Establishment of a high-sensitivity reporter system in mammalian cells for detecting juvenoids using juvenile hormone receptors of Daphnia pulex
Establishment of a high-sensitivity reporter system in mammalian cells for detecting juvenoids using juvenile hormone receptors of Daphnia pulex
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利用水溞保幼激素受体在哺乳动物细胞中建立高灵敏度检测类保幼体的报告系统
DOI：
10.1002/jat.3713
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发表时间：
2018
影响因子：
3.3
通讯作者：
Miyakawa Hitoshi
中科院分区：
文献类型：
--
作者：
Tanaka Takahiro;Iguchi Taisen;Miyakawa Hitoshi
环境沃茨受到人类活动产生的各种化学物质的污染。近年来，保幼激素（JHS）对水生微甲壳动物的环境风险已被风险评估人员和研究人员所认识。JH是一种主要的节肢动物激素，调节蜕皮和繁殖，并已被用作昆虫生长调节剂的类似物。已知JH会干扰水蚤的性别决定系统，水蚤是湖沼生态系统中的关键动物，不是灭绝的目标。为了评估污染化学品的风险和保护生物多样性，检测此类化学品的可靠方法至关重要。本研究试图建立一个实用的体外报告基因检测系统，用于检测具有JH活性的化学物质。使用新构建的报告载体（从赤拟谷盗Krüppel同源物1的JH反应元件修饰，这是昆虫中主要的JH反应基因），在表达蚤状蚤JH受体的中国仓鼠卵巢细胞中发现了强的JH依赖性转录活性（>40倍激活）。对几种JH和非JH化学品进行的剂量反应分析表明，建立的报告基因测定系统对JH化学品具有严格的特异性，半数最大有效浓度（EC50）在10− 7和10− 9 m之间。这些结果表明，新系统是一种快速，经济的方法来评估JH活性化学品的环境风险。
Environmental waters are polluted by various chemicals originating from human activities. Recently, the environmental risk of juvenile hormones (JHs) to aquatic microcrustaceans has been recognized by risk assessors and researchers. JH is a major arthropod hormone that regulates molting and reproduction and has analogs that have been used as insect growth regulators. JHs are known to disturb the sex determination system ofDaphnia, which is a keystone animal in limnetic ecosystems and is not the target of extermination. To assess the risk of contaminant chemicals and to protect biodiversity, reliable methods for detecting such chemicals are essential. In this study, we attempted to establish a practical in vitro reporter assay system for detecting chemicals with JH activity. Using a newly constructed reporter vector (modified from the JH response element ofTribolium castaneumKrüppel homolog 1, which is a major JH responsive gene in insects), strong JH‐dependent transcriptional activity (>40‐fold activation) was found in Chinese hamster ovary cells that express JH receptors ofDaphnia pulex. Dose‐response analysis conducted on several JH and non‐JH chemicals revealed that the established reporter assay system has strict specificity to JH chemicals, and the half maximum effective concentration (EC50) was between 10−7and 10−9m. These results suggest that the new system is a rapid and economical method for assessing the environmental risk of JH‐active chemicals.