Establishment of the evaluation system for endocrine-disrupting chemimals using stickleback

雄性刺鱼产生一种名为“spiggin”的胶蛋白质，这种胶蛋白质被用作筑巢的粘合剂。Spiggin的合成受雄激素的强烈控制。因此，spiggin是一个有用的生物标志物，以评估雄激素活性的环境化学品，除了卵黄蛋白原，前体的卵黄蛋白从肝脏产生的雌激素刺激。本研究建立了spiggin和vitellogenin的高灵敏定量体系，并利用实时荧光定量PCR技术建立了spiggin mRNA的高灵敏定量体系。该系统能够实现精确和高灵敏度的定量。卵黄蛋白原，雌激素刺激的生物标志物，也纯化了几个色谱技术。还产生了抗卵黄蛋白原的特异性抗体。并采用荧光免疫吸附法建立了卵黄蛋白原定量检测体系 ...更多信息 ent测定。该系统还能够实现与spiggin测定系统相同的高灵敏度定量。除了这些用于生物标志物的定量系统的制备之外，还确定了使用刺鱼进行体内暴露测试的详细条件。以MT作为对照雄激素，进行暴露试验。结果表明，MT浓度为0.1和1 μg/L时，仅暴露3d即可有效促进spiggin mRNA的表达。然后在5、10和15℃三种不同水温下进行MT暴露，以了解水温对spiggin mRNA表达的影响。在15℃时，MT对spiggin mRNA表达的影响最大，而在其他温度下，尤其是5℃时，MT的影响明显减弱。这一结果表明，以spiggin为生物标记物，进行高灵敏度的雄激素活性检测至少需要10℃以上的水温，本研究建立的系统将为评价化学物质的内分泌干扰效力提供有力的工具。少

Male stickleback produce a glue protein named "spiggin" that is used as a cementing substance for the building of its nest. The synthesis of spiggin is strongly controlled by androgen. Therefore, spiggin is a useful biomarker to evaluate androgenic activity of environmental chemicals, in addition to vitellogenin, precursor of yolk protein produced from the liver by the estrogenic stimulation. In this study, we established the highly sensitive quantification system for both spiggin and vitellogenin to evaluate sex-hormonal effect of chemicals.Highly sensitive quantification system for spiggin mRNA was established using real-time RT-PCR technique. This system enables precise and high sensitive quantification. Vitellogenin, biomarker for estrogenic stimulation, was also purified by several chromatographic techniques. Specific antibody against vitellogenin was also raised. Furthermore, vitellogenin quantification system was established using the method of chemiluminescent-linked immunosorb … More ent assay. This system also enables high sensitive quantification same with spiggin assay system. In addition to these preparations for quantification systems for the biomarkers, the detail condition for in vivo exposure test using stickleback was determined. Using MT as a control androgen, we performed exposure test. As a result, only 3 days exposure at a MT concentration 0.1 and 1 μg/L was efficient for spiggin mRNA expression in kidney. Next, MT exposure was performed under three different water temperature (5, 10 and 15℃) to know the effect of water temperature on spiggin mRNA expression. Exposure at 15℃ induced maximal MT effect on spiggin mRNA expression, the others, particular in exposure at 5℃, markedly diminished MT effect. This result suggests that at least water temperature from 10℃ and up is necessary to perform high sensitive detection of androgenic activity using spiggin as a biomarker.The present established systems will be powerful tools for the evaluation of the endocrine disrupting potency of chemicals. Less