Teratogenic and neuro-behavioural toxic effects of bisphenol A (BPA) and B (BPB) on Xenopus laevis development.

Bisphenol A (BPA) is a plastic additive with endocrine disruptive activity, classified in 2017 by EU ECHA as substance of very high concern. A correlation between environmental exposure to BPA and congenital defects has been described in humans and in experimental species, including the amphibian Xenopus laevis. Among BPA analogues, bisphenol B (BPB) is used as alternative in different not-EU countries, including US, but seems to share with BPA its endocrine disruptor properties. Aim of the present work is the evaluation of the effects of BPB versus BPA exposure in a X. laevis developmental model. A windowed exposure (R-FETAX method) was applied covering the developmental phylotypic period (teratogenicity window), or the late tailbud stages (neuro-behavioural toxicity window, corresponding to the spontaneous swimming acquisition period). Samples were monitored for lethal effects during the full test period. External morphology evaluation and deglutition functional test were applied in any group. Abnormal tadpoles were also processed for cartilage staining. In groups exposed during neuro-behavioural toxicity window the swimming test was also applied. Lethality and malformations were obtained only in samples exposed during the teratogenicity window; these data were modelled using PROAST software and BPB relative potency resulted about 3 times higher than BPA. The day-by-day evaluation revealed that lethality was correlated to embryonic abnormal development of gills and apoptosis in gill primordia. Teratogenicity was never detected in groups exposed during the neuro-behavioural toxicity window, where some significant neuro-behavioural deficits were detected in tadpoles exposed to the highest tested concentrations of BPA and BPB.

Biochemical and developmental effects of thyroid and anti-thyroid drugs on different early life stages of Xenopus laevis.

The effects of two drugs containing the synthetic thyroid hormone levothyroxine (LEV) and an anti-thyroid drug containing propylthiouracil (PTU) on the three early life stages of Xenopus laevis were evaluated with the Frog Embryo Teratogenesis Assay-Xenopus, Tadpole Toxicity Test, and Amphibian Metamorphosis Assay using biochemical and morphological markers. Tested drugs caused more effective growth retardation in stage 8 embryos than stage 46 tadpoles. Significant inhibition of biomarker enzymes has been identified in stage 46 tadpoles for both drugs. AMA test results showed that LEV-I caused progression in the developmental stage and an increase in thyroxine level in 7 days exposure and growth retardation in 21 days exposure in stage 51 tadpoles. On the other hand, increases in lactate dehydrogenase activity for both drugs in the AMA test may be due to impacted energy metabolism during sub-chronic exposure. These results also show that the sensitivity and responses of Xenopus laevis at different early developmental stages may be different when exposed to drugs.

Role of TrkA signaling during tadpole tail regeneration and early embryonic development in Xenopus laevis.

Neurotrophic signaling regulates neural cell behaviors in development and physiology, although its role in regeneration has not been fully investigated. Here, we examined the role of neurotrophic signaling in Xenopus laevis tadpole tail regeneration. After the tadpole tails were amputated, the expression of neurotrophin ligand family genes, especially ngf and bdnf, was up-regulated as regeneration proceeded. Moreover, notochordal expression of the NGF receptor gene TrkA, but not that of other neurotrophin receptor genes TrkB and TrkC, became prominent in the regeneration bud, a structure arising from the tail stump after tail amputation. The regenerated tail length was significantly shortened by the pan-Trk inhibitor K252a or the TrkA inhibitor GW-441756, but not by the TrkB inhibitor ANA-12, suggesting that TrkA signaling is involved in elongation of regenerating tails. Furthermore, during Xenopus laevis embryonic development, TrkA expression was detected in the dorsal mesoderm at the gastrula stage and in the notochord at the neurula stage, and its knockdown led to gastrulation defects with subsequent shortening of the body axis length. These results suggest that Xenopus laevis TrkA signaling, which can act in the mesoderm/notochord, plays a key role in body axis elongation during embryogenesis as well as tail elongation during tadpole regeneration.

Bisphenol A, Bisphenol AF, di-n-butyl phthalate, and 17ß-estradiol have shared and unique dose-dependent effects on early embryo cleavage divisions and development in Xenopus laevis.

