Sugawara Teruo, Nakajima Ayako, Nomura Eiji
Department of Biochemistry, Hokkaido University School of Medicine, Sapporo, Hokkaido, Japan.
Med Sci Monit. 2002 Nov;8(11):BR431-8.
An endocrine disruptor is a synthetic chemical, which causes adverse effects in an organism, or its progeny, after causing perturbations in the endocrine system. It is important to know which synthetic chemicals have endocrine-disrupting action. However, an increasing number of synthetic chemicals are being produced by modern synthetic chemistry, and the examination of endocrine disruptor potential has not yet caught up with the advances in synthetic chemistry. In this study, we have developed such a screening system for detecting synthetic chemicals with estrogen-like effects.
MATERIAL/METHODS: The system was based on the yeast one-hybrid system. Both HIS3 and lacZ reporter genes connected to three tandem copies of the estrogen response element were prepared. Gal4-estrogen receptor is a fusion protein made from the activation domain (AD) of the yeast GAL4 transactivator gene and then incorporated into a plasmid, which was transfected into the YM4271 yeast cell strain. The estrogen effect was judged by this developed screening system.
A dual reporter assay-system was established by transfection of the both HIS3 and lacZ reporter genes into the yeast cells. This screening system enabled the detection of as little as 10-12 mol of beta-estradiol.
These results show that this newly developed dual assay is useful for the screening of endocrine-disruptors that have estrogen-like action.
内分泌干扰物是一种合成化学物质，它在扰乱内分泌系统后会对生物体或其后代产生不良影响。了解哪些合成化学物质具有内分泌干扰作用非常重要。然而，现代合成化学产生的合成化学物质数量不断增加，而内分泌干扰物潜力的检测尚未跟上合成化学的发展。在本研究中，我们开发了一种用于检测具有雌激素样作用的合成化学物质的筛选系统。
材料/方法：该系统基于酵母单杂交系统。制备了连接到三个串联雌激素反应元件拷贝的HIS3和lacZ报告基因。Gal4-雌激素受体是一种由酵母GAL4反式激活基因的激活域（AD）制成的融合蛋白，然后整合到质粒中，该质粒被转染到YM4271酵母细胞株中。通过这种开发的筛选系统判断雌激素效应。
通过将HIS3和lacZ报告基因转染到酵母细胞中建立了双报告基因检测系统。这种筛选系统能够检测低至10-12摩尔的β-雌二醇。
这些结果表明，这种新开发的双检测方法可用于筛选具有雌激素样作用的内分泌干扰物。
