Effect of 17β-estradiol and 17β-ethynilestradiol on RTG-2 fish cell line

Brojne antropogene kemikalije iz kućanstava, industrije i poljoprivredne proizvodnje ulaze u vodene sustave izravno ili putem otpadnih voda. Primjena in vitro sustava sa stanicama riba ima veliku vaţnost u određivanju toksičnih učinaka takvih tvari. Cilj rada bio je odrediti učinke 17ß-estradiola (E2) i 17α-etinilestradiola (EE2) na proliferaciju i staničnu smrt RTG-2 stanica. Stanice su tretirane s E2 (10-300 μM) i EE2 (0,1-100 μM), a citotoksični učinak određen je metodama tripan plavo, MTT i Neutral red (NR). Primjenom tripan plavo i NR metoda utvrđeno je da E2 i EE2 induciraju citotoksični učinak ovisan o koncentraciji. MTT metodom pokazan je dvostruki učinak E2 i EE2 na rast RTG-2 stanica, koji je ovisno o koncentraciji stimulativan, odnosno citotoksičan. Fluorescentnom mikroskopijom utvrđena je prisutnost pokoje apoptozne te više nekroznih stanica u uzorcima tertiranim s E2, odnosno EE2. Analizom protočnim citometrom nije dokazano da je zapažena citotoksičnost E2 i EE2 posljedica indukcije procesa apoptoze ili nekroze.Numerous anthropogenic chemicals from household, industry and agriculture enter aquatic system directly or via wastewaters. In vitro systems with fish cell lines have become of growing importance in ecotoxicology in the last decade. The aim was to determine the effects of 17ß-estradiol (E2) and 17α-ethynilestradiol (EE2) on proliferation and cell death of RTG-2 cells. Cells were treated with E2 (10-300 μM) and EE2 (0,1-100 μM) and cytotoxic effect was determined using trypan blue, MTT and Neutral red (NR) methods. E2 and EE2 induced cytotoxic effects on concentration-dependent manner when Trypan blue and NR were used. In MTT assay E2 and EE2 showed duble effect on the stimulation of RTG-2 cells which was either stimulating or cytotoxic, depending on the applied concentration. Fluorescence microcopy showed the presence of a few apoptotic and more necrotic cells in the samples treated with E2 and EE2, respectively. Flow cytometry analysis did not show that the observed cytotoxicity of E2 and EE2 is the consequence of apoptotic or necrotic processes