Bisphenol A (BPA), Bisphenol AF (BPAF), and di-n-butyl phthalate (DBP) are widespread compounds used in the production of plastics. We used Xenopus laevis to compare their effects on early embryo cell division and development. Directly after in vitro fertilizations, embryos were exposed to BPA, BPAF, DBP, or 17ß-estradiol (E2) for up to 96 h. BPA (1-50 µM) and BPAF (0.003-25 µM) caused disrupted cleavage divisions, slowed cytokinesis, and cellular dissociation within 1-6 h. Flexures of the spinal cord, shorter body axis/tail, craniofacial malformations, and significant mortality occurred with environmentally relevant doses of BPAF (LC50 = 0.013 µM). DBP (10-200 µM) showed similar effects, but with severe ventral edema. There were both shared and unique effects of all compounds, with BPAF having the greatest potency and toxicity (BPAF > BPA > estradiol > DBP). These findings underscore the pleiotropic effects of widespread toxicants on early development and highlight the need for better toxicological characterization.

Chronic exposures to monomethyl phthalate in Western clawed frogs.

Polymer flexibility and elasticity is enhanced by plasticizers. However, plasticizers are often not covalently bound to plastics and thus can leach from products into the environment. Much research effort has focused on their effects in mammalian species, but data on aquatic species are scarce. In this study, Western clawed frog (Silurana tropicalis) embryos were exposed to 1.3, 12.3, and 128.7mg/L monomethyl phthalate (MMP) until the juvenile stage (11weeks) and to 1.3mg/L MMP until the adult stage (51weeks). MMP decreased survival, hastened metamorphosis, and biased the sex ratio toward males (2M:1F) at the juvenile stage without altering the expression of a subset of thyroid hormone-, sex steroid-, cellular stress- or transcription regulation-related genes in the juvenile frog livers. At the adult stage, exposure to MMP did not have significant adverse health effects, except that females had larger interocular distance and the expression of the heat shock protein 70 was decreased by 60% in the adult liver. In conclusion, this study shows that MMP is unlikely to threaten amphibian populations as only concentrations four orders of magnitude higher than the reported environmental concentrations altered the animal physiology. This is the first complete investigation of the effects of phthalates in a frog species, encompassing the entire life cycle of the organisms.

Organophosphate pesticides induce morphological abnormalities and decrease locomotor activity and heart rate in Danio rerio and Xenopus laevis.

Organophosphate pesticides (OPs), a class of acetylcholinesterase inhibitors, are used widely in agriculture to reduce insect populations. Because of the conservation of acetylcholinesterase between invertebrates and vertebrates, OPs also can adversely affect nontarget species, such as aquatic and terrestrial animals. This study used uniform conditions to analyze the morphological and physiological effects caused by developmental exposure to 3 commonly used OPs-chlorpyrifos, dichlorvos, and diazinon-on 2 aquatic vertebrate species, Danio rerio (zebrafish) and Xenopus laevis. Survival, locomotor activity, heart rate, and gross anatomical abnormalities, including kyphosis and edema, were observed over a 5-d period in response to OP concentrations ranging from 0 µM to 1000 µM. Both zebrafish and Xenopus showed decreased survival for all 3 OPs at higher concentrations. However, Xenopus showed higher mortality than zebrafish at lower chlorpyrifos and dichlorvos concentrations. Both models showed a dose-dependent decrease in heart rate and free-swimming larval activity in response to chlorpyrifos and dichlorvos. In addition, kyphosis and decreased spine length were prominent in Xenopus in response to 10 µM of chlorpyrifos and 0.1 µM dichlorvos. Although diazinon induced no effects on skeletal and cardiac motor activity in either species, it did induce cardiac edemas in zebrafish. Differences in the biological actions of OPs and their differential effects in these 2 vertebrate models demonstrate the importance of using common protocols and multiple models to evaluate the ecotoxicology of OPs.

Essential roles of LEM-domain protein MAN1 during organogenesis in Xenopus laevis and overlapping functions of emerin.

Mutations in nuclear envelope proteins are linked to an increasing number of human diseases, called envelopathies. Mutations in the inner nuclear membrane protein emerin lead to X-linked Emery-Dreifuss muscular dystrophy, characterized by muscle weakness or wasting. Conversely, mutations in nuclear envelope protein MAN1 are linked to bone and skin disorders. Both proteins share a highly conserved domain, called LEM-domain. LEM proteins are known to interact with Barrier-to-autointegration factor and several transcription factors. Most envelopathies are tissue-specific, but knowledge on the physiological roles of related LEM proteins is still unclear. For this reason, we investigated the roles of MAN1 and emerin during Xenopus laevis organogenesis. Morpholino-mediated knockdown of MAN1 revealed that MAN1 is essential for the formation of eye, skeletal and cardiac muscle tissues. The MAN1 knockdown could be compensated by ectopic expression of emerin, leading to a proper organ development. Further investigations revealed that MAN1 is involved in regulation of genes essential for organ development and tissue homeostasis. Thereby our work supports that LEM proteins might be involved in signalling essential for organ development during early embryogenesis and suggests that loss of MAN1 may cause muscle and retina specific diseases.

