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    Keywords: OPTIMIZATION ; RECEPTOR ; CELLS ; IN-VITRO ; CELL ; COMBINATION ; VITRO ; GENE ; GENES ; BIOLOGY ; ASSAY ; CELL-LINE ; LINE ; NETHERLANDS ; RECEPTORS ; ER ; signaling ; SCIENCE ; methods ; ANDROGEN RECEPTOR ; CHEMICALS ; ANTAGONISTS ; ANDROGEN ; androgens ; in vitro ; reporter gene assay ; RANGE ; ANDROGEN-RECEPTOR ; PANEL ; Prevalidation ; ENDOCRINE DISRUPTORS ; REPORTER ; Hershberger assay ; Androgen reporter gene assay ; BIOASSAYS ; HUMAN CELL-LINE
    Abstract: To date there are no validated methods available to test androgenicity or antiandrogenicity in vitro. A problem with testing androgenicity using reporter genes is the possibility by other steroid receptors than androgen receptors to activate the same reporter gene, thereby lowering selectivity. To avoid this we have established a robust and very selective method, the AR CALUX reporter gene assay, to test androgenic and antiandrogenic activity of compounds in vitro. This assay uses a human U2-OS cell line stably transfected with the human androgen receptor and an androgen receptor responsive reporter gene. We optimized protocols to be used in combination with AR CALUX cells and carried out an in house prevalidation. In addition we successfully transferred this assay to another laboratory, leading to comparable test results with a panel of androgen receptor agonists and antagonists. The assay was able to readily rank a range of chemicals on the basis of their EC50 values. The CALUX assay was found to be selective for androgens and seemed not influenced by signaling through other steroid receptors.
    Type of Publication: Journal article published
    PubMed ID: 20438827
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  • 2
    Keywords: RECEPTOR ; CELLS ; IN-VITRO ; CELL ; Germany ; MODEL ; MODELS ; VITRO ; screening ; TOOL ; GENE ; validation ; REDUCTION ; DOMAIN ; BIOLOGY ; hormone ; ASSAY ; CELL-LINE ; LINE ; PREDICTION ; RECEPTORS ; PROJECT ; AFFINITY ; ANTAGONIST ; ESTROGEN-RECEPTOR ; CYTOTOXICITY ; FRAMEWORK ; SCIENCE ; development ; ESTROGEN ; estrogen receptor ; CHEMICALS ; in vitro ; reporter gene assay ; RANGE ; ReProTect ; Agonist ; Estrogen activity ; hER transactivation assay ; MELN cell line ; TRANSACTIVATION ASSAY
    Abstract: The need for development and validation of in vitro hormone receptor transactivation assays as important alternative tools to study interactions with sex hormone receptors is outlined by international organisations, as such assays should be included in the OECD conceptual framework for the testing and assessment of endocrine active chemicals. Therefore as part of the European Union (EU)-sponsored 6th framework project ReProTect, the validation study with MELN cells, MCF-7 cells (ER+, estrogen receptor positive) which were stably transfected with the estrogen responsive gene ERE-beta Glob-Luc-SVNeo was set up. Standard operating procedures including a prescreen assay for unknown chemicals, an ER-agonist assay and an ER-antagonist assay were developed at the Flemish Institute for Technological Research, Belgium, and successfully transferred to Bayer Schering Pharma AG, Germany. Test results were obtained for 16 chemicals, and it was demonstrated that the MELN assay is transferable, robust and reproducible which allowed to rank chemical compounds according to their strong to weak affinity for the estrogen-alpha receptor, or identify negative chemicals within the test range up to 10(-5) M. Besides the screening for agonism, we demonstrated the suitability of MELN cells to test for antagonistic activity, which is of added value compared to current validated assays. As the MELN assay successfully passed the first modules of the ECVAM validation procedure, it now should be considered for further steps including the definition of a prediction model and application domain to get it accepted as an alternative screening assay, contributing to the 3R's with a reduction of animal experiments. (C) 2010 Elsevier Inc. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 20362049
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