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Evidence of Cytotoxic and Apoptotic Effects of 17α-ethynylestradiol and diethylstilbestrol on CHO-K1 Cells

The rising number of endocrine disrupting chemicals (EDCs) present in the environment is a growing concern, not only for researchers but also for policy makers, because negative effects on developmental and reproductive processes are observed in wildlife and humans. Those substances of natural and man-made origin have hormonal, mainly estrogenic activity and thus interfere with endocrine system of vertebrates. The molecular and cellular effects of EDCs are best studied under well-defined in vitro conditions applying systems such as mammalian and fish cell lines. Cytotoxicity induced by 17α-ethynilestradiol (EE2) and diethylstilbestrol (DES) was studied on CHO-K1 cell line. Cell viability was determined by Trypan blue exclusion method. Cytotoxicity of EE2 and DES (1-10 µg/ml) was found to be concentration- dependent with IC50 values of 3.8 µg/ml and 2.8 µg/ml, respectively, after 72 h of exposure. Most of chemical agents that cause cell death in certain doses and times of exposure induce either apoptosis or necrosis. To determine the type of cell death in treated CHO-K1 cells hematoxilyn- eosin staining was applied to examine morphological changes. In both EE2 and DES (5 µg/ml) treated cells monolayer integrity was disrupted, rounding and reduction in number was observed and more intensively stained cells could be seen. In DES-treated cells vacuolization of cytoplasm was also visualized. Labeling of DNA strand breaks in treated cells by TUNEL assay revealed a few TUNEL-positive nuclei relative to total DAPI-stained nuclei. The effect of EE2 and DES on CHO-K1 cell survival was determined by flow cytometry using FITC labeled Annexin-V and propidium iodide. Increased number of apoptotic cells up to 9.3% was detected in CHO-K1 cell population treated with 10 µg/ml EE2 for 48h. Although CHO-K1 cells treated with DES (10 µg/ml) for 48h showed an increase in the rate of apoptotic cells (3.4%), necrosis was dominant (26%). Obtained results show that EE2 inhibits proliferation of CHO-K1 cells by inducing apoptosis, while DES predominantly induces necrosis.

CHO-K1 cell line; Cytotoxicity; Diethylstilbestrol; 17α-ethynilestradiol

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