title: Downscaling procedures reduce chemical use in androgen receptor reporter gene assay.
abstract: Bioactivity screening studies often face sample amount limitation with respect to the need for reliable, reproducible and quantitative results. Therefore approaches that minimize sample use are needed. Low-volume exposure and chemical dilution procedures were applied in an androgen receptor reporter gene human cell line assay to evaluate environmental contaminants and androgen receptor modulators, which were the agonist 5α-dihydrotestosterone (DHT); and the antagonists flutamide, bisphenol A, 1-hydroxypyrene and triclosan. Cells were exposed in around 1/3 of the medium volume recommended by the protocol (70μL/well). Further, chemical losses during pipetting steps were minimized by applying a low-volume method for compound dilution in medium (250μL for triplicate wells) inside microvolume glass inserts. Simultaneously, compounds were evaluated following conventional procedures (200μL/well, dilution in 24-well plates) for comparison of results. Low-volume exposure tests produced DHT EC50 (3.4-3.7×10(-10)M) and flutamide IC50 (2.2-3.3×10(-7)M) values very similar to those from regular assays (3.1-4.2×10(-10) and 2.1-3.3×10(-7)M respectively) and previous studies. Also, results were within assay acceptance criteria, supporting the relevance of the downscaling setup for agonistic and antagonistic tests. The low-volume exposure was also successful in determining IC50 values for 1-hydroxypyrene (2.1-2.8×10(-6)M), bisphenol A (2.6-3.3×10(-6)M), and triclosan (1.2-1.9×10(-6)M) in agreement with values obtained through high-volume exposure (2.3-2.8, 2.5-3.4 and 1.0-1.3×10(-6)M respectively). Finally, experiments following both low-volume dosing and exposure produced flutamide and triclosan IC50 values similar to those from regular tests. The low-volume experimental procedures provide a simple and effective solution for studies that need to minimize bioassay sample use while maintaining method reliability. The downscaling methods can be applied for the evaluation of samples, fractions or chemicals which require minimal losses during the steps of pipetting, transference to medium and exposure in bioassays.
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany. Electronic address: carolina.dipaolo@bio5.rwth-aachen.de.
authors.forename: Carolina
authors.initials: C
authors.lastname: Di Paolo
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Kristina
authors.initials: K
authors.lastname: Kirchner
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Fabian Gerhard Peter
authors.initials: FG
authors.lastname: Balk
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany; UFZ-Helmholtz Centre for Environmental Research, Leipzig, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Muschket
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany; UFZ-Helmholtz Centre for Environmental Research, Leipzig, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Brack
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany; College of Resources and Environmental Science, Chongqing University, 1 Tiansheng Road, Beibei, Chongqing 400715, China; College of Environmental Science and Engineering and State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, 1239 Siping Road, Shanghai, China; State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, China.
authors.forename: Henner
authors.initials: H
authors.lastname: Hollert
authors.affiliation: Department of Ecosystem Analysis, Institute for Environmental Research, ABBt - Aachen Biology and Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Thomas-Benjamin
authors.initials: TB
authors.lastname: Seiler
mesh_terms: D059002:Androgen Receptor Antagonists / Q000494:pharmacology*; D000728:Androgens / Q000494:pharmacology*; D002460:Cell Line; D017930:Genes, Reporter / Q000187:drug effects*; D006801:Humans; D011944:Receptors, Androgen / Q000378:metabolism*
chemical_list: C410798:AR protein, human; D059002:Androgen Receptor Antagonists; D000728:Androgens; D011944:Receptors, Androgen
keywords: (Anti)androgens; Androgen receptor binders; Downscaling; Low-volume; Miniaturization; Reporter gene bioassay