The influence of fullerenol on the frequency of chromosome aberrations and micronuclei in ovarian hamster cell line CH0-K1 and in human peripheral blood lymphocites cell culture

Beograd : [J. Ž. Mrđanović]

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Biologija - genotoksikologija / Biology - genotoxicology

Nanomaterijali zahvaljujući svojim malim dimenzijama i raznovrsnim fizičkohemijskim osobinama imaju velik potencijal za primenu u različitim oblastima života. Među nanočestice sa širokom upotrebom u biomedicini ubraja se fuleren C60. Njegova slaba vodorastvorljivost može se prevazići derivatizacijom, pri čemu fuleren stiče nova svojstva koja utiču na njegovu funkcionalnost. Najpoznatiji vodorastvorni derivati C60 su fulerenoli (С60(ОН)n, n=2-40), koji imaju velik potencijal za primenu u medicini. Taj potencijal je baziran na njihovoj sposobosti da u biološkim sistemima skupljaju slobodne radikale nastale u oksidativnom stresu. Međutim, sa druge strane, fulerenoli imaju sposobnost da pod određenim uslovima deluju prooksidativno. Zbog toga je neophodno definisanje koncentracija i uslova pod kojima fulerenol i njegovi derivati deluju kao antioksidanti odnosno prooksidanti. Taj aspekt je veoma važan zbog uticaja ovih nanočestica na DNK i određuje u velikoj meri njihova genotoksična odnosno antigenotoksična svojstva. Pored uticaja koncentracije nanočestica na oksidativni status, na genotoksičnost utiču i fizičkohemijske karakteristike samih nanočestica kao što su: veličina, oblik, površinsko naelektrisanje, čistoća, rastvorljivost, površinske modifikacije i agregacija, koje je takođe potrebno precizno definisati. Konačno, ispitivanje uticaja novosintentisanih supstanci sa potencijalom za medicinsku aplikaciju na DNK, predstavlja neophodan korak pre faze kliničkih studija. Zbog svega navedenog, primarni cilj ovog rada jeste ispitivanje genotoksičnih odnosno antigenotoksičnih svojstava vodorastvornog fulerenskog derivata fulerenola C60(OH)24, na dva različita sisarska modela. Prvi model je ćelijska kultura ovarijuma kineskog hrčka CHO-K1, a drugi primarna ćelijska kultura humanih limfocita. U radu su evaluirane fizičkohemijske karakteristike nanočestice fulerenola C60(OH)24 u medijumu za ćelijske kulture sa serumom i to u uslovima koji odgovaraju 6 eksperimentalnim uslovima u testovima mikronukleusa i hromozomskih aberacija. Zatim je ispitan uticaj fulerenola na preživljavanje ćelija u cilju utvrđivanja subcitotoksičnih koncentracija fulerenola, potrebnih za ispitivanje genotoksičnosti i antigenotoksičnosti fulerenola. Nakon toga je izvršena evaluacija incidencije biomarkera oštećenja DNK radi utvrđivanja genotoksičnog potencijala fulerenola. U poslednjem segmentu rada je evaluacijom istih biomarkera analiziran i antigenotoksičan potencijal fulerenola, na ćelijskim kulturama koje su prethodno oštećene poznatim citostatskim agensom mitomicinom C. Rezultati analiza su pokazali da je C60(OH)24 dimenzija od 40-180 nm u zavisnosti od kosolventa, kao i da gradi stabilne nanočestice u medijumu za ćelijsku kulturu sa fetalnim goveđim serumom. Citotoksičnost fulerenola kreće se u opsegu koncentracija od 88,6 μM-443,2 μM. Analizom genotoksičnosti fulerenola testom mikronukleusa i hromozomskih aberacija pokazano je da fulerenol nije bio genotoksičan u ispitivanim ćelijskim kulturama, pri ispitivanim subcitotoksičnim koncentracijama i pri datim vremenskim tretmanima. Ispitivanje antigenotoksičnog potencijala je pokazalo da fulerenol utiče na snižavanje ispitivanih biomarkera oštećenja DNK.

Nanomaterials, due to their small dimensions and manifold physicochemical characteristics have great potential for application in various areas of life. One of nanoparticles with wide implementation in biomedicine is fullerene C60. Its weak water solubility can be overcome by derivatization after which fullerene acquires new characteristics, which affect its functionality. The most famous water soluble C60 derivatives are fullerenols (С60(ОН)n, n=2-40), which have great potential for implementation in medicine. This potential is based on their ability to collect free radicals occurring in oxidative stress in biological systems. However, on the other hand, fullerenols are able to act prooxidatively under certain conditions. Thus, it is necessary to define concentrations and conditions under which fullerenol and its derivatives act as antioxidants, i.e. prooxidants. This aspect is very important because of the impact of these nanoparticles to the DNA, which largely defines their genotoxic i.e. antigenotoxic characteristics. Besides the impact of nanoparticles’ concentration to oxidative status, genotoxicity is also affected by physicochemical characteristics of nanoparticles themselves: their size, shape, surface charge, purity, solubility, surface modifications and aggregation, which are all necessary to be precisely defined. Finally, the testing of the impact of newly synthesized substances with potential for medical application to DNA, represents a necessary step before the phase of clinical trials. Due to all the above state, the primary aim of this paper was to test genotoxic i.e. antigenotoxic characteristic of water-soluble fullerene derivative fullerenol C60(OH)24, in two different mammalian models. The first model was an ovarian cell culture of a Chinese hamster CHO-K1, and the other was a primary humane lymphocyte cell culture. The paper evaluated physicochemical characteristics of fullerenol nanoparticle C60(OH)24 in the cell culture medium with serum under the conditions which were in accordance with the experimental conditions in micronuclei and chromosomal 8 aberrations tests. Furthermore, the affect of fullerenol to cell survival was tested with the aim of determination of fullerenol subcytotoxic concentrations, necessary for testing of fullerenol genotoxicity and antigenotoxicity. After that, the DNA damage biomarkers incidence evaluation was performed for determination of fullerenol genotoxicity potential. In the last segment of the paper, the antigenotoxic potential of fullerenol was analyzed by evaluation of the same biomarkers, in cell cultures, which were previously damaged by the known cytostatic agent mitomycin C. The results of the analysis showed that C60(OH)24 dimensions of 40-180nm depended on the cosolvent, and that it crated stable nanoparticles in the cell culture medium with fetal bovine serum. Cytotoxicity of fullerenol varied in the range of concentrations from 88.6 μM-443.2 μM. The analysis of fullerenol genotoxicity by the micronuclei test and chromosomal aberrations test showed that fullerenol was not genotoxic in the tested cell cultures, at given subcytotoxic concentrations and treatment duration. Testing of antigenotoxic potential showed that fullerenol affected the decrease of the tested DNA damage biomarkers.

Nanočestice, fulereni, fulerenol, genotoksičnost, mikronukleusi, hromozomske aberacije

Nanoparticles, fullerenes, fullerenol, genotoxicity, micronuclei, chromosomal aberrations

575.224.6:66.017 (043)

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