Highly sensitive detection of endocrine disrupter in natural environment and its evaluation

在自然环境中，我们可以发现许多化学合成的化合物，其中一些被认为是内分泌干扰物。它们的生态活动给全球生态系统带来了严重的问题。本研究采用GC/MS分析技术，对海洋环境中有机锡化合物污染对腹足类性畸变的影响进行了研究，首次在棒形泰国螺（Thais clavigera）的睾丸和卵巢中分别检测到睾酮和17β-雌二醇。此外，在日本东风螺的相应组织中还发现了雄酮、雌酮和乙炔基炔雌醇。建立了甾体类化合物的提取和精制工艺，C_8-SPE对甾体类化合物的净化效果较好。虽然建立了良好的回收率，GC/MS需要大量的腹足类（超过100）的测定。在此基础上，建立了一种用酶联免疫吸附法（ELISA）测定单头螺生殖器官中睾酮含量的方法。通过选择ELISA试剂盒和在预处理步骤中对待分析的组分进行广泛纯化，克服了ELISA中经常遇到的分析值的大偏差，从而使ELISA值与GC/MS的值相当。基于这种方法，证实了从有机锡污染的海岸收集的雌性imposex-sufficient贝壳中的睾酮含量略高于正常贝壳。本文对日本东风螺睾丸进行了组织学和免疫化学染色，观察了间质细胞的存在，间质细胞是哺乳动物中已知的产生睾酮的细胞。

In natural environment, we can find so many chemically synthesized compounds, some of which are thought as endocrine disrupter. They have the hormone-like activities resulting the serious problems to the global ecosystem. In this project, in order to clarify the effect of the contamination of organotin compounds in the marine environment on the imposex of the gastropods, the speciation and levels of steroid hormones of the organisms were investigated by means of the GC/MS. Testosterone and 17β-estradiol were first identified and quantitated in the testes and ovaries of Thais clavigera, respectively. In addition, androsterone, estorone and ethynylestradiol were also found in the respective tissues of Babylonia japonica. The extraction and refining procedures were constructed, and the passage of C_8-SPE was quite effective for clean-up of the steroid fractions. Though good recoveries were established, GC/MS required lot of gastropods(more than 100) to a determination. Then, a determination method for testosterone in the reproductive organ of an individual gastropod, Thais clavigera , was established by ELISA. Big deviation in analytical values often encountered in ELISA was overcome both by selecting the ELISA kit used and by extensive purification of the fraction-to-be analyzed in the pretreatment step, which resulted in the ELISA values comparable to those of GC/MS. Based on this method, slightly higher testosterone content was confirmed in the female imposex-suffered shells collected from organotin-contaminated seashores than normal ones. The testes of Babylonia japonica were stained both histologically and immunohistochemically to observe the presence of inter-stitial cells, testosterone producing cells known in mammals.