Lethal and sublethal effects of three insecticides on two developmental stages of Xenopus laevis and comparison with other amphibians.

It has been suggested that Xenopus laevis is less sensitive than other amphibians to some chemicals, and therefore, that the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) may have limited use in risk assessments for other amphibians. However, comparisons are based mostly on results of FETAX, which emphasizes embryos. Larval X. laevis may be more sensitive to chemicals than embryos and may serve as a better life stage in risk assessments. The present study was conducted to determine the lethal and sublethal effects of 3 insecticides (malathion, endosulfan, and α-cypermethrin) on X. laevis embryos and larvae and to compare toxicity of X. laevis with that of other amphibians. All 3 insecticides have different modes of action, and they caused mortality, malformations, and growth inhibition in both developmental stages. Compared with embryos, larvae were more sensitive to endosulfan and α-cypermethrin but not to malathion. Xenopus laevis larvae had low sensitivity to endosulfan, median sensitivity to malathion, and high sensitivity to α-cypermethrin/cypermethrin relative to other larval amphibians. Our results suggest that X. laevis larvae may generate more protective toxicity estimates in risk assessments than embryos. Xenopus laevis may have limited use in evaluating risk of organochlorine insecticides to other amphibians but may provide useful toxicity thresholds for pyrethroid and perhaps organophosphorus insecticides.

The embryonic development of Xenopus laevis under a low frequency electric field.

The aim of this study was to determine the effects of a low frequency electric field on the early embryonic development of frogs. The embryos of African clawed toads, Xenopus laevis, were exposed to a 20-µA electric current during the cleavage stages. The developmental processes of embryos during and after electric field exposure were monitored for teratogenic effects. All the embryos continuously exposed to the electric field died without undergoing any developmental processes. However, when the embryos were exposed to the electric field for 20-min periods (four times/over 2 d), the embryos developed into both normal tadpoles (70 %) and malformed tadpoles with light edema, reduced pigmentation, or axial anomalies, such as crooked tails. After exposure, the control embryos were at development stage 35.5 (2 d 2 h), while the normal embryos of the assay group were at developmental stage 41(3 d 4 h). There was a 1 d 2 h difference between the two developmental stages, revealing the importance of that time period for embryogenesis. In conclusion, the effects of electric current on Xenopus embryos are dependent on the initial developmental stage and the duration of exposure.

Domain specific overexpression of TIMP-2 and TIMP-3 reveals MMP-independent functions of TIMPs during Xenopus laevis development.

Extracellular matrix remodelling mediates many processes including cell migration and differentiation and is regulated through the enzymatic action of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). TIMPs are secreted proteins, consisting of structurally and functionally distinct N- and C-terminal domains. TIMP N-terminal domains inhibit MMP activity, whereas their C-terminal domains may have cell signalling activity. The in vivo role of TIMP N- and C-terminal domains in regulating developmental events has not previously been demonstrated. Here we investigated the roles of TIMP-2 and TIMP-3 N- and C-terminal domains in Xenopus laevis embryos. We show that overexpression of TIMP-2 N- and C-terminal domains results in severe developmental defects and death, as well as unique changes in MMP-2 and -9 expression, indicating that the individual domains may regulate MMPs through distinct mechanisms. In contrast, we show that only the N-terminal, but not the C-terminal domain of TIMP-3, results in developmental defects.

Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes.

