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

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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.