NR-binding activation/inhibition effects were evaluated applying a panel of quantitative Chemical Activated Luciferase gene eXpression (CALUX) gene assays developed by BDS (Amsterdam, the Netherlands [25 (link),26 (link)]. Licensed reporter cell lines were used: the Estrogen Receptor alpha (ERα) and the Androgen Receptor (AR) with the respective antagonistic version (Anti-ERα and Anti-AR) and the Aryl hydrocarbon Receptor (AhR). The tests were performed by LAB D and LAB C, adapting the OECD No. 455 [27 (link)] as well as OECD No. 458 for AR CALUX [28 (link)].
Briefly, cells were seeded in a 96-well plate (VWR, Dietikon, Switzerland) and incubated for 24 h at 37 °C, 5% CO2 and 100% humidity. After the incubation, cells were exposed in triplicates to concentration series representing a full dose–response curve of reference compounds for each test: 17β-Estradiol (E2) for ERα CALUX, Dihydrotestosterone (DHT) for AR CALUX and Benzo(a)pyrene (B(a)P) for AhR CALUX. In addition, a serial dilution of each of the different migrates were exposed in triplicates. After 24 h of exposure for cytotoxicity, ERα, AR and 4 h of exposure for poly-aromatic hydrocarbons (PAH) of exposure, the luciferase activity is measured with a luminometer plate reader (Mithras, Berthold, Germany).
Evaluation of antagonistic effect was performed at fixed concentration (EC50) of agonist in presence of serial dilutions of the antagonistic reference. For the Anti-AR CALUX, the agonist is DHT at the EC50 0.3 nM and the antagonist is Flutamide. For the Anti-ERα CALUX, the agonist is E2 at the EC50 6.0 nM and the antagonist is Tamoxifen. Blank, negative control and cell control were tested on each plate.
To discriminate between activity induction and potential cytotoxic effect, two approaches were applied: (LAB C) used an in-well multiplex method RealTime-Glo™ MT Cell Viability Assay (Promega, Dübendorf, Switzerland) [29 (link)] and (LAB D) used a cell line designed for that purpose (Cytotox CALUX) using the tributyltin acetate substance as reference [30 (link)].
Briefly, cells were seeded in a 96-well plate (VWR, Dietikon, Switzerland) and incubated for 24 h at 37 °C, 5% CO2 and 100% humidity. After the incubation, cells were exposed in triplicates to concentration series representing a full dose–response curve of reference compounds for each test: 17β-Estradiol (E2) for ERα CALUX, Dihydrotestosterone (DHT) for AR CALUX and Benzo(a)pyrene (B(a)P) for AhR CALUX. In addition, a serial dilution of each of the different migrates were exposed in triplicates. After 24 h of exposure for cytotoxicity, ERα, AR and 4 h of exposure for poly-aromatic hydrocarbons (PAH) of exposure, the luciferase activity is measured with a luminometer plate reader (Mithras, Berthold, Germany).
Evaluation of antagonistic effect was performed at fixed concentration (EC50) of agonist in presence of serial dilutions of the antagonistic reference. For the Anti-AR CALUX, the agonist is DHT at the EC50 0.3 nM and the antagonist is Flutamide. For the Anti-ERα CALUX, the agonist is E2 at the EC50 6.0 nM and the antagonist is Tamoxifen. Blank, negative control and cell control were tested on each plate.
To discriminate between activity induction and potential cytotoxic effect, two approaches were applied: (LAB C) used an in-well multiplex method RealTime-Glo™ MT Cell Viability Assay (Promega, Dübendorf, Switzerland) [29 (link)] and (LAB D) used a cell line designed for that purpose (Cytotox CALUX) using the tributyltin acetate substance as reference [30 (link)].
Free full text: Click here