The upper lip and primary palate form an essential separation between the brain, nasal structures and the oral cavity. Surprisingly little is known about the development of these structures, despite the fact that abnormalities can result in various forms of orofacial clefts. We have uncovered that retinoic acid is a critical regulator of upper lip and primary palate development in Xenopus laevis. Retinoic acid synthesis enzyme, RALDH2, and retinoic acid receptor gamma (RARγ) are expressed in complementary and partially overlapping regions of the orofacial prominences that fate mapping revealed contribute to the upper lip and primary palate. Decreased RALDH2 and RARγ result in a median cleft in the upper lip and primary palate. To further understand how retinoic acid regulates upper lip and palate morphogenesis we searched for genes downregulated in response to RARγ inhibition in orofacial tissue, and uncovered homeobox genes lhx8 and msx2. These genes are both expressed in overlapping domains with RARγ, and together their loss of function also results in a median cleft in the upper lip and primary palate. Inhibition of RARγ and decreased Lhx8/Msx2 function result in decreased cell proliferation and failure of dorsal anterior cartilages to form. These results suggest a model whereby retinoic acid signaling regulates Lhx8 and Msx2, which together direct the tissue growth and differentiation necessary for the upper lip and primary palate morphogenesis. This work has the potential to better understand the complex nature of the upper lip and primary palate development which will lead to important insights into the etiology of human orofacial clefts.

FETAX assay for evaluation of developmental toxicity.

The Frog Embryo Teratogenesis Assay Xenopus (FETAX) test is a development toxicity screening test. Due to the small amount of compound needed and the capability to study organogenesis in a short period of time (96 h), FETAX test constitutes an efficient development toxicity alert test when performed early in drug safety development. The test is conducted on fertilized Xenopus laevis mid-blastula stage eggs over the organogenesis period. Compound teratogenic potential is determined after analysis of the mortality and malformation observations on larva. In parallel, FETAX test provides also information concerning embryotoxic effect based on larva length.

Xclaudin 1 is required for the proper gastrulation in Xenopus laevis.

Claudin 1 is one of the tight junctional proteins involved in the tight sealing of the cellular sheets and plays a crucial role in the maintenance of cell polarity. Although its structure and physiological function in intercellular adhesion is relatively well understood, we have little information about its possible involvement in early development of vertebrates. We found Xclaudin 1 is expressed maternally in the oocyte of Xenopus laevis and the zygotic expression initiates stage 9 in the animal hemisphere but not in the vegetal hemisphere, limited on the ectoderm and mesoderm until the end of gastrulation. We have investigated a potential role for claudin 1 at gastrulation by gain and loss-of-function studies. Over-expression of Xclaudin 1 resulted in gastrulation defect in a dose-dependent manner. Knockdown of Xclaudin 1 by antisense morpholino oligonucleotides (MOs) blocked convergent extension, whereas ectopic expression of Xclaudin 1-myc mRNA rescued these defects. However, altered expression of Xclaudin 1 did not inhibit mesodermal gene expression. Taken together, our results suggest that Xclaudin 1 is required for proper convergent extension movement during Xenopus gastrulation.

EYA1 mutations associated with the branchio-oto-renal syndrome result in defective otic development in Xenopus laevis.

BACKGROUND INFORMATION: The BOR (branchio-oto-renal) syndrome is a dominant disorder most commonly caused by mutations in the EYA1 (Eyes Absent 1) gene. Symptoms commonly include deafness and renal anomalies. RESULTS: We have used the embryos of the frog Xenopus laevis as an animal model for early ear development to examine the effects of different EYA1 mutations. Four eya1 mRNAs encoding proteins correlated with congenital anomalies in human were injected into early stage embryos. We show that the expression of mutations associated with BOR, even in the presence of normal levels of endogenous eya1 mRNA, leads to morphologically abnormal ear development as measured by overall otic vesicle size, establishment of sensory tissue and otic innervation. The molecular consequences of mutant eya1 expression were assessed by QPCR (quantitative PCR) analysis and in situ hybridization. Embryos expressing mutant eya1 showed altered levels of multiple genes (six1, dach, neuroD, ngnr-1 and nt3) important for normal ear development. CONCLUSIONS: These studies lend support to the hypothesis that dominant-negative effects of EYA1 mutations may have a role in the pathogenesis of BOR.

Antioxidant metabolism in Xenopus laevis embryos is affected by stratospheric balloon flight.

To test the effects of low levels of radiation from space on living organisms, we flew Xenopus laevis embryos at different stages of development on a stratospheric balloon (BI.R.BA mission). After recovery, different parameters were analyzed to assess the effects of flight, with particular regard to oxidative stress damage. Because of failed temperature control during flight, the flight shielded embryos (FC) could not be used for biochemical or morphological comparisons. In contrast, the incubation conditions (i.e. temperature, containers, volumes) for the flight embryos (F) were parallel to those for the ground controls. Mortality data show that younger embryos (16 h) flown on the balloon (F) are more sensitive to radiation exposure than older ones (40 h and 6 days). Exposure during flight lowered the antioxidant potential in all embryos, particularly older ones. These preliminary data demonstrate that flight on a stratospheric balloon might affect antioxidant metabolism, though it is not yet possible to correlate these results with low radiation exposure during flight.

