Edwards Thea M, Morgan Howard E, Balasca Coralia, Chalasani Naveen K, Yam Lauren, Roark Alison M
Department of Biology, University of the South; School of Biological Sciences, Louisiana Tech University.
School of Biological Sciences, Louisiana Tech University; School of Medicine, Louisiana State University Health Sciences Center.
J Vis Exp. 2018 Jan 1(131):55754. doi: 10.3791/55754.
The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade "Environmental Estrogens" (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ERα) and/or beta (ERβ). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-β-D-galactopyranoside (ONPG) or chlorophenol red-β-D-galactopyranoside (CPRG)) that are cleaved by β-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.
酵母雌激素筛选（YES）用于检测环境样品中的雌激素配体，并已广泛应用于内分泌干扰研究。雌激素配体包括在许多消费品中发现的天然和人造“环境雌激素”（EEs），这些消费品包括个人护理产品（PCPs）、塑料、农药和食品。环境雌激素会干扰人类和其他动物的激素信号，可能降低生育能力并增加疾病风险。尽管环境雌激素和其他内分泌干扰化学物质（EDCs）对公众健康很重要，但内分泌干扰通常不包括在本科课程中。这一缺陷部分是由于缺乏相关的实验室活动，这些活动既能够说明其中涉及的原理，又能让本科生参与。本文介绍了一种优化的YES方法，用于定量个人护理产品中与雌激素受体α（ERα）和/或β（ERβ）结合的配体。该方法采用了两种比色底物之一（邻硝基苯基-β-D-吡喃半乳糖苷（ONPG）或氯酚红-β-D-吡喃半乳糖苷（CPRG）），它们可被β-半乳糖苷酶切割，有一个6天的冷藏孵育步骤以便于在本科实验室课程中使用，有一个用于LacZ计算的自动化应用程序，以及用于相关四参数逻辑回归分析的R代码。该方案旨在让本科生能够开展并进行实验，在实验中他们可以筛选自己选择的产品中的雌激素模拟物。在此过程中，他们学习内分泌干扰、细胞培养、受体结合、酶活性、基因工程、统计学和实验设计。同时，他们还练习基本且广泛适用的实验室技能，例如：计算浓度；配制溶液；演示无菌技术；连续稀释标准品；构建和内插标准曲线；识别变量和对照；收集、整理和分析数据；构建和解释图表；以及使用常见的实验室设备，如微量移液器和分光光度计。因此，实施该测定法鼓励学生在探索环境科学与健康领域的新问题时参与基于探究的学习。
