Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

作者：Edwards Thea M; Morgan Howard E; Balasca Coralia; Cha****ni Naveen K; Yam Lauren; Roark Alison M^*

来源：Jove-Journal of Visualized Experiments, 2018, (131): e55754.

DOI：10.3791/55754

• Yeast estrogen screen
• undergraduate
• endocrine disruption
• estrogenic
• personal care products
• environmental estrogens
• ESR1
• ESR2
• estrogen receptor
• ortho-nitrophenyl-beta-D-galactopyranoside
• chlorophenol red-beta-D-galactopyranoside

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 (ERa) and/or beta (ER beta). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) or chlorophenol red-beta-D-galactopyranoside (CPRG)) that are cleaved by beta-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.

• 出版日期2018-1