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

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

The frequency of sister chromatid exchange and micronuclei in evaluation of cytogenetic activity of Kombucha on human peripheral blood lymphocytes

Kombucha is a refreshing beverage obtained by the fermentation of sweetened black tea with a “tea fungus” (symbiotic culture of acetic acid bacteria and yeasts). It is consumed due to its potential beneficial effects on human health. The aim of this study was to investigate activity of Kombucha on human peripheral blood lymphocytes in vitro. We analyzed Kombucha made from different substrates: Camellia sinensis and Satureja montana, and effects of substrates alone. The frequencies of sister chromatid exchange (SCE) and micronuclei (MN) were scored as genetic endpoints and mitomycin C was used as model mutagen. Kombucha from Camellia sinensis and Camellia sinensis substrate increased frequency of MN and SCE on mitomycin C-treated and -untreated peripheral blood lymphocytes. However, Kombucha from Satureja montana reduced incidence of MN on mitomycin C-treated and -untreated peripheral blood lymphocytes, while SCE frequency was higher than control value. In our pilot study we showed for the first time that Kombucha from different substrates induced different effects on mitomycin C-treated and -untreated peripheral blood lymphocytes

Novel insights to the anti-proliferative activity of rosemary (Rosmarinus officinalis L.) co-treatment

The aim of this study was to characterize volatile and non-volatile compounds of rosemary from the North Adriatic region and to determine its antiproliferative activity, alone or in combination with radiomimetic bleomycin (BLM) on three malignant and one non-transformed human cell lines. Chemi-cal analysis of the volatile compounds revealed the presence of monoterpenes (93.8%), among which 1.8-cineol (32.9%) and camphor (15.5%) were the dominant compounds. Also, obtained results showed that the major polyphenolic constituents in rosemary extract were phenolic acids (rosmarinic acid and its derivatives up to 69.2 mg 100 g -1), as well as flavones and flavonols in the following order: lute-olin>isorhamnetin>quercetin>kaempferol>apigenin. Cell growth tests showed that rosemary extract alone exerted moderate antiproliferative activity, as well as a synergistic antiproliferative effect with bleomycin (EC 50 344.3-461.5 µg mL -1 and 58.6-292 µg mL -1 , respectively). The anti-tumor effect of rosemary extract in combination with BLM was much stronger, compared to BLM itself on the breast cancer cells. Through its proposed sensitizing effect, rosemary extract, in combination with the standard chemotherapeutics, could be used for the investigations of possible therapeutic modalities

The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs) are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC) treatment on superoxide-dismutase (SOD) activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH)24) at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET). The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL) after 3 and 24 h of cell incubation. Increasing of C60(OH)24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL) + MMC was noticed boost in total SOD activity in comparison with controls. Treatment with fullerenol decreased SOD activity in rest of samples treated with MMC. Decreased activity of superoxide-dismutase in almost all samples treated with fullerenol and MMC might be contributed to antioxidative properties of fullerenol. Increased enzyme level at concentration of 0.0625 mg/mL may be due to its prooxidative activity.U ovom radu ispitivani su efekti fulerenola (C60(OH)24) na preživljavanje, kao i tretmana fulerenolom i mitomicinom c (MMC) na aktivnost ukupne superoksid-dismutaze u CHO-K1 (ovarijalnih ćelija hrčka) ćelijskoj liniji. U uzorcima ćelija tretiranim fulerenolom koncentracija 0,01-0,5 mg/mL, praćeno je preživljavanje testom odbacivanja boje (DET) u 3 i 24 h tretmanu. Aktivnost superoksid-dismutaze (SOD) merena je u uzorcima tretiranim fulerenolom izabranih koncentracija i mitomicinom c (0,5 i 0,1 mg/mL) nakon 3 i 24 h. Sa porastom koncentracije (C60(OH)24) opada procenat preživelih ćelija tokom 3 i 24 h. Aktivnost SOD raste sa porastom koncentracije fulerenola i u najvećoj koncentraciji opada u obe vremenske tačke eksperimenta. U uzorcima tretiranim fulerenolom i MMC došlo je do smanjenja aktivnosti SOD, izuzev pri koncentraciji fulerenola od 0,0625 mg/mL, kada je zapažen porast aktivnosti SOD u odnosu na kontrolne grupe.Projekat ministarstva br. 14207

