Study on the development of bioassay system for organic halogenated compounds with thyroid-disrupting activity

在脊椎动物中，甲状腺激素对于胚胎后发育是必不可少的，例如在哺乳动物中建立中枢神经系统，在两栖动物中变态。目前的研究集中在环境化学物质已知或怀疑以甲状腺激素信号通路为靶点的甲状腺外过程。我们开发了三种体外和一种体内生物检测方法，分别检测干扰甲状腺激素与血浆反式甲状腺激素结合(TTR法)、甲状腺激素与核受体结合(TR法)、诱导甲状腺激素依赖的报告基因表达(LUC法)以及甲状腺激素诱导的两栖动物变态过程中的基因表达(变形法)的化学物质。几种有机卤素影响甲状腺外甲状腺激素信号通路的不同方面，但几乎不影响甲状腺激素与受体的结合。在特定的甲状腺激素相关发育窗口期，两栖类蝌蚪对环境化学物质的影响最为敏感。通过这些生物测定，调查了日本和泰国水环境中污染物对甲状腺激素的干扰活性。我们的分析表明，废水处理有效地去除了造纸厂和污水处理厂废水中的TH干扰污染物。LUC分析表明，日本的污水处理厂流出物含有Ta样活性，而泰国的污水处理厂流出物含有抗Ta样活性。这三种体外生物测定很可能以依赖于水样的方式检测到具有TH干扰活性的相同或不同的污染物。我们的结果表明，几种体外生物测定的组合有助于快速筛选环境水样，以确定具有TH干扰活性的污染物的位置。

In vertebrates, thyroid hormones are essential for post-embryonic development, such as establishing the central nervous system in mammals and metamorphosis in amphibians. The present study focused on the extra-thyroidal processes that environmental chemicals are known to or suspected to target in the thyroid hormone-signaling pathway. We developed three in vitro and one in vivo bioassays, which detect chemicals interfering with thyroid hormone binding to plasma transthyretin (TTR assay), thyroid hormone binding to nuclear receptor (TR assay), induction of thyroid hormone-dependent reporter gene expression (Luc assay, and gene expression during thyroid hormone-induced amphibian metamorphosis (metamorphosis assay). Several organohalogens affect different aspects of the extra-thyroidal thyroid-hormone-signaling pathway but hardly affect thyroid hormone binding to receptors. Amphibian tadpoles are most sensitive to the effects of environmental chemicals during specific thyroid-hormone-related developmental windows. The thyroid hormone-disrupting activity of contaminants in aquatic environments in Japan and Thailand was surveyed by these bioassays. Our assays demonstrated that wastewater treatment effectively removed the TH-disrupting contaminants from wastewater in paper manufacturing plants and sewage-treatment plants. The Luc assay indicated that sewage-treatment plant effluents in Japan contained Ta-like activity whereas that in Thailand contained anti-Ta-like activity. It is likely that the three in vitro bioassays detected the same or different contaminants with TH-disrupting activity in a water sample-dependent manner. Our results indicate that a combination of several in vitro bioassays facilitates the rapid screening of environmental water samples to identify the locations of contaminants with TH-disrupting activity.