A novel high throughput screening assay for binding affinities of perfluoroalkyl iodide for estrogen receptor alpha and beta isoforms
A novel high throughput screening assay for binding affinities of perfluoroalkyl iodide for estrogen receptor alpha and beta isoforms
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一种新的高通量筛选方法，用于测定全氟烷基碘与雌激素受体α和β亚型的结合亲和力
DOI：
10.1016/j.talanta.2017.07.068
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发表时间：
2017-12-01
期刊：
影响因子：
6.1
通讯作者：
Jiang, Guibin
中科院分区：
文献类型：
--
作者：
Song, Wenting;Zhao, Lixia;Jiang, Guibin
随着环境污染物数量的不断增加，发展高通量筛选技术以评价其潜在的生物学效应，尤其是内分泌干扰效应，对环境中的种群动态产生直接影响。建立了一种新的基于酶片段互补技术的竞争结合试验，以筛选新兴化学品与雌激素受体（ER）α或β亚型的结合亲和力。外源化合物可以与基因工程β-半乳糖苷酶（β-gal）的片段（ED-ES）竞争结合ER α或β，从而定量地改变酶活性β-gal的形成和发光底物的水解。通过对荧光曲线的监测和对ER α或ER β浓度的优化，发现荧光信号可持续发射9 h，体系中40 nM的ER α或ER β将导致最灵敏的发光响应。以17 β-雌二醇（E-2）和染料木黄酮为代表性雌激素，评价了它们与ER α和ER β的结合亲和力。与传统方法测定结果一致，证实了基于β-gal的竞争结合试验的可靠性。筛选了四种在碘取代和碳链长度方面具有特定结构特征的多氟碘烷（PFIs），结果表明这些化学品对ER α或β亚型具有不同的结合亲和力和不同的偏好性。PFI与ER α的结合亲和力与MVLN转录报告基因测定的结果一致。总体而言，本研究中提出的竞争性结合试验为高通量筛选具有雌激素效应的新兴化学品提供了一种有前途的替代方法，这对于解释其潜在的毒理学效应和人类暴露风险非常重要。
Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) alpha or beta isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered beta-galactosidase enzyme (beta-gal) for the binding to ER alpha or beta, thus quantitatively altering the formation of enzymatically active beta-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ER alpha or beta concentrations, it was found that luminescent signals were sustainably emitted for 9 h, and 40 nM ER alpha or beta in the system would lead to the most sensitive luminescence response. Using 17 beta-estrodiol (E-2) and genistein as the representative estrogenic hormones, their binding affinities for ER alpha and beta were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on beta-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ER alpha or beta isoforms. The binding affinities of PFIs for ER alpha were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure risks.