| Bisphenol A(BPA)is an industrial raw material for the production of polycarbonate,epoxy resins and the resin linings for beverage and food cans,which is widely used in the plastic production industry.BPA is widely found not only in water,air and dust,but also in the contents of canned food and drink.BPA is a xenoestrogenic endocrine disrupting compound(EDC),which is able to bind to various membrane receptors and nuclear receptors as well as affects multiple developmental pathways.BPA has embryonic developmental toxicity and genetic toxicity,which can impair embryos and offspring development via maternal exposure.BPA impaired the embryo development of porcine and bovine,which resulted in dead embryos and arrested development in blastocyst stage.Pregnant sheep exposed to BPA can cause metabolic disorder in offspring.Furthermore,laying hens exposed to BPA can reduce the production performance.Therefore,BPA is toxic to the embryonic development of vertebrates,harmful to environment and livestock production,and has potential hazards to human health through accumulation in the environment and livestock products.Many researches have reported the embryonic toxicity of BPA in model animals and domestic animals,but the systematic analysis of the embryo deformities is still lacking,and the teratogenic mechanism of BPA is not well known.In this study,the teratogenic effect of BPA was systematically analyzed,and the molecular mechanism of teratogenesis were alos studied,which providing valuable reference for the study of embryonic toxicity and teratogenic mechanism of BPA.Xenopus laevis is a well known embryonic development and environmental toxicology model.In this study,2-cell stage embryos of X.Laevis were exposed to BPA at different concentrations(0.1,1,10,20 μM)for 96 h.The effect of BPA on embryo development was studied by monitoring embryonic development and its indexes(full length,head length,eye distance,eye diameter,heart rate)as well as behavioral ability(touch response,autonomous swimming ability).Results showed that at the early stage of embryonic development(NF St.9-St.24),BPA(≥ 1 μM)significantly delayed embryonic development(P < 0.01),while the retardation effect of 20 μM BPA lasted to the end of 96 h acute exposure experiment.Most embryos exposed to 30 μM BPA were retarded at blastocyst stage.At NF st.45,the eye diameter of embryos exposed to BPA(≥ 0.1 μM)was significantly decreased than that of the control group(P < 0.01).The full length of embryos exposed to BPA(≥ 1 μM)was significantly reduced(P < 0.05).The head length,ratio of head length to full length and eye distance of embryos treated with 20 μM BPA were significantly decreased than those of the control group(P < 0.01),the heart rate and behavior of embryos exposed to this concentration were also impaired,which suggested that BPA is toxic to X.laevis embryonic development and 20 μM BPA affected head development.The teratogenicity of BPA was studied by score of malformation(SOM)to comprehensively evaluate the teratogenicity of BPA.Result showed that BPA(≥ 1 μM)exhibited significant teratogenic effect(P < 0.05)including short body axis,bend body axis,cardiac edema,misociled gut,abdominal stretching or edema,etc.Embryos exposed to 20 μM BPA showed the most types of and the most serious of malformations,which covered almost every aspect of development.These results suggest that BPA can cause a variety of abnormalities in embryos,and the notochord,eyes,brain,intestine and heart are the main target organs.Furthermore,a series of histological and molecular biological methods including hematoxylin-eosin(HE)staining,TUNEL staining,single-cell gel electrophoresis(SCGE),western bolt,q RT-PCR and immunofluorescent staining(IF)were used to investigate the mechanism of BPA teratogenicity.Single cell gel electrophoresis showed that even 0.1 μM BPA could cause DNA damage(P < 0.05).HE and TUNEL staining of body axis section showed that the notochord vacuolar cells of X.laevis embryos exposed to 20 μM BPA were inadequate and significantly apoptotic(P < 0.05),and the notochord was significantly thinner than that of control(P < 0.01).Furthermore,Immunofluorescence(IF)staining