Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
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.Javnost sve više zabrinjavaju brojne tvari u okolišu koje mogu utjecati na endokrini sustav kralježnjaka. Među tzv. endokrinim modulatorima, koji mogu poremetiti ili potpuno narušiti razvojne i reproduktivne procese u organizmu, najveću pozornost privlače tvari što imaju estrogenu aktivnost. Ta je zabrinutost potaknula razvoj novih metoda za njihovo praćenje i određivanje te izbjegavanje neželjenih učinaka. Testovi in vitro sa staničnim linijama sisavaca i riba postali su važni modelni sustavi u ispitivanju toksičnosti tih spojeva. Citotoksični i apoptotički učinci 17α-etinilestradiola i dietilstilbestrola ispitani su na CHO-K1 staničnoj liniji. Za određivanje preživljavanja stanica upotrebljena je metoda tripan plavo. Citotoksičnost 17α-etinilestradiola (0,34-34 µM) i dietilstilbestrola (0,37-37 µM) ovisi o koncentraciji, a izračunate IC50 vrijednosti nakon 72 sata nakon tretmana iznosile su pojedinačno 12,8 i 10,4 µM. Smrt CHO-K1 stanica praćena je na temelju uočenih morfoloških promjena, bojenjem hematoksilinom i eozinom, zatim promjena u izgledu jezgre, bojenjem fluorescein diacetatom/propidijevim jodidom i fluorescentnom mikroskopijom, te prema fragmentaciji DNA TUNEL metodom i translokaciji fosfatidilserina protočnom citometrijom. Dobiveni rezultati upućuju na to da 17α-etinilestradiol uzrokuje apoptozu u CHO-K1 stanicama, a dietilstilbestrol njihovo odumiranje procesom nekroze
