Division of Neurology, Department of Medicine, University of Hong Kong, Hong Kong SAR, China ; Research Centre of Heart, Brain, Hormone and Healthy Aging, University of Hong Kong, Hong Kong SAR, China.
PLoS One. 2013 Sep 6;8(9):e74065. doi: 10.1371/journal.pone.0074065. eCollection 2013.
Xenoestrogens are either natural or synthetic compounds that mimic the effects of endogenous estrogen. These compounds, such as bisphenol-A (BPA), and phthalates, are commonly found in plastic wares. Exposure to these compounds poses major risk to human health because of the potential to cause endocrine disruption. There is huge demand for a wide range of chemicals to be assessed for such potential for the sake of public health. Classical in vivo assays for endocrine disruption are comprehensive but time-consuming and require sacrifice of experimental animals. Simple preliminary in vitro screening assays can reduce the time and expense involved. We previously demonstrated that catechol-O-methyltransferase (COMT) is transcriptionally regulated by estrogen via estrogen receptor (ER). Therefore, detecting corresponding changes of COMT expression in estrogen-responsive cells may be a useful method to estimate estrogenic effects of various compounds. We developed a novel cell-based ELISA to evaluate cellular response to estrogenicity by reduction of soluble-COMT expression in ER-positive MCF-7 cells exposed to estrogenic compounds. In contrast to various existing methods that only detect bioactivity, this method elucidates direct physiological effect in a living cell in response to a compound. We validated our assay using three well-characterized estrogenic plasticizers - BPA, benzyl butyl phthalate (BBP), and di-n-butyl phthalate (DBP). Cells were exposed to either these plasticizers or 17β-estradiol (E2) in estrogen-depleted medium with or without an ER-antagonist, ICI 182,780, and COMT expression assayed. Exposure to each of these plasticizers (10(-9)-10(-7)M) dose-dependently reduced COMT expression (p<0.05), which was blocked by ICI 182,780. Reduction of COMT expression was readily detectable in cells exposed to picomolar level of E2, comparable to other in vitro assays of similar sensitivity. To satisfy the demand for in vitro assays targeting different cellular components, a cell-based COMT assay provides useful initial screening to supplement the current assessments of xenoestrogens for potential estrogenic activity.
外源性雌激素是天然或合成的化合物，可模拟内源性雌激素的作用。这些化合物，如双酚 A（BPA）和邻苯二甲酸酯，通常存在于塑料器皿中。由于这些化合物具有潜在的内分泌干扰作用，因此对人类健康构成重大风险。为了公众健康，需要对广泛的化学物质进行评估，以确定其潜在的风险。经典的体内内分泌干扰检测试验全面，但耗时且需要牺牲实验动物。简单的初步体外筛选检测可以减少时间和费用。我们之前证明儿茶酚-O-甲基转移酶（COMT）通过雌激素受体（ER）转录调控。因此，检测雌激素反应细胞中 COMT 表达的相应变化可能是评估各种化合物雌激素作用的有用方法。我们开发了一种新的基于细胞的 ELISA 方法，通过检测暴露于雌激素化合物的 ER 阳性 MCF-7 细胞中可溶性-COMT 表达的减少来评估细胞对雌激素的反应。与仅检测生物活性的各种现有方法不同，该方法阐明了化合物在活细胞中的直接生理作用。我们使用三种经过充分表征的雌激素性增塑剂 - 双酚 A（BPA）、苯佐丁基邻苯二甲酸酯（BBP）和二丁基邻苯二甲酸酯（DBP）验证了我们的检测方法。将细胞暴露于这些增塑剂或 17β-雌二醇（E2）中，在雌激素耗尽的培养基中，有或没有 ER 拮抗剂 ICI 182,780，并检测 COMT 表达。暴露于这些增塑剂（10(-9)-10(-7)M）剂量依赖性地降低 COMT 表达（p<0.05），ICI 182,780 可阻断该作用。在暴露于皮摩尔水平的 E2 的细胞中，COMT 表达的降低很容易检测到，与其他类似灵敏度的体外检测方法相当。为了满足针对不同细胞成分的体外检测的需求，基于细胞的 COMT 检测提供了有用的初步筛选，以补充对外源性雌激素潜在雌激素活性的当前评估。