Tire debris organic extract affects Xenopus development.

Tire debris (TD) and its organic components were identified as a main source of PM10 atmospheric and water pollution. Because few data are available on the embryotoxic effects of TD organic components, the lethal and teratogenic potential of tire debris organic extract (TDOE) was evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. From stage 8 to stage 47, Xenopus laevis embryos were exposed to TDOE at concentrations of 50, 80, 100, 120 and 140 mg/L. The results showed 50 mg/L TDOE to be the non-observable effect concentration (NOEC). TDOE mortality at 80 mg/L was significantly higher than the control, but did not increase further with higher concentrations. A good concentration-response was observed for percentages of malformed larva and from 80 mg/L on these percentages were significantly higher than the control. Therefore, probit analysis gave a 144.6 mg/L TC50. At 120 and 140 mg/L, many larvae were plurimalformed. The most frequent alterations observed were abnormal gut coiling, microphthalmia, monolateral anophthalmia, and narrowing eyes. The histological screening mainly revealed ocular malformations such as double retina, retina nervous cell layer coiling, and altered lens. Moreover severe vacuolisation and necrosis were scored in liver and axial musculature. These results strongly support the assumption that TDOE is a powerful teratogen for X. laevis.

Bisphenol A causes malformation of the head region in embryos of Xenopus laevis and decreases the expression of the ESR-1 gene mediated by Notch signaling.

Bisphenol A (BpA) is widely used in industry and dentistry. Its effects on the embryonic development of Xenopus laevis were investigated. Xenopus embryos at stage 10.5 were treated with BpA. Developmental abnormalities were observed at stage 35; malformation of the head region including eyes and scoliosis. The expression of several markers of embryonic development was investigated by reverse transcription-polymerase chain reaction (RT-PCR). The pan-neural marker SOX-2, the neural stem cell marker nrp-1, the mesodermal marker MyoD, and the endodermal marker sox17alpha, were used. Although the expression of marker genes was not changed by treatment with BpA, that of Pax-6, a key regulator of the morphogenesis of the eyes, was decreased by BpA. Pax-6 is a downstream factor of Notch signaling. So, the expression of a typical Notch-dependent factor, ESR-1, was investigated in the presence of BpA. The expression of ESR-1 was efficiently suppressed by BpA. In whole mount in situ hybridization (WISH), Pax-6 was expressed in the central nervous system and eyes. The expression was lost completely on treatment with BpA. The expression of ESR-1 in the central nervous system and eyes also disappeared with BpA treatment. Injection of the intracellular domain of Notch efficiently recovered ESR-1 expression in the presence of BpA although injection of a ligand for notch, Delta, did not. These results suggest that BpA decreased the expression of ESR-1 by disrupting the Notch signal.

Toxicity and modulations of biomarkers in Xenopus laevis embryos exposed to polycyclic aromatic hydrocarbons and their N-heterocyclic derivatives.

Effects of a newly identified group of organic environmental pollutants of concern (N-heterocyclic derivatives of polycyclic aromatic hydrocarbons, NPAHs) were investigated using the 96 h FETAX (Frog Embryo Teratogenesis Assay - Xenopus). Beside standard FETAX parameters (mortality, malformations), changes in several biochemical markers were studied as early signs of intoxication. Biomarkers included determination of glutathione (GSH) levels and lipid peroxidation as well as activities of important detoxification and antioxidant enzymes (glutathione-S-transferase, glutathione peroxidase, glutathione reductase). 1,10-Phenathroline was the most toxic of all tested compounds (96 h LC(50) = 4 microM). All tested NPAHs induced malformations in the frog embryos. The data suggest that the exposure to NPAHs can induce oxidative stress in amphibians; most biochemical markers were modulated at concentrations lower than those resulting in significant mortality. Results document mortality and teratogenicity of all studied NPAHs to amphibian embryos while no significant mortality, teratogenicity or modulations in biochemical markers could be observed with unsubstituted polycyclic aromatic hydrocarbons (PAHs) at concentrations up to their water solubility. This information along with the significantly greater solubility and thus bioavailability compared to their nonsubstituted parent compounds suggests that NPAHs could contribute significantly to the overall aquatic toxicity of mixtures of PAHs and their derivatives.

