| Popis: |
Tirozin kinazni inhibitorji (TKI) so majhne molekule zasnovane za tarčno zdravljenje raka. Neposredno inhibirajo specifične mutirane tirozin kinaze, ki so prekomerno izražene v določenih vrstah rakavih celic. Ker se pojavnost rakavih obolenj povečuje, in ker so ta zdravila uspešna v boju proti določenim vrstam rakavih obolenj, poraba teh zdravil v zadnjih 20 letih nenehno narašča. Posledično narašča tudi količina ostankov teh snovi v vodnem okolju. Trenutne meritve in predvidevanja kažejo, da se ostanki teh zdravil pojavljajo v koncentracijah nekaj ng/L ali manj. Kljub stalni prisotnosti v okolju pa zaradi tarčnega načina delovanja ne predstavljajo resne grožnje posredno izpostavljenim organizmom v okolju, saj ti ne izražajo tarče za ta zdravila. Kljub temu pa so obstoječe raziskave pokazale, da določeni TKI povzročajo genotoksične učinke na netarčnih okoljskih orgnizmih. V magistrskem delu smo na in vitro modelu jetrnih celic rib cebric (Danio rerio ZFL) ocenili toksičnost petih pogosto uporabljenih TKI: erlotinib, sorafenib, regorafenib, dasatinib in nilotinib. Citotoksične učinke posameznih TKI smo ocenili s testom MTS, njihovo genotoksično delovanje pa s testom komet. S testom MTS smo ugotovili od koncentracije in časa odvisen vpliv sorafeniba, regorafeniba in dasatiniba, medtem ko erlotinib in nilotinib nista značilno vplivala na živost celic ZFL niti po daljšem času izpostavitve (72 ur). S testom komet pri erlotinibu genotoksičnega učinka oz. poškodb DNA nismo zaznali, smo pa zaznali povečano količino poškodb DNA pri drugih preizkušenih TKI. Pri sorafenibu (⡥ 0,8 µM), regorafenibu (⡥ 0,16 µM) in dasatinibu (⡥ 0,006 µM) smo poškodbe DNA zaznali le po 24 urni izpostavitvi, pri nilotinibu pa tako pri krajšem (24 ur ⡥ 2 µM) kot pri daljšem času izpostavitve (72 ur 2 in 10 µM). Rezultati nakazujejo, da regorafenib in nilotinib pri ne-citotoksičnih koncentracijah, ki so sicer višje od pričakovanih v okolju, povzročata poškodbe DNA. Tyrosine kinase inhibitors (TKIs) are small molecules designed for targeted cancer therapy. They directly inhibit certain mutated tyrosine kinases that are overexpressed in certain types of cancer cells, preventing the spread of the disease. As the number of cancer cases increases, the consumption of these pharmaceuticals has steadily increased over the past 20 years. As a result, the amount of these substances in the aquatic environment is also increasing. Current measurements and predictions indicate that residues of these drugs occur at concentrations of a few ng/L or even less. Due to the specific mechanism of action of TKIs, they do not pose a serious threat to indirectly exposed environmental organisms because they do not express targets for these drugs. Nevertheless, existing studies have shown that certain TKIs have genotoxic effects on non-target organisms in the environment. In this master’s thesis, we investigated the toxicity of five commonly used TKIs: erlotinib, sorafenib, regorafenib, dasatinib, and nilotinib using an in vitro model of zebrafish liver cells (Danio rerio ZFL). The cytotoxic effects of each TKIs were assessed by the MTS assay, and their genotoxic effects were assessed by the Comet assay. The MTS assay showed dose- and time-dependent effects of sorafenib, regorafenib, and dasatinib, whereas erlotinib and nilotinib did not significantly affect ZFL cell viability even after prolonged exposure (72 hours). No genotoxic effect or DNA damage was detected with erlotinib using the comet assay, but an increased amount of DNA damage was detected with the other TKIs tested. For sorafenib (⡥ 0.8 µM), regorafenib (⡥ 0.16 µM) and dasatinib (⡥ 0.006 µM), DNA damage was detected only after 24 hours of exposure, whereas nilotinib induced DNA damage at both short (24 hours ⡥ 2 µM) and longer exposure (72 hours 2 and 10 µM). The results suggest that regorafenib and nilotinib cause genotoxic effects at non-cytotoxic concentrations that are, however, higher than would be expected in the environment. |
