双酚A长期暴露对斑马鱼子代骨发育的影响

Effects of long-term exposure to bisphenol A in zebrafish on offspring bone development

目的本实验以斑马鱼为研究对象探究双酚A(BPA,bisphenol A)长期暴露对斑马鱼子代骨发育的影响。方法将亲代斑马鱼从受精卵开始分别暴露于环境相关浓度的BPA(0、15和225μg/L)120 d,通过阿尔新蓝和茜素红染色及荧光定量PCR检测其子代骨骼发育情况。结果225μg/L BPA暴露亲代斑马鱼明显抑制了子代胚胎孵化率(0.28倍,F=28.495,P<0.05)。15和225μg/L BPA暴露亲代斑马鱼均导致子代斑马鱼角腮骨下移、角舌骨夹角变小、腮骨异常弯曲以及舌续骨畸形,并呈现剂量依赖效应。BPA对子代的骨矿化抑制作用也随暴露浓度增加而加重。qRT-PCR结果显示子代斑马鱼骨发育相关基因表达也出现异常,且呈现剂量依赖方式;15μg/L BPA分别明显上调子代软骨发育相关基因bmp2和mmp13a的表达1.16和1.13倍(F=105.300和180.772,P<0.05),下调col2a1的表达0.34倍(F=31.048,P<0.05)。225μg/L BPA分别明显上调子代软骨发育相关基因bmp2、bmp15、mmp13a和sparc的表达1.64、0.29、1.88和0.42倍(F=105.300、12.298、180.772和6.626,P<0.05),下调了sox9、dlx2和col2a1的表达0.45、0.50和0.53倍(F=22.827、9.613和31.048,P<0.05)。子代硬骨发育相关基因runx2和gap43在15μg/L BPA组明显下调0.20和0.39倍(F=11.106、9.786,P<0.05),runx2、wnt16和gap43在225μg/L BPA组分别明显下调0.13、0.22和0.53倍(F=11.106、9.779和9.786,P<0.05)。结论本研究表明亲代斑马鱼长期暴露于BPA可导致子代软骨和硬骨发育异常,这可能与BPA诱导子代斑马鱼骨发育相关基因表达的失调有关。

Objective In this experiment,taking zebrafish as the research model to explore the effects of long-term BPA exposure on bone development of zebrafish offspring.Methods Adult zebrafish were exposed to environmentally relevant concentrations of BPA(0,15,and 225μg/L)from fertilization onward for a duration of 120 days.The skeletal development of their offspring was assessed using Alcian Blue and Alizarin Red staining,as well as real-time quantitative PCR(RT-qPCR).Results The result showed that 225μg/L BPA exposure of the parent significantly inhibited the hatching rate of the offspring embryos.Alcian blue and alizarin red staining showed that parental 15 and 225μg/L BPA exposure could lead to the descending of the ceratobranchial,the reduction of the angle of the chratohyal,the abnormal curvature of the ceratobranchial and the deformity of the palatoquadrate in the offspring of zebrafish,with a dose-dependent effect.The inhibitory effect of BPA on offspring's bone mineralization also intensifies with increasing exposure concentration.The qRT-PCR result revealed abnormal expression of offspring zebrafish bone development-related genes,which displayed a dose-dependent pattern.At 15μg/L BPA,there was a significant upregulation in the expression of offspring cartilage development-related genes bmp2 and mmp13a by 1.16 and 1.13 times(F=105.300,180.772,P<0.05),while col2a1 expression was downregulated by 0.34 times(F=31.048,P<0.05).At 225μg/L BPA,there was a significant upregulation in the expression of offspring cartilage development-related genes bmp2,bmp15,mmp13a,and sparc by 1.64,0.29,1.88,and 0.42 times(F=105.300,12.298,180.772,6.626,P<0.05),while the expression of sox9,dlx2,and col2a1 was downregulated by 0.45,0.50,and 0.53 times(F=22.827,9.613,31.048,P<0.05).Offspring bone development-related genes runx2 and gap43 were significantly downregulated by 0.20 and 0.39 times(F=11.106,9.786,P<0.05)in the 15μg/L BPA group,whereas runx2,wnt16,and gap43 were significantly downregulated by 0.13,0.22,and 0.53 times(F=11.106,9.779,9.786,P<0.05)in the 225μg/L BPA group.Conclusion This study demonstrates that prolonged exposure of parental zebrafish to BPA can lead to abnormal development of cartilage and bone in their offspring,which may be associated with the dysregulation of BPA-induced gene expression related to zebrafish offspring bone development.