed for immunolocalization analysis on formalin-fixed paraffin-embedded sections, and isolated intestine were processed for whole-mount preparations. RESULTS: We observed a differentially regulated expression of TLR4 and TLR7 during embryogenesis and an overall increased expression of both receptors during development. While TLR4 was detectable in neurons of DRG and PVG starting from E14 and only from E18 in the ENS, TLR7 was already expressed in scattered neurons of all the investigated regions at E12. CONCLUSIONS: TLR4 and TRL7 expression temporal patterns suggest a morphogenetic role for these receptors in the development of neural crest derivatives in mammals.

Development of an adverse outcome pathway for cranio-facial malformations: A contribution from in silico simulations and in vitro data.

Mixtures of substances sharing the same molecular initiating event (MIE) are supposed to induce additive effects. The proposed MIE for azole fungicides is CYP26 inhibition with retinoic acid (RA) local increase, triggering key events leading to craniofacial defects. Valproic acid (VPA) is supposed to imbalance RA-regulated gene expression trough histone deacetylases (HDACs) inhibition. The aim was to evaluate effects of molecules sharing the same MIE (azoles) and of such having (hypothetically) different MIEs but which are eventually involved in the same adverse outcome pathway (AOP). An in silico approach (molecular docking) investigated the suggested MIEs. Teratogenicity was evaluated in vitro (WEC). Abnormalities were modelled by PROAST software. The common target was the branchial apparatus. In silico results confirmed azole-related CYP26 inhibition and a weak general VPA inhibition on the tested HDACs. Unexpectedly, VPA showed also a weak, but not marginal, capability to enter the CYP 26A1 and CYP 26C1 catalytic sites, suggesting a possible role of VPA in decreasing RA catabolism, acting as an additional MIE. Our findings suggest a new more complex picture. Consequently two different AOPs, leading to the same AO, can be described. VPA MIEs (HDAC and CYP26 inhibition) impinge on the two converging AOPs.

Relative potency ranking of azoles altering craniofacial morphogenesis in rats: An in vitro data modelling approach.

Facial malformations represent one of the most frequent abnormality in humans. The adverse outcome pathway involved in facial defects seems to be related to retinoic acid (RA) pathway imbalance. Environmental agents inducing craniofacial malformations in experimental models include pesticides (especially azole fungicides). By using the in vitro alternative method postimplantation rat whole embryo culture (WEC), we evaluated the intrinsic embryotoxic activity of some azole antifungals (cyproconazole, CYPRO; triadimefon, FON; flusilazole, FLUSI; and prochloraz, PCZ), in comparison to RA. All the tested molecules induced in a dose-related manner specific defects of the craniofacial structures (fused branchial arches), similar to those induced by RA. Collected data were modelled using PROAST 65.5 software to characterise the relative potency factors (RPFs) versus RA. In comparison to RA, all the evaluated azoles were less potent, showing among them a similar potency. Our data suggest a possible azole-related RA signalling perturbation to be further investigated. Moreover, the present results indicate the approach used in this work to be an interesting tool applicable to the hazard evaluation of novel compounds or the assessment of combined exposure to azoles or other dismorphogens.

Antenatal Microbial Colonization of Mammalian Gut.

The widely accepted dogma of intrauterine sterility and initial colonization of the newborn during birth has been blurred by recent observations of microbial presence in meconium, placenta, and amniotic fluid. Given the importance of a maternal-derived in utero infant seeding, it is crucial to exclude potential environmental or procedural contaminations and to assess fetal colonization before parturition. To this end, we analyzed sterilely collected intestinal tissues, placenta, and amniotic fluid from rodent fetuses and tissues from autoptic human fetuses. Total bacterial DNA was extracted from collected samples and analyzed by Next Generation Sequencing (NGS) techniques using hypervariable 16S ribosomal RNA (rRNA) regions (V3-V4). Colonizing microbes were visualized in situ, using labeled probes targeting 16S ribosomal DNA by fluorescent in situ hybridization. The NGS analysis showed the presence of pioneer microbes in both rat and human intestines as well as in rodent placentas and amniotic fluids. Microbial communities showed fetus- and dam-dependent clustering, confirming the high interindividual variability of commensal microbiota even in the antenatal period. Fluorescent in situ hybridization analysis confirmed the microbes' presence in the lumen of the developing gut. These findings suggest a possible antenatal colonization of the developing mammalian gut.

The Ascidian Embryo Teratogenicity assay in Ciona intestinalis as a new teratological screening to test the mixture effect of the co-exposure to ethanol and fluconazole.

The aim of this work was to evaluate the Ascidian Embryo Teratogenicity assay (AET) as new alternative invertebrate model to test the developmental effects of the co-exposure to ethanol and fluconazole. Ciona intestinalis embryos were exposed to the azolic fungicide fluconazole, (FLUCO, 7.8-250µM), to ethanol (Eth, 0.01-0.5%) and to their mixture (0.01% Eth+FLUCO 7.8-250µM) from neurula to larval stage. At the end of the exposure period, larvae were morphologically evaluated and benchmark analysis performed by using the PROAST modelling software. Both compounds were teratogenic in a concentration-related manner, particularly affecting the pigmented organs. The co-exposure to Eth enhanced the effects of FLUCO, the additive hypothesis was not rejected by the modelling. The results demonstrated that AET could be considered a good vertebrate-free alternative model for toxicological investigation in embryos.

Gene expression in Xenopus laevis embryos after Triadimefon exposure.

The triazole derivative Triadimefon (FON) is a systemic fungicide used to control powdery mildews, rusts, and other fungal pests. Some data have already been published about the teratogenic activity of this compound: craniofacial malformations were found in mouse, rat, and Xenopus laevis embryos exposed to FON. These alterations were correlated to defective branchial arch development possibly caused by abnormal neural crest cell (NCC) migration into the branchial mesenchyme. As the migration of NCCs is controlled by the HOX code and by an anteroposterior retinoic acid (RA) gradient, we analyzed the expression of CYP26, a key enzyme in RA metabolism, following FON exposure. The increased expression of this gene and the ability of citral (a RA inhibitor) to reduce the teratogenic effects of the fungicide support the notion that endogenous RA is involved in the mechanism of action of FON. Moreover, by in situ hybridization, we studied the effects of FON exposure at gastrula stage on the expression of some genes involved in craniofacial development, hindbrain patterning, and NCC migration. We observed abnormal localization of xCRABP, Hoxa2 and Xbap signal expression at the level of migrating NCC domains, whereas in the hindbrain, we did not find any alteration in Krox20 and Hoxa2 expression.