20.500.12556/RUL-117790 Vpliv izbranih inhibitorjev tirozinskih kinaz na genomsko stabilnost ribjih jetrnih celic ZFL in vitro The influence of selected tyrosine kinase inhibitors on genomic stability of zebrafish liver cells ZFL in vitro Tirozin kinazni inhibitorji (TKI-ji) so majhne molekule za tarčno zdravljenje rakavih celic. Po zaužitju se TKI-ji izločijo preko fekalij in urina v vodno okolje ter imajo morebitne škodljive učinke na ne-tarčne organizme, ki v takem okolju živijo. V magistrski nalogi smo raziskovali citotoksično in genotoksično delovanje šestih TKI-jev: erlotiniba (ERL), sorafeniba (SOR), regorafeniba (REG), dasatiniba (DAS), imatinib mezilata (IM) in nilotiniba (NIL) na in vitro modelu jetrnih celic rib cebric (Danio rerio; ZFL). Citotoksičnost smo raziskovali z barvanjem celic s propidijevim jodidom in pretočno citometrijo, vpliv na celični cikel z barvanjem s Hoechst 33342 in pretočno citometrijo, vpliv na genomsko stabilnost pa z dvema različicama testa mikrojeder, in sicer s testom mikrojeder s pretočno citometrijo ter testom mikrojeder z ustavitvijo citokineze. Celice ZFL so bile v vseh poskusih TKI-jem izpostavljene 72 ur pri različnih koncentracijah posameznega TKI-ja. Pri testu mikrojeder z ustavitvijo citokineze, s katerim smo potrjevali rezultate dobljene s testom mikrojeder s pretočno citometrijo, so bile celice TKI-jem izpostavljene le eni koncentraciji. Rezultati so pokazali, da so določeni TKI-ji, in sicer SOR (4 µM), DAS (0,015, 0,03 in 0,06 µM) in NIL (5, 10 in 20 µM) značilno vplivali na živost celic ZFL. Po 72 urah izpostavitve TKI-ji niso značilno vplivali na celični cikel celic ZFL kot tudi ne na genomsko stabilnost celic ZFL pri pogojih izpostavitve. Slednje smo potrdili tudi s klasično metodo testa mikrojeder z ustavitvijo citokineze. Noben izmed proučevanih TKI-jev ni značilno vplival na število mikrojeder, nukleoplaznmatskih mostičkov in jedrnih brstov, medtem ko so na jedrni delitveni indeks značilno vplivali SOR (4 µM), REG (3 µM) ter NIL (20 µM). Tyrosine kinase inhibitors (TKIs) are small molecules for the targeted treatment of cancer cells. After ingestion, TKIs are excreted via faeces and urine into aquatic environment and have potential adverse effects on non-target organisms. In the thesis we investigated the cytotoxic and genotoxic activity of six TKIs: erlotinib (ERL), sorafenib (SOR), regorafenib (REG), dasatinib (DAS), imatinib mesylate (IM) and nilotinib (NIL) on an in vitro model of zebrafish liver cell line (Danio rerio; ZFL cells). Cytotoxicity was investigated by staining of cells with propidium iodide and flow cytometry, effect on the cell cycle by staining with Hoechst 33342 and flow cytometry, and effect on genomic stability by two variants of micronucleus test; micronucleus test with flow cytometry and cytokinesis-block micronucleus (CBMN) assay. ZFL cells were exposed to TKIs for 72 hours at different concentrations of individual TKI in all experiments. In the CBMN assay, which confirmed the results obtained by flow cytometry micronucleus assay, cells were exposed to TKIs at only one concentration. The results showed that certain TKIs, namely SOR (4 µM), DAS (0.015, 0.03, and 0.06 µM) and NIL (5, 10, and 20 µM), significantly influenced the viability of ZFL cells. After 72 hours of exposure, TKIs did not significantly affect the cell cycle of ZFL cells as well as the genomic stability of ZFL cells under exposure conditions. The latter were also confirmed by the classical method of CBMN assay. None of the studied TKIs had a significant effect on the number of micronuclei, nucleoplasmic bridges, and nuclear buds, while the nuclear division index was significantly influenced by SOR (4 µM), REG (3 µM) and NIL (20 µM). tirozin kinazni inhibitorji TKI ne-tarčni organizmi odpadne vode citotoksičnost genotoksičnost erlotinib sorafenib regorafenib dasatinib imatinib mezilat nilotinib Danio rerio jetrne celice rib cebric celice ZFL pretočna citometrija test mikrojeder tyrosine kinase inhibitors TKI non-target organisms wastewater cytotoxicity genotoxicity erlotinib sorafenib regorafenib dasatinib imatinib mesylate nilotinib Danio rerio zebrafish liver cells ZFL cells flow cytometry micronucleus test true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-07-26 07:16:05 2020-07-26 07:16:09 2022-08-30 04:00:16 0000-00-00 00:00:00 2020 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 176573 26721539 10497.pdf 10497.pdf 1 DA6897845A08B4D77B550E54FE8ACD2E 563aaeb0d43164ea95716c6d23dc8f2df821a4daaf104e49d9c64ac7ead82b51 435fb862-a1b9-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=131693 Biotehniška fakulteta 0 0 0