Ma Xiaoju, Yang Jing, Jia Hong, Li Xiaohua, Wang Dawei, Fu Hongxia, Yuan Jie, Li Yun, Zheng Guangmei, Huang Xiaoming
Department of Nutrition , School of Public Health , Chengdu University of T.C.M. , Chengdu , Sichuan , P.R. China 611137.
Department of Infectious Disease , Renmin Hospital , Hubei University of Medicine , Shiyan , Hubei , P.R. China 442000.
Toxicol Res (Camb). 2019 Mar 21;8(4):522-530. doi: 10.1039/c9tx00008a. eCollection 2019 Jul 1.
Recent reports have concentrated on some androgens/antiandrogens and confirmed that certain chemicals have demonstrated androgenic/antiandrogenic activities . However, it is still unknown whether more chemicals in the human environment possess endocrine toxicity. 58A novel AR-mediated reporter gene assay based on LLC-MK2 cells was established by transiently co-transfecting with pARE-sv40-Luc, hAR-pcDNA3.1 and pRL-tk. pARE-sv40-Luc was constructed using a pGL3-promoter plasmid with three repeated androgen responsive elements. hAR-pcDNA3.1 was constructed using pcDNA3.1 with a hAR sequence. After transfection for 12 h, the culture medium was exposed to various concentrations of dihydrotestosterone (DHT) and other test chemicals (phthalic acid esters and dexamethasone) in order to measure the androgenic/antiandrogenic activity. The assay possessed a concentration-dependent response to DHT from 10 M to 10 M. Nilutamide concentrations of over 10 M completely blocked the luciferase expression induced by 10 M DHT. Other data showed that DBP, DEHP and MEHP possessed weak androgenic activity for certain concentration ranges, while DMP, DINP and DIBP did not show any androgenic activity. Moreover, five PAEs (DBP, DEHP, DINP, DIBP and MEHP) showed corresponding antiandrogenic activities for certain concentrations with an approximate tendency (MEHP > DBP > DEHP > DIBP > DINP). The assay is high-throughput, specific, and sensitive for the detection of androgenic/antiandrogenic chemicals. In addition, PAEs (especially transitional PAEs) exhibited corresponding androgenic/antiandrogenic activities for certain concentration ranges.
最近的报告集中在一些雄激素/抗雄激素上，并证实某些化学物质已表现出雄激素/抗雄激素活性。然而，人类环境中的更多化学物质是否具有内分泌毒性仍不清楚。通过将pARE-sv40-Luc、hAR-pcDNA3.1和pRL-tk瞬时共转染，建立了一种基于LLC-MK2细胞的新型雄激素受体（AR）介导的报告基因检测方法。pARE-sv40-Luc是使用带有三个重复雄激素反应元件的pGL3-启动子质粒构建的。hAR-pcDNA3.1是使用带有hAR序列的pcDNA3.1构建的。转染12小时后，将培养基暴露于不同浓度的二氢睾酮（DHT）和其他测试化学物质（邻苯二甲酸酯和地塞米松）中，以测量雄激素/抗雄激素活性。该检测方法对10⁻⁹ M至10⁻⁶ M的DHT具有浓度依赖性反应。尼鲁米特浓度超过10⁻⁶ M完全阻断了10⁻⁸ M DHT诱导的荧光素酶表达。其他数据表明，邻苯二甲酸二丁酯（DBP）、邻苯二甲酸二（2-乙基己基）酯（DEHP）和邻苯二甲酸单（2-乙基己基）酯（MEHP）在某些浓度范围内具有弱雄激素活性，而邻苯二甲酸二甲酯（DMP）、邻苯二甲酸二异壬酯（DINP）和邻苯二甲酸二异丁酯（DIBP）未表现出任何雄激素活性。此外，五种邻苯二甲酸酯（DBP、DEHP、DINP、DIBP和MEHP）在某些浓度下表现出相应的抗雄激素活性，具有近似的趋势（MEHP > DBP > DEHP > DIBP > DINP）。该检测方法对雄激素/抗雄激素化学物质的检测具有高通量、特异性和敏感性。此外，邻苯二甲酸酯（尤其是过渡性邻苯二甲酸酯）在某些浓度范围内表现出相应的雄激素/抗雄激素活性。
