Development of an improved E-screen

The existing E-screen was developed to measure estrogenicity of environmental pollutants. The proliferative effect of estrogens on MCF7 cells as an assay end point has not been standardised for water industry testing and the cells are difficult to maintain to high passage number. A comparative study of 5 cell lines, reported as expressing estrogen receptor alpha (ESR1), was conducted to improve the E-screen. An MTT assay was used to determine that the attachment time and optimum seeding density in a 96 well plate format were 4 hours and 20000 cells/well for the cell lines ZR75-1, Ovcar, RL-95, H23, T47D and MCF7. Three replicates of the assay showed ZR75-1 (3.00%) and T47D (3.67%) to have a lower inter-assay coefficient of variation than MCF7 (12.87%) when seeded at 20 000 cells/well for 72 hours. Conversely, the intra-assay coefficient of variation for the same conditions was high for T47D (41.02%) and ZR75-1 (29.18%) but low for MCF7 (8.33%). Based on total cell number at 72 hours, RL95-2 (8.6x105 cells) had the highest proliferation, T47D (5.8X10 5 cells) and ZR75-1 (4.0x105 cells) had the lowest proliferation. Phenol Red has been reported as being estrogenic, but had no significant proliferative effect (p>0.05) when cells were cultured for 72h in 1% dextrancharcoal treated (DC) serum. Minimal growth was maintained in 1% DC serum. Western blotting confirmed the presence of the 66kDa ESR1 in ZR75-1, T47D and MCF7 but not Ovcar, H23 and RL95 cells. Quantitative RT-PCR was used to compare expression of ESR1 mRNA across the cell lines. MCF7 had the highest level (100%) followed by T47D (97.76%), ZR75 (85.75%) and Ovcar (67.15%). ESR1 mRNA was not detected in H23 and RL95 consistent with Western Blotting results. These initial data suggest that MCF7 and T47D are suitable candidates for developing an improved E-screen, although additional information regarding Estrogen Receptor beta (ESR2) and estrogenic responsiveness will also be taken into account.