Vpliv kombiniranega delovanja citostatikov na živost celic ZFL in nastanek poškodb DNA

Modic, Barbara (avtor), Žegura, Bojana (mentor)

Citostatiki, ki se uporabljajo za zdravljenje raka, z urinom in/ ali blatom rakavih bolnikov pridejo v odpadne vode, in se posledično v primeru, da le-te niso ustrezno prečiščene, znajdejo v okolju. Zaradi specifičnega mehanizma delovanja lahko citostatiki že v zelo nizkih koncentracijah predstavljajo nevarnost za netarčne organizme, s katerim pridejo v stik v okolju. V magistrski nalogi smo raziskovali citotoksično in genotoksično delovanje izbranih citostatikov in njihovih kompleksnih mešanic pri koncentracijah, ki so relevantne za okolje na in vitro modelu jetrnih celic rib cebric (Danio rerio; ZFL). Na osnovi literaturnih podatkov o pojavljanju citostatikov v odpadnih vodah Onkološkega inštituta v Ljubljani smo izbrali štiri, ki so eni od najpogosteje uporabljenih citostatikov in so tudi najpogosteje zaznani v odpadnih vodah. Izbrali smo naslednje citostatike: 5-fluorouracil (5-FU), cisplatin (CDDP), ciklofosfamid (CP) in ifosfamid (IF). Citotoksično delovanje posameznih citostatikov in njihovih kompleksnih mešanic smo ugotavljali s testom MTS, njihovo genotoksično delovanje pa s testom komet in testom mikrojeder. S testom MTS smo ugotovili od koncentracije in časa odvisen vpliv 5-FU in CDDP na živost celic, medtem ko CP in IF pri koncentracijh ≤ 150 mg/L nista značilno vplivala na živosti celic ZFL niti po daljšem času izpostavitve (72 ur). Kompleksna mešanica štirih citostatikov pri okoljskih koncentracijah (0.9 µg/L 5FU, 6 µg/L CDDP, 120 µg/L CP in 100 µg/L IF) ni vplivala na živost celic, medtem ko smo pri kompleksni mešanici, ki je vsebovala 10-krat višje koncentracije citostatikov, zaznali statistično značilen vpliv na živost celic ZFL, ki pa v primerjavi s kontrolo ni bila znižana za več kot 30 %. Po 72 urah izpostavitve so čisti citostatiki povzročili poškodbe DNA, kar smo ugotavljali s testom komet. LOEC (iz angl low observed effect concentration) vrednosti za posamezne citostatike so bile 0.01 mg/L za 5-FU, 0.1 mg/L za CDDP, 1 mg/L za CP in 0.01 mg/L za IF. Rezultati so pokazali, da je kompleksna mešanica povzročila od koncentracije odvisno povišanje poškodb DNA. Medtem ko pri testiranih koncentracijah čistih citostatikov (≤ 1 mg/L za 5-FU in CDDP ter ≤ 10 mg/L za CP in IF) kot tudi pri kompleksnih mešanicah nismo zaznali vpliva na genomsko stabilnost celic ZFL, kar smo ugotavljali s testom mikrojeder. Rezultati nakazujejo, da kompleksne mešanice citostatikov lahko predstavljajo nevarnost za vodne organize pri okoljsko relevantnih koncentracijah, kot tudi potrjujejo dejstvo, da ne moremo negativnih učinkov kompleksnih mešanic predvideti na podlagi toksikoloških podatkov za posamezne čiste snovi, ki se v takšni mešanici nahajajo.

• Jezik: Slovenski jezik
• Ključne besede: citotoksičnost/genotoksičnost/citostatiki/5-fluorouracil/cisplatin/ciklofosfamid/ifosfamid/ Danio rerio/jetrne celice rib cebric/celice ZFL/test MTS/test komet/test mikrojeder
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: BF - Biotehniška fakulteta
• Leto izida: 2017
• PID: 20.500.12556/RUL-94217
• COBISS.SI-ID: 4418895
• Datum objave v RUL: 20.07.2017

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: The combined effects of cytostatic drugs on cell viability and induction of DNA damage

Izvleček

Cytostatic drugs (cytostatics) used in cancer treatments are excreted by patients via urine or/and faeces. From there they get to hospital or municipal wastewaters, through which, if not properly cleaned, they enter surface waters. Due to their mechanism of action, cytostatics can pose a threat to non-target organisms in the environment even in very low concentrations. For this reason we decided to research cytotoxicity and genotoxicity of an environmentally relevant mixture and concentrations of cytostatics on in vitro zebrafish liver cell model (Danio rerio; ZFL). In reference to the analyses done on hospital wastewater from Institute of Oncology in Ljubljana and the range of their usage we chose to research following four cytostatics: 5-fluorouracil (5-FU), cisplatin (CDDP), cyclophosphamide (CP) and ifosfamide (IF). We tested cytotoxic activity of individual cytostatics and their complex mixtures wits MTS assay. Comet assay and micronucleus assay were used to test the cytostatics' genotoxicity. MTS assay determined both the concentration-related and time-related influence of 5-FU and CDDP on cell viability. CP and IF at concentrations below 150 mg/L had no relevant impact on ZFL cell viability even after prolonged exposure (72 hours). A complex mixture of four cytostatics at environmentally relevant concentrations (0.9 µg/L 5FU, 6 µg/L CDDP, 120 µg/L CP in 100 µg/L IF) had no effect on cell viability. However, the 10 times higher concentration of cytostatics in the complex mixture had statistically relevant effect of ZFL cell viability; in comparison with the control the viability was reduced by less than 30 %. Comet assay was used to identify DNA damage caused by pure cytostatic drugs after a 72-hour exposure. LOEC (low observed effect concentration) values of each individual cytostatic were as follows: 0.01 mg/L for 5-FU, 0.1 mg/L for CDDP, 1 mg/L for CP and 0.01 mg/L for IF. Results showed a correlation between the complex mixture concentration and DNA damage. Genomic stability of ZFL cells was tested using both pure cytostatic drugs (≤ 1 mg/L for 5-FU and CDDP and ≤ 10 mg/L for CP and IF) and complex mixture; neither had any effect on the genomic stability. This was also tested using micronucleus assey. Our results indicate that environmentally relevant cytostatic mixtures may pose a threat to aquatic organisms. It also seems that negative effects of complex mixtures cannot be predicted based on toxicity of each individual element.

• Ključne besede: cytotoxicity/genotoxicity/cytostatics/5-fluorouracil/cisplatin/cyclophosphamide/ifosfamide/Danio rerio/zebrafish liver cells/ZFL cells/MTS assay/comet assay/micronucleus assay