Validation of an estrogen receptor dimerization(α-α/α-β/β-β) BRET-based biosensors for screening estrogenic endocrine-disrupting chemicals

Abstract

We validated the reproducibility and accuracy of a previously developed assay for screening endocrine-disrupting chemicals (EDCs) based on estrogen receptor (ER) dimerization (α-α/α-β/β-β), following OECD GD34 guidelines, to assess its applicability across various laboratories. The inter- and intra-laboratory accuracy was evaluated using 22 validation substances (ICCVAM-recommended substances for validating in vitro ER-binding assays) with confirmed estrogenic activity in four independent laboratories. Intra-laboratory reproducibility for 22 chemicals was at least 95.5 % for ER α-α and β-β and 100 % for ER α-β, with mean values of 98.9 % (ER α-α), 100 % (ER α-β), and 98.9 % (ER β-β), respectively. The inter-laboratory qualitative reproducibility for ER α-α, ER α-β, and ER β-β was 100 %, 100 %, and 95.5 %, respectively. The validated results for the ER dimerization (α-α/α-β/β-β) assays were compared with the results (17 test chemicals) from the National Toxicology Program Interagency Center for Evaluation of Alternative Toxicological Methods (NICEATM), confirming the validity of the assay. The accuracies for ER α-α, α-β, β-β dimerization in cell were 88.2 %, 94.1 %, 88.2 %, respectively. Thus, ER dimerization assays demonstrated high intra- and inter-laboratory reproducibility and accuracy through this validation study. This suggests that the assay is a robust method for detecting ER dimerization within cells.