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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., (© 2011 Wiley-Liss, Inc.)

The embryotoxicity and teratogenicity of Cr(VI) on the survival and morphology of the anuran Xenopus laevis have been assessed by frog embryo teratogenesis assay-Xenopus (FETAX). The lethal median (LC(50)) and teratogenic median (TC(50)) concentration values of Cr(VI) were 890 microM and 260 microM, respectively. The calculated teratogenic index (TI) value was 3.42, suggesting that hexavalent chromium has a teratogenic potential. Malformations of embryos included lifting of the body, coiling of the tail and body oedema. Furthermore, the chromium salt caused significant growth retardation at 25 microM exposure concentrations. The use of radiolabelled (51)Cr(VI) allowed the determination of the time course uptake of Cr in Xenopus exposed to concentrations ranging from 0.025 to 500 microM. The evaluation of its distribution into the body (head-abdomen-tail) was evaluated at different exposure times. Chromium is taken up at 24 h by Xenopus embryos for all concentrations tested. At 48 h post fertilization (stage of larva) the amount of Cr accumulated by the two-day-old larva ranged from 0.42 to 580 pg mg(-1) wet weight at 0.025 and 500 microM respectively. These amounts were lower than those at 24 h (2.77 to 11016 pg mg(-1) wet weight embryo) reaching values of the same order of magnitude at 120 h (five-days-old larva). Since at 48 h Xenopus development leads to a swimming embryo, the observed uptake at 24 h could be the result of the binding of Cr to jelly coat compounds surrounding the embryo body as confirmed by gel filtration experiments on (51)Cr-jelly coat. The interaction of Cr with jelly coat is in agreement with the role of jelly coat in protecting the embryo against pathogen and chemical toxins to ensure fertilization. This work further supports the hypothesis that Cr contamination of surface waters could contribute to explain the reported worldwide depletion of frog population.

GD3, a minor ganglioside in most normal tissues, is involved in important biological events and its expression could increase in pathological conditions. Organism integrity requires a tight balance between the anabolic and catabolic processes, thus it is important to control the intracellular expression of those "key" enzymes, which act at the "branching point" of ganglioside metabolism; one of these is the GD3-synthase (ST8Sia I). In this paper, we report the sequences of two ST8Sia I mRNAs found in Xenopus laevis and their genomic organization; the canonical form resulted constituted of 5 exons and 4 introns, while the "short" mRNA lacks of the exon 2. The expression of the two ST8Sia I mRNAs during embryo development and their tissue distribution in adult animals showed the single or simultaneous presence of the two forms. Experiments of in vitro expression and evaluation of enzymatic activity of the two hypothetical proteins turned out to be ST8Sia I. In the end, considering the growing interest toward the specie Xenopus tropicalis, due to its diploid genome that render it more suitable for genetic studies, we also cloned X. tropicalis ST8Sia I.

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.

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.

Since the level of platinum in the environment is destined to increase, because of its use in vehicle catalytic converters, the toxicity of platinum needs further investigation. In this study, the frog embryo teratogenesis assay-Xenopus (FETAX) was used to compare the embryotoxicity and teratogenicity of two common platinum species, (NH4)2PtCl4 and (NH4)2PtCl6. The uptake rates of the two platinum species were studied, and also their effects on the expression of genes encoding metallothionein and heat-shock protein 70, which are known to be induced by several stress factors. In addition, the differential display technique was used to search for genes that were specifically induced by platinum. A gene for the type I collagen alpha-chain and a novel gene were identified.

The evaluation of the effect of trace metals on health can be difficult, because of their presence in the environment in various chemical forms. Exposure to arsenic compounds is an example of this complexity, as it can be present in the environment in inorganic and organic forms. The effects of arsenic in vertebrates are complicated by several variables, such as speciation of the element, the exposure route, and the susceptibility of the particular animal species. The embryotoxicity and teratogenicity of three arsenic species - sodium arsenite (NaAsO(2)), disodium hydrogen arsenate (Na(2)HAsO(4)) and dimethylarsinic acid [(CH3)2AsOOH] - were evaluated by the modified frog embryo teratogenic assay on Xenopus (FETAX). We also show how the classical FETAX endpoints, based on morphological and morphometrical analysis, can conveniently be integrated with the study of molecular markers. Possible changes in the expression of the mRNA for the heat-shock protein HSP70, following exposure to NaAsO(2), were examined by using the reverse transcriptase polymerase chain reaction. HSP70 mRNA is strongly induced by arsenic.

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran amphibian Xenopus laevis. The FETAX can detect xenobiotics that affect embryonic development, when mortality, teratogenicity and growth inhibition are used as endpoints. The FETAX was used to compare the embryotoxic and teratogenic potentials of two chemical species of mercury, inorganic mercury(II) chloride (HgCl2) and organic methylmercury chloride (MeHgCl). A higher toxicity of MeHgCl (the estimated median lethal concentration [LC50] and median teratogenic concentration [TC50] were 0.313microM and 0.236microM, respectively) over HgCl2, with estimated LC50 and TC50 values of 0.601microM and 0.513microM, respectively). On the basis of these results, HgCl2 and MeHgCl can be classified as "slightly teratogenic compounds", as the ratio of LC50/TC50 is less than 1.5. There was a significant deviation from the commonly described monotonic behaviour of the concentration-response curves, suggesting a hormetic effect of both species of mercury. Uptake experiments, followed by neutron activation analysis, showed a higher incorporation of mercury in embryos exposed to MeHgCl compared with those exposed to HgCl2. Interestingly, Hg- exposed embryos showed a higher content of selenium and zinc than did control embryos.

The frog embryo teratogenesis assay-Xenopus (FETAX) is a powerful and flexible bioassay that makes use of the embryos of the anuran Xenopus laevis. FETAX satisfies the requirements of low cost, reliability and reproducibility and, thanks to its three endpoints (i.e., mortality, teratogenicity and growth inhibition) can detect the xenobiotics that affect embryonic development. In this paper, we have used FETAX to evaluate samples of soils collected in an oil-contaminated area. Embryos were exposed directly to the soil to be tested. Particular attention was devoted to provide a statistical procedure for analysing mortality and malformation data as well as growth retardation.