A robotic MCF-7:WS8 cell proliferation assay to detect agonist and antagonist estrogenic activity.
A robotic MCF-7:WS8 cell proliferation assay to detect agonist and antagonist estrogenic activity.
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DOI：
10.1093/toxsci/kft250
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发表时间：
2014-02
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影响因子：
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通讯作者：
Chun Z. Yang;W. Casey;M. Stoner;G. Kollessery;A. W. Wong;G. Bittner
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作者：
Chun Z. Yang;W. Casey;M. Stoner;G. Kollessery;A. W. Wong;G. Bittner
具有雌激素活性（EA）或抗雌激素活性（AEA）的内分泌干扰化学物质已被广泛报道可能对健康产生许多不利影响。我们利用MCF-7:WS8细胞增殖（或抑制）开发了自动化检测方法，以检测78种测试物质的EA（或AEA），这些物质由替代方法验证机构间协调委员会和国家毒理学计划的替代毒理学方法评估机构间中心提供，用于验证研究。我们还检测了ICI 182780，一种强雌激素拮抗剂。要测定的化学物质首先检查溶解度和挥发性，以确定最佳测定条件。对于EA和AEA的测定，采用测距法测定检测浓度范围，然后进行综合测定。在EA综合试验中获得潜在阳性结果的测试物质进行EA确认试验，评估ICI 182780逆转化学诱导的MCF-7细胞增殖的能力。相对于ICI或雷洛昔芬（也是一种强雌激素拮抗剂），AEA试验检测了化学物质降低17β-雌二醇（E2）不饱和浓度诱导的MCF-7细胞增殖的能力。要被归类为具有AEA，饱和浓度的E2必须显著逆转非饱和E2中被试物质产生的细胞增殖下降。我们的结论是，我们的自动化MCF-7 EA和AEA分析具有至少等同于美国环境保护署和经济合作与发展组织认可的验证测试方法的准确性，灵敏度和特异性值。
Endocrine-disrupting chemicals with estrogenic activity (EA) or anti-EA (AEA) have been extensively reported to possibly have many adverse health effects. We have developed robotized assays using MCF-7:WS8 cell proliferation (or suppression) to detect EA (or AEA) of 78 test substances supplied by the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Toxicology Program's Interagency Center for the Evaluation of Alternative Toxicological Methods for validation studies. We also assayed ICI 182,780, a strong estrogen antagonist. Chemicals to be assayed were initially examined for solubility and volatility to determine optimal assay conditions. For both EA and AEA determinations, a Range-Finder assay was conducted to determine the concentration range for testing, followed by a Comprehensive assay. Test substances with potentially positive results from an EA Comprehensive assay were subjected to an EA Confirmation assay that evaluated the ability of ICI 182,780 to reverse chemically induced MCF-7 cell proliferation. The AEA assays examined the ability of chemicals to decrease MCF-7 cell proliferation induced by nonsaturating concentrations of 17β-estradiol (E2), relative to ICI or raloxifene, also a strong estrogen antagonist. To be classified as having AEA, a saturating concentration of E2 had to significantly reverse the decrease in cell proliferation produced by the test substance in nonsaturating E2. We conclude that our robotized MCF-7 EA and AEA assays have accuracy, sensitivity, and specificity values at least equivalent to validated test methods accepted by the U.S. Environmental Protection Agency and the Organisation for Economic Co-operation and Development.