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。它们具有荷尔蒙样活性，对全球生态系统造成严重问题。为了阐明海洋环境中有机锡化合物污染对腹足动物性畸变的影响，采用GC/MS法研究了这些生物体内类固醇激素的形态和含量，首次在泰国棒虫的睾丸和卵巢中鉴定和测定了睾酮和17β-雌二醇的含量。此外，在日本东风螺的组织中还发现了雄酮、雌酮和乙炔雌二醇。建立了提取和精制工艺，C8-SPE对甾体组分有较好的净化效果。虽然有良好的回收率，但GC/MS需要大量腹足类(100多只)才能进行测定。在此基础上，建立了用酶联免疫吸附试验(EL ISA)测定克氏腹足类个体生殖器官中睾酮的方法。通过选用试剂盒和对前处理步骤中待分析组分的大量纯化，克服了常规分析中常出现的较大偏差，使ELISA值达到了与GC/MS相当的水平。用组织学和免疫组织化学方法对日本东风螺的睾丸进行染色，以观察间质细胞的存在。间质细胞是哺乳动物中已知的产生睾丸激素的细胞。

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.