Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Toxicol Sci. 2014 May;139(1):198-209. doi: 10.1093/toxsci/kfu023. Epub 2014 Feb 4.
Endocrine disrupting chemicals (EDCs) interfere with the biosynthesis, metabolism, and functions of steroid hormones, including estrogens and androgens. Aromatase enzyme converts androgen to estrogen. Thus, EDCs against aromatase significantly impact estrogen- and/or androgen-dependent functions, including the development of breast cancer. The current study aimed to develop a biologically relevant cell-based high-throughput screening assay to identify EDCs that act as aromatase inhibitors (AIs), estrogen receptor (ER) agonists, and/or ER antagonists. The AroER tri-screen assay was developed by stable transfection of ER-positive, aromatase-expressing MCF-7 breast cancer cells with an estrogen responsive element (ERE) driven luciferase reporter plasmid. The AroER tri-screen can identify: estrogenic EDCs, which increase luciferase signal without 17β-estradiol (E2); anti-estrogenic EDCs, which inhibit the E2-induced luciferase signal; and AI-like EDCs, which suppress a testosterone-induced luciferase signal. The assay was first optimized in a 96-well plate format and then miniaturized into a 1536-well plate format. The AroER tri-screen was demonstrated to be suitable for high-throughput screening in the 1536-well plate format, with a 6.9-fold signal-to-background ratio, a 5.4% coefficient of variation, and a screening window coefficient (Z-factor) of 0.78. The assay suggested that bisphenol A (BPA) functions mainly as an ER agonist. Results from screening the 446 drugs in the National Institutes of Health Clinical Collection revealed 106 compounds that modulated ER and/or aromatase activities. Among these, two AIs (bifonazole and oxiconazole) and one ER agonist (paroxetine) were confirmed through alternative aromatase and ER activity assays. These findings indicate that AroER tri-screen is a useful high-throughput screening system for identifying ER ligands and aromatase-inhibiting chemicals.
内分泌干扰化学物质 (EDCs) 干扰甾体激素(包括雌激素和雄激素)的生物合成、代谢和功能。芳香酶将雄激素转化为雌激素。因此,针对芳香酶的 EDC 会显著影响雌激素和/或雄激素依赖性功能,包括乳腺癌的发展。本研究旨在开发一种具有生物学相关性的基于细胞的高通量筛选测定法,以鉴定作为芳香酶抑制剂 (AIs)、雌激素受体 (ER) 激动剂和/或 ER 拮抗剂的 EDC。AroER 三联筛选测定法通过将具有雌激素反应元件 (ERE) 驱动的荧光素酶报告质粒的 ER 阳性、芳香酶表达 MCF-7 乳腺癌细胞进行稳定转染而开发。AroER 三联筛选可以鉴定:增加荧光素酶信号而无需 17β-雌二醇 (E2) 的雌激素性 EDC;抑制 E2 诱导的荧光素酶信号的抗雌激素性 EDC;以及类似 AI 的 EDC,其抑制睾酮诱导的荧光素酶信号。该测定法首先在 96 孔板格式中进行优化,然后小型化为 1536 孔板格式。证明 AroER 三联筛选适用于 1536 孔板格式的高通量筛选,具有 6.9 倍的信号与背景比、5.4%的变异系数和 0.78 的筛选窗口系数 (Z 因子)。该测定法表明双酚 A (BPA) 主要作为 ER 激动剂发挥作用。对国立卫生研究院临床收藏中的 446 种药物进行筛选的结果表明,有 106 种化合物调节了 ER 和/或芳香酶活性。其中,两种 AI(比佛拉唑和奥昔康唑)和一种 ER 激动剂(帕罗西汀)通过替代芳香酶和 ER 活性测定得到证实。这些发现表明,AroER 三联筛选是一种用于鉴定 ER 配体和芳香酶抑制性化学物质的有用高通量筛选系统。