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

自然环境中内分泌干扰物的高灵敏检测及评价

• 批准号: 10555293
• 负责人: OKUBO Akira
• 金额: $ 8.38万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10555293/
• 关键词: organotin compound; endocrine disrupter; gastropod; imposex

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.

在自然环境中，我们可以找到如此多的化学合成化合物，其中一些被认为是内分泌的。他们进行了类似马的活动，从而给全球生态系统带来了严重的问题。在该项目中，为了阐明有机素化合物在海洋环境中污染对腹足动物的污染的影响，通过GC/MS研究了生物体的类固醇骑兵的规格和水平。首先在Thais clavigera的测试和卵巢中首先鉴定并定量睾丸激素和17β-雌二醇。此外，在Japonica的相应组织中还发现了雄激素，雌酮和乙烯甲二醇。构建了提取和炼油程序，C_8-SPE的通过对于清理类固醇分数非常有效。尽管建立了良好的恢复，但GC/MS仍需要大量的腹足动物（超过100）来确定。然后，ELISA建立了单个腹足类动物Thais Clavigera的生殖器官中睾丸激素的测定方法。通过选择所使用的ELISA试剂盒和在预处理步骤中分析的分数进行广泛纯化，可以克服ELISA经常遇到的分析值的很大偏差，从而导致ELISA值与GC/MS相当。基于这种方法，在有机污染的海洋中收集的女性摄影壳中确认了睾丸激素含量略高，而不是正常睾丸激素。在组织学和免疫组织化学上染色了巴比伦氏菌的测试，以观察存在胸骨间细胞，睾丸激素在哺乳动物中已知的细胞。

项目成果

陸明, 堀口敏宏, 白石寛明, 柴田康行, 安保充, 大久保明, 山崎素直: "ガスクロマトグラフィー/質量分析法による海産巻貝類におけるステロイドホルモンの同定と定量"分析化学. 50・No.4. 247-255 (2001)

Akira Riku、Toshihiro Horiguchi、Hiroaki Shiraishi、Yasuyuki Shibata、Mitsuru Yasubo、Akira Okubo、Motonao Yamazaki：“通过气相色谱/质谱法鉴定和定量海洋蜗牛中的类固醇激素”《分析化学》，第 50 期，第 4. 247-255 期。 (2001)

Ming Lu, Toshihiro Horiguchi, Hiroaki Shiraishi, Yasuyuki Shibata, Mitsuru Abo, Akira Okubo and Sunao Yamazaki: "Identification and quantitation of steroid hormones in marine gastropods By GC/MS"Bunseki Kagaku. Vol. 50, No. 4 (in Japanese). 247-255 (2001)

Ming Lu、Toshihiro Horiguchi、Hiroaki Shiraishi、Yasuyuki Shibata、Mitsuru Abo、Akira Okubo 和 Sunao Yamazaki：“通过 GC/MS 鉴定和定量海洋腹足动物中的类固醇激素”Bunseki Kagaku。