Hypericum perforatum

St. John’s wort is a widely used medicinal plant. The quality of herbal drug, which is in most of the cases collected from nature, varies. Therefore, the aim of the present study was detailed chemical characterization of Hypericum perforatum subsp. perforatum samples collected in close time intervals during flowering and fruitification with the purpose to state the phenological stage characterized by maximum levels of active principles. The antioxidant potential and potential to inhibit biologically important enzymes, as well as the cytotoxicity and genotoxicity of the sample collected during the full flowering period, were evaluated. Data showed that the optimal period for the achieving of maximum level of active principles is the phenophase between floral budding and flowering stage. Significant antioxidant potential and the ability to inhibit biologically important enzymes (especially α-glucosidase) were recorded. The extract exhibited no genotoxicity in subcytotoxic concentrations, while increased cytotoxicity recorded in cotreatment with bleomycin on malignant cell lines was especially significant

The Puzzling Potential of Carbon Nanomaterials: General Properties, Application, and Toxicity

Being a member of the nanofamily, carbon nanomaterials exhibit specific properties that mostly arise from their small size. They have proved to be very promising for application in the technical and biomedical field. A wide spectrum of use implies the inevitable presence of carbon nanomaterials in the environment, thus potentially endangering their whole nature. Although scientists worldwide have conducted research investigating the impact of these materials, it is evident that there are still significant gaps concerning the knowledge of their mechanisms, as well as the prolonged and chronic exposure and effects. This manuscript summarizes the most prominent representatives of carbon nanomaterial groups, giving a brief review of their general physico-chemical properties, the most common use, and toxicity profiles. Toxicity was presented through genotoxicity and the activation of the cell signaling pathways, both including in vitro and in vivo models, mechanisms, and the consequential outcomes. Moreover, the acute toxicity of fullerenol, as one of the most commonly investigated members, was briefly presented in the final part of this review. Thinking small can greatly help us improve our lives, but also obliges us to deeply and comprehensively investigate all the possible consequences that could arise from our pure-hearted scientific ambitions and work

Activity of lactate dehydrogenase and superoxide dismutase in the circulation of patients with breast carcinoma

Background: A rapid increase in the turnover of malignant cells modulates the enzymes level in circulation and may be a prognostic sign of disease progression. We evaluated the activity of serum lactate dehydrogenase and superoxide dismutase in erythrocytes of breast cancer patients immediately after surgery and before adjuvant therapy. Methods: Fourteen newly operated breast cancer patients were included in the study. Lactate dehydrogenase and superoxide dismutase activity were estimated using standard tests for evaluation of enzyme activity. Results: Activity of lactate dehydrogenase was in normal range in all but one sample obtained from patient at clinical status IIIB. Superoxide dismutase activity was elevated in 11 out of 14 patients, and was 2 to 8 fold higher compare to control values. The highest activity of superoxide dismutase was found in samples of two patients at clinical status I. Conclusion: Determination of lactate dehydrogenase and superoxide dismutase activity might be useful in clinical follow up of breast cancer patients.nul

The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs) are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC) treatment on superoxide-dismutase (SOD) activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH)24) at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET). The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL) after 3 and 24 h of cell incubation. Increasing of C60(OH)24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL) + MMC was noticed boost in total SOD activity in comparison with controls. Treatment with fullerenol decreased SOD activity in rest of samples treated with MMC. Decreased activity of superoxide-dismutase in almost all samples treated with fullerenol and MMC might be contributed to antioxidative properties of fullerenol. Increased enzyme level at concentration of 0.0625 mg/mL may be due to its prooxidative activity

The activity of superoxide-dismutase in animal cell culture CHO-K1 after treatment with fullerenol and mytomicine C

