2.5. Stably Transfected Transactivation (STTA) Assay for Estrogen Receptor Alpha

For the estrogen receptor agonist and antagonist tests, the ERa transactivation assay was performed based on the OECD TG 455 using cultured HeLa 9903 cells, which stably express the ERα gene containing a firefly luciferase gene as the reporter gene [31]. After subculturing the cells to a density of 75~90% in a 100 mm culture dish, the cells were transferred to a 96-well plate at 1 × 10⁴ cells/100 μL/well, stabilized for 3 h, and then treated with the test chemicals for 24 h. After exposure, the luciferase activity was measured using a commercial luciferase assay reagent (E2510, Promega, Maidison, WI, USA). For the ERα transcription agonist assay, the normalized mean value of the luminescence signal (Luciferase activity) of the test wells was divided by that of the positive chemical (1 nM 17β-estradiol) to obtain relative transcriptional activity. The concentration–response curves of the test chemicals were processed to calculate the PC₁₀ and PC₅₀ values, which show 10% and 50% of the transcriptional activity of positive chemicals, respectively, using software provided by the OECD Test guideline. 17β-estradiol, 17α-estradiol, 17α-methyltestosterone, and corticosterone were used as the reference chemicals for the ERα transactivation assay to monitor the stability of the assay system. For the antagonist assay, test wells were spiked with 25 pM 17β-estradiol. Tamoxifen and flutamide were used as reference chemicals. The normalized mean value of the luminescence signal (Luciferase activity) of the test wells was divided by that of spiked 25 pM 17β-estradiol (100%) to obtain relative transcriptional activity. RPC_Max for the agonist and IC₃₀ values for the antagonist were used for the positive and negative decision criteria, according to the OECD TG 455. The calculations of PC₁₀, PC_50, and RPC_Max in ERα agonist assay and IC₃₀ and IC₅₀ in ERα antagonist assay can be made by using the spreadsheet available together with the Test Guideline on the OECD public website [32,33].