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

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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