Eukaryotic cell survives in predominantly reduced conditions. Homeostasis of cellular redox system is an imperative of cell surviving and its normal metabolism. ROS are well recognized for playing a dual role as both deleterious and beneficial species, since they can be either harmful or beneficial to living systems. These species are mutagenic compounds known to lead to DNA damage, favor cell transformation, and contribute to the development of a variety of malignant diseases. All the effects of oxidants are influenced by the cellular antioxidant defenses. This multilayer system consists of low molecular weight components and several antioxidant enzymes. Superoxide dismutases (SODs) are the only enzymes dismuting superoxide radicals. Mitomycin C, a cross-linking agent, demonstrated genotoxicity in all in vitro and in vivo test systems in mammalian cells and animals. Water-soluble fullerenes are well known as cytotoxic agents for many cell lines in vitro. At the other side, fullerenols are good free radical scavengers and antioxidants both in vitro and in vivo. This paper investigates the effects of fullerenol on survival and fullerenol/ /mytomicine (MMC) treatment on superoxide-dismutase (SOD) activity in CHO-K1 cells. Samples were treated 3 and 24 h with fullerenol (C60(OH)24) at concentration range 0.01-0.5 mg/mL and survival was monitored with dye exclusion test (DET). The activity of total SOD was estimated in samples treated with chosen concentrations of fullerenol and MMC (0.5 and 0.1 mg/mL) after 3 and 24 h of cell incubation. Increasing of C60(OH)24 concentration leads to decreasing of percent of surviving cells 3 and 24 h after incubation. The activity of total SOD enhanced with higher concentration of fullerenol, while decreased in the highest concentration at both experimental points. In samples treated with MMC, as well as in samples treated with fullerenol (0.0625 mg/mL) + MMC was noticed boost in total SOD activity in comparison with controls. Treatment with fullerenol decreased SOD activity in rest of samples treated with MMC. Decreased activity of superoxide-dismutase in almost all samples treated with fullerenol and MMC might be contributed to antioxidative properties of fullerenol. Increased enzyme level at concentration of 0.0625 mg/mL may be due to its prooxidative activity.U ovom radu ispitivani su efekti fulerenola (C60(OH)24) na preživljavanje, kao i tretmana fulerenolom i mitomicinom c (MMC) na aktivnost ukupne superoksid-dismutaze u CHO-K1 (ovarijalnih ćelija hrčka) ćelijskoj liniji. U uzorcima ćelija tretiranim fulerenolom koncentracija 0,01-0,5 mg/mL, praćeno je preživljavanje testom odbacivanja boje (DET) u 3 i 24 h tretmanu. Aktivnost superoksid-dismutaze (SOD) merena je u uzorcima tretiranim fulerenolom izabranih koncentracija i mitomicinom c (0,5 i 0,1 mg/mL) nakon 3 i 24 h. Sa porastom koncentracije (C60(OH)24) opada procenat preživelih ćelija tokom 3 i 24 h. Aktivnost SOD raste sa porastom koncentracije fulerenola i u najvećoj koncentraciji opada u obe vremenske tačke eksperimenta. U uzorcima tretiranim fulerenolom i MMC došlo je do smanjenja aktivnosti SOD, izuzev pri koncentraciji fulerenola od 0,0625 mg/mL, kada je zapažen porast aktivnosti SOD u odnosu na kontrolne grupe.Projekat ministarstva br. 14207

Bioimpact of Carbon Nanomaterials

The unique size-dependent properties of carbon nanomaterials (CNMs)- graphene, nanotubes (CNTs) and fullerenes- make them very attractive for diagnostic and therapeutic application. This chapter presents possible application of CNMs. Graphene with extraordinary chemical and physical properties has already revealed a great number of potential applications such as environmental toxic material removal, drug delivery, tissue engineering, and fl uorescence-based biomolecular sensing. The CNT derivatives have many interesting properties which make them potentially useful in a living system as biosensors, bioelectronic devices based on enzyme-nanotube or antibody-nanotube conjugates, chemotherapeutic agents, hyperthermia therapy and immunotherapy agents, agent in treatment of central nervous system disorders, and tissue engineering agent. Fullerene C60 derivatives have been used as: drug and gene delivery vectors, magnetic resonance imaging agents, radio protectors, antioxidants, HIV-1 protease inhibitors, antigenotoxic agents, and phototherapy agents. Fullerenols are polyhydroxylated derivatives of fullerene (C60(OH)n) with remarkable antioxidant, xenobiotic-protective, radioprotective, nanodrug and endohedral gadolinium carrier properties