RESUMO
Folate supplementation prevents up to 70% of neural tube defects (NTDs), which result from a failure of neural tube closure during embryogenesis. The elucidation of the mechanisms underlying folate action has been challenging. This study introduces Xenopus laevis as a model to determine the cellular and molecular mechanisms involved in folate action during neural tube formation. We show that knockdown of folate receptor 1 (Folr1; also known as FRα) impairs neural tube formation and leads to NTDs. Folr1 knockdown in neural plate cells only is necessary and sufficient to induce NTDs. Folr1-deficient neural plate cells fail to constrict, resulting in widening of the neural plate midline and defective neural tube closure. Pharmacological inhibition of folate action by methotrexate during neurulation induces NTDs by inhibiting folate interaction with its uptake systems. Our findings support a model in which the folate receptor interacts with cell adhesion molecules, thus regulating the apical cell membrane remodeling and cytoskeletal dynamics necessary for neural plate folding. Further studies in this organism could unveil novel cellular and molecular events mediated by folate and lead to new ways of preventing NTDs.
Assuntos
Polaridade Celular , Receptor 1 de Folato/metabolismo , Placa Neural/citologia , Placa Neural/metabolismo , Tubo Neural/citologia , Tubo Neural/embriologia , Organogênese , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Caderinas/metabolismo , Polaridade Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endocitose/efeitos dos fármacos , Feminino , Receptor 1 de Folato/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Marcação de Genes , Humanos , Morfolinos/farmacologia , Tubo Neural/metabolismo , Neurulação/efeitos dos fármacos , Organogênese/efeitos dos fármacos , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMO
BACKGROUND: Valproic acid (VPA) is prescribed therapeutically for multiple conditions, including epilepsy. When taken during pregnancy, VPA is teratogenic, increasing the risk of several birth and developmental defects including neural tube defects (NTDs). The mechanism by which VPA causes NTDs remains controversial and how VPA interacts with folic acid (FA), a vitamin commonly recommended for the prevention of NTDs, remains uncertain. We sought to address both questions by applying untargeted metabolite profiling analysis to neural tube closure (NTC) stage mouse embryos. METHODS: Pregnant SWV dams on either a 2 ppm or 10 ppm FA supplemented diet were injected with a single dose of VPA on gestational day E8.5. On day E9.5, the mouse embryos were collected and evaluated for NTC status. Liquid chromatography coupled to mass spectrometry metabolomics analysis was performed to compare metabolite profiles of NTD-affected VPA-exposed whole mouse embryos with profiles from embryos that underwent normal NTC from control dams. RESULTS: NTDs were observed in all embryos from VPA-treated dams and penetrance was not diminished by dietary FA supplementation. The most profound metabolic perturbations were found in the 10ppm FA VPA-exposed mouse embryos, compared with the other three treatment groups. Affected metabolites included amino acids, nucleobases and related phosphorylated nucleotides, lipids, and carnitines. CONCLUSION: Maternal VPA treatment markedly perturbed purine and pyrimidine metabolism in E9.5 embryos. In combination with a high FA diet, VPA treatment resulted in gross metabolic changes, likely caused by a multiplicity of mechanisms, including an apparent disruption of mitochondrial beta-oxidation. Birth Defects Research 109:106-119, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Suplementos Nutricionais , Ácido Fólico/administração & dosagem , Defeitos do Tubo Neural/metabolismo , Neurulação/efeitos dos fármacos , Teratogênicos/toxicidade , Ácido Valproico/toxicidade , Aminoácidos/metabolismo , Animais , Carnitina/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Lipídeos/análise , Masculino , Metaboloma , Camundongos , Tubo Neural/anormalidades , Tubo Neural/efeitos dos fármacos , Tubo Neural/metabolismo , Defeitos do Tubo Neural/induzido quimicamente , Defeitos do Tubo Neural/patologia , Penetrância , Gravidez , Purinas/metabolismo , Pirimidinas/metabolismoRESUMO
The aim of this study was to explore the direct embryonic teratogenicity of vinyl chloride monomer (VCM), especially the toxic effects on the early development of the nervous system and its underlying mechanisms. Pregnant mice at embryonic day 6.5 (E6.5) were injected with different doses of VCM (200, 400 and 600 mg/kg) and embryos were harvested at E10.5. Our results showed that doses higher than 400 mg/kg of VCM increased the incidence of malformed embryos, especially the neural tube defects (NTDs). In addition, high-dose of VCM decreased mitotic figure counts in the neuroepithelium and enhanced the percentage of cells in G0/G1 phase, while they were reduced in S phase. The more VCM was injected into mice, the fewer positive PCNA cells were seen and the more positive TUNEL cells were observed in the neuroepithelium. Moreover, significant increases in the levels of caspase-3 protein were observed in NTD embryos. Our results demonstrate that during early pregnancy, exposure to doses higher than 400 mg/kg of VCM increases the incidence of malformations and particularly the rate of NTDs. High-dose of VCM inhibits the proliferation of neural cells and induces cell apoptosis, leading to an imbalance in the ratio of proliferation and apoptosis. Meanwhile, the apoptosis of neuroepithelial cells might be accelerated by the activation of the caspase-3 pathway, and it might be a reason for NTDs.
Assuntos
Encéfalo/efeitos dos fármacos , Defeitos do Tubo Neural/induzido quimicamente , Neurônios/efeitos dos fármacos , Neurulação/efeitos dos fármacos , Cloreto de Vinil/toxicidade , Animais , Apoptose/efeitos dos fármacos , Encéfalo/embriologia , Feminino , Marcação In Situ das Extremidades Cortadas/métodos , Camundongos , Neurônios/metabolismo , GravidezRESUMO
The whole embryo culture (WEC) model serves as a potential alternative for classical in vivo developmental toxicity testing. In the WEC, cultured rat embryos are exposed during neurulation and early organogenesis and evaluated for morphological effects. Toxicogenomic-based approaches may improve the predictive ability of WEC by providing molecular-based markers associated with chemical exposure, which can be compared across multiple parameters (e.g., exposure duration, developmental time, experimental model). Additionally, comparisons between in vitro and in vivo models may identify objective relevant molecular responses linked with developmental toxicity endpoints in vivo. In this study, using a transcriptomic approach, we compared all-trans retinoic acid (RA)-exposed and nonexposed Wistar rat embryos derived using WEC (RA, 0.5 µg/ml) or in vivo (RA, 50 mg/kg, oral gavage) to identify overlapping and nonoverlapping effects of RA on RNA expression in parallel with morphological changes. Across six time points (gestational day 10 + 2-48 h), we observed strong similarities in RA response at the gene (directionality, significance) and functional (e.g., embryonic development, cell differentiation) level which associated with RA-induced adverse morphological effects, including growth reduction as well as alterations in neural tube, limb, branchial, and mandible development. We observed differences between models in the timing of RA-induced effects on genes related to embryonic development and RA metabolism. These observations on the gene expression level were associated with specific differential morphological outcomes. This study supports the use of WEC to examine compound-induced molecular responses relative to in vivo and, furthermore, assists in defining the applicability domain of the WEC in determining complementary windows of sensitivity for developmental toxicological investigations.