Assessment Of The Embryotoxicities Of Bisphenol A And Cadmium Chloride Using Modified Embryonic Stem Cell Test

Objective: The embryonic stem cell test（EST）,a validated method,is used to assess the embryotoxic hazard of test substances in vitro.However,as the misclassifications of methylmercury,the generic limitation of EST became clear to us.This study was aimed to establish a modified embryonic stem cell test system using the neuronal differentiation end point to substitute myocardial differentiation end point,and to apply the established experimental system to determine the developmental toxicity of bisphenol A and cadmium chloride.Methods:（1）The establishment and validation of neural differentiation method: The m RNA expression level of neural cell specific markers MAP2,nestin,and KCC2 during neural differentiation were detected by fluorescence quantitative PCR to explore the best embryonic stem cells inoculum density and culture time in nerve cell culture fluid;the protein expression level of neural cell specific markers MAP2,nestin,KCC2 and β-Tubullin III were detected by immunofluorescence to determine the feasibility of neural differentiation method.（2）The establishment of modified EST: The established neural differentiation method was combined with the classical EST and was applied to determine the embryonic developmental toxicity of bisphenol A and cadmium chloride.In order to detect the credibility of the system,the positive control 5-Fu and negative control Penicillin-G were set up as quality control group.Results:（1）The most suitable embryonic stem cells inoculum density was 15×104/ well and the culture time in nerve cell culture fluid was determined as 12 days.Compared to MAP2 and KCC2,Nestin was more suitable as the detection biomarker of neural differentiation inhibition test.The results of immunofluorescence showed that the nerve cell specific markers were almost expressed in all cells,which showed that the neural differentiation method was feasible.（2）The modified EST with neural differentiation as terminal was used to determine the developmental toxicity of BPA and CdCl₂.The results indicatedthat BPA had weak embryo developmental toxicity and CdCl₂ had strong embryo developmental toxicity.Positive control 5-Fu was judged to have strong embryonic developmental toxicity,while the negative control Penicillin-G was judged to have no embryonic developmental toxicity correctly,which showed that the modified EST was credible.Conclusion: In this study,the neural differentiation method of embryonic stem cells was eplored and the modified EST with neural differentiation ID50 was established.Using the modified EST,the developmental toxicity of BPA and CdCl₂ were predicted successfully.Further improvement of the experimental system would be beneficial to expand the application scope and to improve the effectiveness of EST.