Cytotoxic and Apoptotic Effects of 17a-Ethynylestradiol and Diethylstilbestrol on CHO-K1 Cells

Kristina Radošević¹, Ruđer Novak², Igor Slivac¹, Mirna Mihajlović¹, Jerka Dumić², Zlatko Kniewald¹ and Višnja Gaurina Srček¹*

¹Laboratory of Cell Culture Technology and Biotransformation, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, HR-10000 Zagreb, Croatia
²Department for Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, HR-10000 Zagreb, Croatia

Article history:
Received December 2, 2010
Accepted September 23, 2011

Key words:
apoptosis, CHO-K1 cells, cytotoxicity, diethylstilbestrol, 17α-ethynylestradiol, necrosis

Summary:
There is considerable concern about the substances present in the environment and their potential to interfere with the endocrine system of vertebrates. Among these, the so-called endocrine-disrupting compounds, which can modulate or disrupt developmental and reproductive processes, substances with estrogenic activity have attracted most attention. Concerns about the presence of these compounds in the environment have led to the development of screening and testing assays that are able to detect such substances and evaluate their potential to induce adverse effects. In vitro systems such as mammalian and fish cell lines have become of growing importance in toxicity testing of such compounds. The cytotoxic and apoptotic effects induced by 17α-ethynylestradiol and diethylstilbestrol were studied on CHO-K1 cell line. Trypan blue exclusion method was used to determine the cell viability. Cytotoxicity of 17α-ethynylestradiol (0.34–34 μM) and diethylstilbestrol (0.37–37 μM) was found to be concentration-dependent with IC₅₀ values of 12.8 and 10.4 μM after 72 h of exposure, respectively. In treated CHO-K1 culture cell death was assessed by determining morphological changes by haematoxilyn and eosin staining, nuclear morphology
by fluorescein diacetate/propidium iodide staining and fluorescence microscopy, DNA fragmentation by TUNEL method and translocation of phosphatidyl serine by flow cytometry. The obtained results showed that 17α-ethynylestradiol induced apoptosis, while diethylstilbestrol induced necrosis in the treated CHO-K1 cells.

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