Abstract
The fluorescence-based multi-analyte chip platform for the analysis of estrogenic and anti-estrogenic substances is a new in vitro tool for the high throughput screening of environmental samples. In contrast to existing tools, the chip investigates the complex action of xenoestrogens in a human cell model by characterizing protein expression. It allows for the quantification of 10 proteins secreted by MCF-7 cells, representing various biological and pathological endpoints of endocrine action and distinguishing between estrogen- and anti-estrogen-dependent secretion of proteins. Distinct protein secretion patterns of the cancer cell line after exposure to known estrogen receptor agonists ß-estradiol, bisphenol A, genistein, and nonylphenol as well as antagonists fulvestrant and tamoxifen demonstrate the potential of the chip. Stimulation of cells with Interleukin-1ß shifts concentrations of low abundant biomarkers towards the working range of the chip. In the non-stimulated cell culture, Matrix Metalloproteinase 9 (MMP-9) and Vascular Endothelial Growth Factor (VEGF) show differences upon treatment with antagonists and agonists of the estrogen receptor. In stimulated MCF-7 cells challenged with receptor agonists secretion of Monocyte Chemoattractant Protein (MCP-1), Interleukin-6 (IL-6), Rantes, and Interleukin-8 (IL-8) significantly decreases. In parallel, the proliferating effect of endocrine-disrupting substances in MCF-7 cells is assessed in a proliferation assay based on resazurin. Using ethanol as a solvent for test substances increases the background of proliferation and secretion experiments, while using dimethyl sulfoxide (DMSO) does not show any adverse effects. The role of the selected biomarkers in different physiological processes such as cell development, reproduction, cancer, and metabolic syndrome makes the chip an excellent tool for either indicating endocrine-disrupting effects in food and environmental samples, or for screening the effect of xenoestrogens on a cellular and molecular level.
Highlights
The World Health Organization defines endocrine-disrupting chemicals (EDCs) as “exogenous substances or mixtures that alter functions of the endocrine system and cause adverse health effects in an intact organism, or its progeny, or populations” [1]
In 200 μL DMEM supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acid (NEAA), 2 mM L-glutamine and pen/step for one day at 37 ◦ C, 95% humidity, and 5% CO2
The sensitivity and specificity of assays processed in buffer were compared to those transferred to the standard medium of MCF-7 cell culture, DMEM
Summary
The World Health Organization defines endocrine-disrupting chemicals (EDCs) as “exogenous substances or mixtures that alter functions of the endocrine system and cause adverse health effects in an intact organism, or its progeny, or (sub) populations” [1]. EDCs are able to inhibit or activate these receptors and to disrupt the normal endocrine function by altering circulating hormone levels. They act together with endogen hormones and show low dose, additive, and synergistic effects [7,8,9,10].
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