Molecular contrast optical coherence tomography: A pump-probe scheme using indocyanine green as a contrast agent.

The use of indocyanine green (ICG), a U.S. Food and Drug Administration approved dye, in a pump-probe scheme for molecular contrast optical coherence tomography (MCOCT) is proposed and demonstrated for the first time. In the proposed pump-probe scheme, an optical coherence tomography (OCT) scan of the sample containing ICG is first acquired. High fluence illumination (approximately 190 kJ/cm2) is then used to permanently photobleach the ICG molecules--resulting in a permanent alteration of the overall absorption of the ICG. A second OCT scan is next acquired. The difference of the two OCT scans is used to determine the depth resolved distribution of ICG within a sample. To characterize the extent of photobleaching in different ICG solutions, we determine the cumulative probability of photobleaching, phi(B,cum), defined as the ratio of the total photobleached ICG molecules to the total photons absorbed by the ground state molecules. An empirical study of ICG photobleaching dynamics shows that phi(B,cum) decreases with fluence as well as with increasing dye concentration. The quantity phi(B,cum) is useful for estimating the extent of photobleaching in an ICG sample (MCOCT contrast) for a given fluence of the pump illumination. The paper also demonstrates ICG-based MCOCT imaging in tissue phantoms as well as within stage 54 Xenopus laevis.

Triadimefon causes branchial arch malformations in Xenopus laevis embryos.

BACKGROUND, AIMS AND SCOPE: Triazole-derivatives are potent antifungal agents used as systemic agricultural fungicides and against fungal diseases in humans and domestic animals. They act by inhibiting the cytochrome P-450 conversion of lanosterol to ergosterol, thus resulting in faulty fungal cell wall synthesis. Some data have been published about the teratogenic activity of triazoles on rodent embryos: Hypoplasias, abnormal shape, agenesis of the branchial arches, for example, were reported as typical induced malformations. Unfortunately, no data are available on the embryotoxicity of these compounds in amphibians, despite the increasing concern among the scientific community about the phenomenon of global amphibian population declines. The aim of the present work is to evaluate the embryo-lethal and teratogenic potentials of Triadimefon (FON), a triazole-derivative widely used as an antimycotic in agriculture, by the test FETAX (Frog Embryos Teratogenic Assay, Xenopus) with particular attention being paid to the analysis of branchial arch malformations. METHODS: Xenopus laevis embryos were exposed continuously from stage 9 to increasing concentrations of FON and analyzed at stage 47 for mortality and teratogenicity (group I) to determine the median lethal (LC50) and teratogenic (TC50) concentrations. Another two pools of larvae were exposed to FON for a 2 hour period at early gastrula (Group II) or neurula (Group III) stages to verify which period of development is the most sensitive to FON. The malformations observed were further investigated by histological section and cartilage staining with Alcian blue. RESULTS AND DISCUSSION: The assay has estimated LC50 and TC50 values of 63.8 microM and 2.73 microM, respectively; the resulting TI (Teratogenic Index = LC50/TC50) value of 23.4 has underlined the very high teratogenic risk associated with this compound. Neurulation was more sensitive to FON exposure than gastrulation, since the TC50 estimated values for group III (neurula exposed) specimens was 7.6 times lower than those of group II (gastrula exposed). Interestingly, for each group analyzed, 100% of malformed embryos showed alterations at branchial arch derived cartilages: Anterior cartilages were reduced, missing, fused or incorrectly positioned while gill cartilages were altered only in the most severely affected specimens. In some cases these malformations were associated with hyperpigmentation. Our results support the hypothesis that FON can interfere with Neural Crest Cell (NCC) migration, since craniofacial components and melanophores are derived from neural crest material. CONCLUSION: In conclusion, our data show Triadimefon to be a potent teratogen able to induce specific craniofacial malformation in Xenopus laevis embryos, probably interfering with the NCC migration into the branchial mesenchyme. These results are also interesting for ecotoxicological reasons as FON, as well as other pesticides, are likely to be present in water systems near agricultural or urban areas which may serve as habitats for developing amphibians and fishes. RECOMMENDATION AND OUTLOOK: Our results are in agreement with the data obtained on in vitro cultured rat embryos suggesting that the FON mechanism of action involves strongly conserved molecules. The choice of Xenopus laevis as the model organism allows us to extend the toxicological and teratological observations to a molecular level, in order to search for novel genes regulated by FON exposure.