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.
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.
