Development of a highly sensitive reporter gene cell line for detecting estrogenic activity (the ER Isjaki assay)

Monitoring of estrogens in water sources faces significant challenges, as proposed changes in the European Union regulation for environmental protection of water bodies, compromise the ability of conventional analytical methods to detect low concentrations of estrogens. The proposed changes involve the decrease of the environmental quality standards for 17β-estradiol, estrone and 17α-ethinyl estradiol in surface waters and the obligation to monitor estrogenic substances in water bodies, using effect-based methods. In this study, the optimal experimental conditions for developing a novel and highly sensitive reporter gene assay were established. For this purpose, optimization of transfection plasmid concentration, exposure time and basement membrane matrix effect as well as assessment of assay reproducibility and relative effect potency of natural estrogens and estrogenic substances were conducted. With the optimal experimental conditions set as 5 ng per well in 96-well uncoated plates for plasmid transfection and 24 h exposure to treatments, the assay yielded an average sensitivity, measured as effect level 20 % for 17β-estradiol, estrone and 17α-ethinyl estradiol of 0.29, 1.36 and 0.02 pM, respectively. The assay showed a reproducibility variation of approximately 20 % and was able to differentiate the relative effect potency between 17α-ethinyl estradiol and 17β-estradiol with the capacity of detecting 17α-ethinyl estradiol with a high relative effect potency. Moreover, this assay is approximately 10-100 times more sensitive compared to the current state-of-the-art in vitro assays used to measure estrogenicity, indicating that the assay can be used to detect 17β-estradiol, estrone and 17α-ethinyl estradiol at the low levels needed to meet regulatory standards.