title: Differentiating true androgen receptor inhibition from cytotoxicity-mediated reduction of reporter-gene transactivation in-vitro.
abstract: In vitro effect-based reporter assays are applied as biodetection tools designed to address nuclear receptor mediated-modulation. While such assays detect receptor modulating potential, cell viability needs to be addressed, preferably in the same well. Some assays circumvent this by co-transfecting a second constitutively-expressed marker gene or by multiplexing a cytotoxicity assay. Some assays, such as the CALUX®, lack this feature. The cytotoxic effects of unknown substances can confound in vitro assays, making the interpretation of results difficult and uncertain, particularly when assessing antagonistic activity. It's necessary to determine whether the cause of the reporter signal decrease is an antagonistic effect or a non-specific cytotoxic effect. To remedy this, we assessed the suitability of multiplexing a cell viability assay within the CALUX® transcriptional activation test for anti-androgenicity. Tests of both well-characterized anti-androgens and cytotoxic compounds demonstrated the suitability of this approach for discerning between the molecular mechanisms of action without altering the nuclear receptor assay; though some compounds were both cytotoxic and anti-androgenic. The optimized multiplexed assay was then applied to an uncharacterized set of polycyclic aromatic compounds. These results better characterized the mode of action and the classification of effects. Overall, the multiplexed protocol added value to CALUX test performance.
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland. Electronic address: maricel.marin-kuan@rdls.nestle.com.
authors.forename: Maricel
authors.initials: M
authors.lastname: Marin-Kuan
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
authors.forename: Karma C
authors.initials: KC
authors.lastname: Fussell
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Riederer
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
authors.forename: Helia
authors.initials: H
authors.lastname: Latado
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
authors.forename: Patrick
authors.initials: P
authors.lastname: Serrant
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
authors.forename: Julie
authors.initials: J
authors.lastname: Mollergues
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
authors.forename: Myriam
authors.initials: M
authors.lastname: Coulet
authors.affiliation: Chemical Food Safety, Nestlé Research Centre, P.O. Box 44, CH-1000 Lausanne 26, Switzerland.
authors.forename: Benoit
authors.initials: B
authors.lastname: Schilter
mesh_terms: D000726:Androgen Antagonists / Q000494:pharmacology; D059002:Androgen Receptor Antagonists / Q000494:pharmacology*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects*; D052244:Endocrine Disruptors / Q000494:pharmacology; D017930:Genes, Reporter / Q000187:drug effects*; D005819:Genetic Markers; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D011084:Polycyclic Aromatic Hydrocarbons / Q000633:toxicity; D011944:Receptors, Androgen / Q000187:drug effects*; D015533:Transcriptional Activation / Q000187:drug effects*
chemical_list: D000726:Androgen Antagonists; D059002:Androgen Receptor Antagonists; D052244:Endocrine Disruptors; D005819:Genetic Markers; D011084:Polycyclic Aromatic Hydrocarbons; D011944:Receptors, Androgen; D008156:Luciferases
keywords: Anti-androgen; Biodetection; CALUX® assay; Cytotoxicity; Endocrine active substance; Endocrine disruptor