Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
Aquat Toxicol. 2013 Apr 15;130-131:31-40. doi: 10.1016/j.aquatox.2012.12.011. Epub 2013 Jan 3.
In this paper, the non-target effects of tebufenozide were evaluated on the estuarine crustacean, the opposum shrimp Neomysis integer (Leach, 1814). Tebufenozide is a synthetic non-steroidal ecdysone agonist insecticide and regarded as potential endocrine-disrupting chemical (EDC). N. integer is the most used crustacean in ecotoxicological research in parallel to Daphnia sp. and has been proposed for the regulatory testing of potential EDCs in the US, Europe and Japan. Major results were: (i) cDNAs encoding the ecdysteroid receptor (EcR) and the retinoid-X-receptor (RXR), were cloned and sequenced, and subsequent molecular phylogenetic analysis (maximum likelihood and neighbor-joining) revealed that the amino acid sequence of the ligand binding domain (LBD) of N. integer EcR (NiEcR) clusters as an outgroup of the Crustacea, while NiRXR-LBD clusters in the Malacostracan clade (bootstrap percentage=75%). (ii) 3D-modeling of ligand binding to NiEcR-LBD demonstrated an incompatibility of the insecticide tebufenozide to fit into the NiEcR-ligand binding pocket. This was in great contrast to ponasterone A (PonA) that is the natural molting hormone in Crustacea and for which efficient docking was demonstrated. In addition, the heterodimerization of NiEcR-LBD with the common shrimp Crangon crangon (Linnaeus, 1758) RXR-LBD (CrcRXR-LBD) was also modeled in silico. (iii) With use of insect Hi5 cells, chimeric constructs of NiEcR-LBD and CrcRXR-LBD fused to either the yeast Gal4-DNA binding domain (DBD) or Gal4-activation domain (AD) were cloned into expression plasmids and co-transfected with a Gal4 reporter to quantify the protein-protein interactions of NiEcR-LBD with CrcRXR-LBD. Investigation of the ligand effect of PonA and tebufenozide revealed that only the presence of PonA could induce dimerization of this heterologous receptor complex. (iv) Finally, in an in vivo toxicity assay, N. integer juveniles were exposed to tebufenozide at a concentration of 100 μg/L, and no effects against the molting process and nymphal development were scored. In conclusion, the in vitro cell reporter assay, based on NiEcR-LBD/CrcRXR-LBD heterodimerization in Hi5 cells and validated with the natural ecdysteroid hormone PonA, represents a useful tool for the screening of putative EDCs. As a test example for non-steroidal ecdysone agonist insecticides, tebufenozide had no negative effects on NiEcR/RXR receptor dimerization in vitro, nor on the molting process and nymphal development of N. integer at the tested concentration (100 μg/L) in vivo.
本文评估了噻嗪酮对港湾甲壳动物鲎虾（Neomysis integer（Leach，1814））的非靶标效应。噻嗪酮是一种合成的非甾体蜕皮激素激动剂杀虫剂，被认为是一种潜在的内分泌干扰化学物质（EDC）。鲎虾是生态毒理学研究中最常用的甲壳类动物，与水蚤属（Daphnia sp.）一起被用于评估潜在的 EDC，已被提议在美国、欧洲和日本进行监管测试。主要结果如下：（i）克隆并测序了编码蜕皮激素受体（EcR）和视黄酸受体（RXR）的 cDNA，并进行了后续的分子系统发育分析（最大似然法和邻接法），结果表明鲎虾 EcR 的配体结合域（LBD）的氨基酸序列（NiEcR）作为甲壳类动物的外群聚类，而 NiRXR-LBD 则在软甲亚纲（Malacostraca）分支中聚类（bootstrap 百分比=75%）。（ii）用噻嗪酮对 NiEcR-LBD 进行配体结合的 3D 建模表明，该杀虫剂与 NiEcR 配体结合口袋不兼容。这与蜕皮激素 PonA 形成了鲜明对比， PonA 是甲壳类动物的天然蜕皮激素，已证明其与 NiEcR 结合的效率很高。此外，还在计算机上模拟了 NiEcR-LBD 与普通虾 Crangon crangon（Linnaeus，1758）RXR-LBD（CrcRXR-LBD）的异源二聚化。（iii）利用昆虫 Hi5 细胞，将 NiEcR-LBD 和 CrcRXR-LBD 的嵌合构建体与酵母 Gal4-DNA 结合域（DBD）或 Gal4 激活域（AD）融合，克隆到表达质粒中，并与 Gal4 报告基因共转染，以定量分析 NiEcR-LBD 与 CrcRXR-LBD 的蛋白-蛋白相互作用。对 PonA 和噻嗪酮的配体效应的研究表明，只有 PonA 的存在才能诱导这种异源受体复合物的二聚化。（iv）最后，在体内毒性试验中，将鲎虾幼体暴露于浓度为 100 μg/L 的噻嗪酮中，未观察到蜕皮过程和若虫发育受到影响。综上所述，基于 NiEcR-LBD/CrcRXR-LBD 异源二聚体在 Hi5 细胞中的细胞报告测定，并用天然蜕皮激素 PonA 进行验证，该测定是筛选潜在 EDC 的有用工具。作为非甾体类蜕皮激素激动剂杀虫剂的测试实例，噻嗪酮在体外对 NiEcR/RXR 受体二聚化、体内 100μg/L 浓度下鲎虾的蜕皮过程和若虫发育均无负面影响。
