Effect of diethylstilbestrol on growth and morphology of CHO-K1 cell line

Kultura životinjskih stanica nezaobilazan je dio biotehnologije i biofarmaceutike, te srodnih znanstvenih područja kao što su molekularna biologija i toksikologija. Alternativni in vitro testovi kao predtestovi ispitivanjima na pokusnim životinjama pogodni su za temeljna znanstvena istraživanja kojima je svrha utvrditi molekularne i stanične mehanizme toksičnog djelovanja ispitivane kemikalije. Posljednjih se nekoliko godina intenzivno provode istraživanja utjecaja endokrinih modulatora, kemijskih spojeva koji oponašaju prirodne hormone i ometaju njihovo djelovanje u organizmu te tako dovode i do patoloških promjena. Sintetski estrogeni, iz raznih farmaceutskih pripravaka, dospijevaju u okoliš i time predstavljaju problem za zdravlje ljudi i životinjskog svijeta. Dietilstilbestrol (DES) je sintetski estrogen, koji ima slično djelovanje i estrogene karakteristike kao prirodni hormon 17ß-estradiol. Cilj ovog rada je utvrditi učinak DEA-a na rast i morfologiju CHO-K1 stanične linije. Stanice su tretirane DES-om u koncentracijama od 1, 5 i 10 µg/mL i utvrđen je citotoksičan učinak ovisan o koncentraciji i trajanju tretmana. Najjaču inhibiciju 72 h nakon tretmana, izazvala je koncentracija od 10 µg/mL DES-a i to gotovo za 95%. Ispitano je i djelovanje bazičnog fibroblastnog faktora rasta (bFGF) koji pozitivno utječe na rast CHO-K1 stanica. Unatoč pokazanom proliferativnom učinku, dodatak bFGF-a nije smanjio citotoksično djelovanje DES-a na CHO-K1 stanice. Uklanjanje medija s DES-om te dodatak svježeg medija potaknuo je revitalizaciju tretiranih stanica te su one postigle nivo rasta kao i kontrolne, netretirane stanice. Svjetlosnom mikroskopijom te bojanjem stanica kristal-ljubičastom bojom i hematoksilin-eozinom vidljive su promjene u nastalom monosloju te morfološke promjene kod stanica izloženih djelovanju 5 µg/mL DES-a. Također je primijećena pojava vakuolizacije citoplazme te kondenzacije jezgre što upućuje na početnu fazu procesa apoptoze.Animal cell culture is inevitable part of biotechnology and biopharmacy as well as related scientific fields such as molecular biology and toxicology. Alternative in vitro tests as preliminary tests to experiments on laboratory animals are suitable for basic scientific researches whose purpose is to determine molecular and cellular mechanisms of toxic effects of the analyzed chemicals. Over recent years there has been intensive research of the influence of endocrine modulators, chemical substances that imitate natural hormones and influence their effects in organism causing pathological changes. Synthetic estrogens, from different pharmaceuticals are reaching environment representing health problems for both human and animal life. Diethylstilbestrol (DES) is synthetic estrogen which has very similar effects and estrogen characteristics as natural hormone 17ß-estradiol. The objective of this study was to determine the effect of DES on growth and morphology of CHO-K1 cell line. Cells are treated with 1, 5 and 10 µg/mL of DES and cytotoxic effect has been detected on time- and concentration- dependent manner. The highest inhibition of 95% compared to control was detected when 10 µg/mL DES was used during 72 h. The influence of the basic fibroblast growth factor (bFGF) has been analyzed as well, showing positive effect on growth of CHO-K1 cells. Despite showed proliferative activity, addition of bFGF did not diminish DES cytotoxic effect on CHO-K1 cells. Removal of medium containing DES and addition of fresh medium has stimulated treated cells revitalization allowing them to achieve level of growth of control cells. Using light microscopy and crystal violet cell staining as well as hematoxylin-eosin staining method it is possible to notice visible changes in the appearing monolayer as well as morphological changes in cells that had been exposed to 5 µg/mL DES. The appearance of cytoplasmic vacuolization has also been noticed as well as nucleus condensation which refers to initial phase of apoptosis