Optimization of an MCF7-E3 Cell Proliferation Assay and Effects of Environmental Pollutants and Industrial Chemicals
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abstract
Environmental contaminants might adversely affect human health by acting as endocrine disruptors and thus need to be identified. Our objective was to optimize the MCF7 cell proliferation assay to screen industrial chemicals for potential oestrogenic effects. Growth conditions, performance of the clone E3 and WT-MCF7 cells and five methods to derive proliferation indices were compared. The E3 cells were further characterized by testing the effects of transforming growth factorbeta (TGFbeta), epidermal growth factor (EGF), insulin, testosterone, the anti-oestrogen ICI 164,384 (ICI) and environmental contaminants with known oestrogenic potential. Industrial chemicals with unknown oestrogenic effects were then tested. As expected, induction of proliferation by estradiol-17beta (E2) was greater and less variable using the clone E3. To generate proliferation indices, the alamarBlue assay had a sensitivity comparable to that of [(3)H]thymidine incorporation ((3)H-TI). The E3 cells were not responsive to EGF (0-100 ng/ml) or insulin (0-313 ng/ml) but their proliferation was decreased (P<0.05) by TGFbeta (45 ng/ml) and testosterone (10(-8)m), which might be typical of highly oestrogen-responsive MCF7 cells. ICI (5x10(-7)m) inhibited the proliferative effects of 10(-10)m E2 and that of 10(-6)m 4-tert-octylphenol (Op) but not the proliferative effect of 10(-5)m Op, suggesting displacement of ICI by Op or induction of oestrogen-receptor independent proliferation. N-oxydiethylene-2-benzothiazole sulfenamide (OBTS) altered (3)H-TI in the MCF7 cells, although not in a dose related manner. OBTS did not induce uterotrophic effects in immature female rats, or any response in a human oestrogen chimeric receptor/reporter gene assay, suggesting that its effects were not mediated through the binding of the oestrogen-receptor. Seven other industrial chemicals were tested and had no effects. In conclusion, the MCF7 cell proliferation assay is one screening tool that permits identification of chemicals with oestrogenic potential which thus require further testing.