of cleaved caspase-3 showed that the vacuolar cells of notochord and the muscle cells around notochord also underwent apoptosis.HE and TUNEL as well as IF staining suggested that apoptosis is the main reason caused the bended embryo axis.q RT-PCR results showed that the ratio of bax/ bcl-2 m RNA in embryos exposed to 10 and 20 μM BPA was significantly increased(P <0.05).The result of WB showed that cleaved caspase-3 expression was significantly up-regulated in the embryos exposed to 10 and 20 μM BPA(P < 0.05).In conclusion,BPA can delay the early development of X.laevis embryos,induce DNA damage and apoptosis in embryos,cause abnormal vacuolar cell in notochord and impaired muscle tissue around notochord,which eventually resulted in the bended the body axis and the impaired behavior of larvae.In order to investigate the mechanism of BPA induced apoptosis in response to DNA damage,the embryonic development related genes which significantly affected by BPA were screened in the comparative toxicogenomics database(CTD).BPA influenced signaling pathways related to embryonic development were analyzed by function enrichment and signal pathway enrichment.Furthermore,the protein-protein interaction network was constructed to explore the pathway involved in DNA damage and cell apoptosis.The results showed that BPA could affect multiple important development pathways such as Hippo,TGF-β,Wnt and Notch pathways.In addition,the significant interactions among YAP(the core effector factor of Hippo pathway),c-Abl,p73 and caspase-3 were observed,which suggested that c-Abl/YAPY357/ p73 signaling pathway is a candidate pathway induced embryo apoptosis in response to DNA damage.P19 cell line is widely used as developmental toxicological model because of its capability of differentiating into ectoderm,mesoderm,and endoderm.In this study,mouse P19 embryonal carcinoma cells were used as in vitro model to investigate the role of c-Abl/YAPY357/ p73 signaling pathway in the process of inducing embryo apoptosis in response to DNA lesion.P19 cells were treated with different concentrations of BPA.The DNA integrity of P19 cells was detected by SCGE.Results showed that BPA(≥ 1 μM)treatment for 12 h significantly induced DNA damage in P19 cells(P < 0.001).Apoptosis of P19 cells was detected by Annexin V-PE and 7-AAD staining and WB.The results showed that 50 and 100μM BPA significantly induced apoptosis in P19 cells(P < 0.05).The influence of BPA on c-ABL /YAPY357/ p73 signaling pathway and its target gene expression were detected by WB and q RT-PCR.Results showed that 50 and 100 μM BPA treated P19 cells for 48 h,c-Abl,phosphorylated YAPY357,phosphorylated p73Y99,total p73,and cleaved caspase-3 protein expression levels were significantly increased(P < 0.05)and total YAP expression level was significantly decreased(P < 0.001).The m RNA expression levels of target genes Bax and Puma were significantly up-regulated(P < 0.05).To confirm the role of c-Abl,P19 cells were co-treated with BPA and Imatinib,an inhibitor of tyrosine kinase c-Abl.The cell index and the expression of c-Abl / YAPY357 / p73 signaling pathway were detected by real-time cellular analysis(RTCA)and WB.The protein expression levels of p-YAPY357,p-p73Y99 and cleaved caspase-3 in the Imatinib and BPA co-treated group were significantly decreased than those in P19 cells treated with BPA alone(P < 0.05).In addition,the normalized cell index(NCI)was significantly improved(P < 0.05).Studies showed that DNA damage triggered by BPA activated the c-ABL /YAPY357/ p73 signaling pathway and induced the expression of Bax,Puma,thus leaded to the apoptosis of embryo cells.In conclusion,BPA induced DNA damage in embryo cells,activated the c-Abl /YAPY357/p73 signaling pathway,and then upregulated the transcriptional expression of target genes Bax and Puma.Furthermore,BPA affected the ratio of bax/bcl-2 in mitochondria and induced apoptosis of embryo cells.Apoptosis triggered the damage of notochord and muscle tissue during embryonic development,and eventually leaded to the bending of the anterior and posterior axis of the embryo,which resulted the deformity of the embryo. |
PDF Full Text Request