TY - JOUR
AU1 - Isakovic,, Aleksandra
AU2 - Markovic,, Zoran
AU3 - Todorovic-Markovic,, Biljana
AU4 - Nikolic,, Nadezda
AU5 - Vranjes-Djuric,, Sanja
AU6 - Mirkovic,, Marija
AU7 - Dramicanin,, Miroslav
AU8 - Harhaji,, Ljubica
AU9 - Raicevic,, Nevena
AU1 - Nikolic,, Zoran
AU1 - Trajkovic,, Vladimir
AB - Abstract The mechanisms underlying the cytotoxic action of pure fullerene suspension (nano-C60) and water-soluble polyhydroxylated fullerene [C60(OH)n] were investigated. Crystal violet assay for cell viability demonstrated that nano-C60 was at least three orders of magnitude more toxic than C60(OH)n to mouse L929 fibrosarcoma, rat C6 glioma, and U251 human glioma cell lines. Flow cytometry analysis of cells stained with propidium iodide (PI), PI/annexin V–fluorescein isothiocyanate, or the redox-sensitive dye dihydrorhodamine revealed that nano-C60 caused rapid (observable after few hours), reactive oxygen species (ROS)–associated necrosis characterized by cell membrane damage without DNA fragmentation. In contrast, C60(OH)n caused delayed, ROS-independent cell death with characteristics of apoptosis, including DNA fragmentation and loss of cell membrane asymmetry in the absence of increased permeability. Accordingly, the antioxidant N-acetylcysteine protected the cell lines from nano-C60 toxicity, but not C60(OH)n toxicity, while the pan-caspase inhibitor z-VAD-fmk blocked C60(OH)n-induced apoptosis, but not nano-C60–mediated necrosis. Finally, C60(OH)n antagonized, while nano-C60 synergized with, the cytotoxic action of oxidative stress–inducing agents hydrogen peroxide and peroxynitrite donor 3-morpholinosydnonimine. Therefore, unlike polyhydroxylated C60 that exerts mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity, pure crystalline C60 seems to be endowed with strong pro-oxidant capacity responsible for the rapid necrotic cell death. cytotoxicity, fullerene, C60, reactive oxygen species, apoptosis, necrosis Water-soluble fullerene derivatives synthesized by attaching various functional groups (−OH, −COOH, −NH2) to the fullerene cage (C60) are promising candidates for many biomedical applications, including cytoprotection, DNA photocleavage, enzyme inhibition, diagnostic imaging, and antimicrobial and anticancer therapy (Bosi et al., 2003). Due to electrochemical features that enable reaction with cell-damaging reactive oxygen species (ROS) such as superoxide \((\mathrm{O}_{2}^{{\bullet}}{-})\) and hydroxyl (•OH) radicals, the fullerene core behaves as a free radical sponge with a protective effect in experimental ROS-dependent neuronal death, both in vitro and in vivo (Dugan et al., 1997, 2001; Lotharius et al., 1999). Polyhydroxylated fullerenes [C60(OH)n], also known as fullerols or fullerenols, are particularly efficient antioxidants, reducing ROS-mediated neuronal death induced by engagement of glutamate receptors (Dugan et al., 1996; Jin et al., 2000). Treatment with fullerols also afforded protection against oxidative stress in the RAW 264.7 macrophage cell line and ischemia-reperfused rat lungs (Chen et al., 2004), and significantly reduced doxorubicin toxicity against human breast cancer cell lines (Bogdanovic et al., 2004). On the other hand, fullerol itself caused RAW 264.7 cell death (Chen et al., 2004) and suppressed proliferation of human breast cancer cells in a cell line–dependent manner (Bogdanovic et al., 2004). The mechanisms responsible for the cytotoxic and cytostatic action of C60(OH)n in these studies were not investigated, while its antiproliferative effect on rat vascular smooth muscle cells was apparently associated with inhibition of protein tyrosine kinase activity (Lu et al., 1998). Investigation of the biological properties of pure, underivatized fullerene has been greatly hampered by its complete lack of solubility in water. However, pure C60 can be brought into water by means of solvent extraction or simply by stirring over time, which results in formation of water-stable aggregates (Cheng et al., 2004; Deguchi et al., 2001). As the unintentional generation of these aggregates in aqueous environments is a possibility, their toxicological effects are of great importance, particularly if C60 finds widespread use in consumer products. Recently, Sayes et al. (2004) described the several orders of magnitude higher toxicity of pure C60 against human dermal fibroblasts and liver carcinoma HepG2 cells, in comparison with fullerol and other water-soluble fullerenes. In a subsequent study, the same group reported that the cytotoxic activity of C60 colloid was mediated through ROS-mediated cell membrane lipid peroxidation (Sayes et al., 2005). In accordance with these data, a study performed in largemouth bass revealed a significant lipid peroxidation in brains of this aquatic species after 48 h of exposure to underivatized C60 (Oberdorster, 2004). While the markedly higher cytotoxicity of pure C60 in comparison with fullerol suggests distinct mechanisms for the induction of cell death, the ability of pure or hydroxylated C60 to induce different types of cell death has not been directly compared. Apoptosis and necrosis are two distinct forms of cell death that have profoundly different implications for the surrounding tissues (reviewed in Edinger and Thompson, 2004). Apoptosis is characterized by chromatin condensation, activation of caspases, and fragmentation of DNA without plasma membrane breakdown, followed by packaging of the deceased cell into apoptotic bodies that are recognized and removed by phagocytic cells in the absence of inflammation. On the other hand, necrosis is typified by vacuolation of the cytoplasm, breakdown of the plasma membrane, and release of cellular contents and pro-inflammatory molecules, resulting in the induction of inflammation around the dying cell. Gaining an insight into the type of cell death (apoptotic or necrotic) is important for designing an effective therapeutic strategy against cytotoxins, as apoptosis and necrosis apparently employ different mechanisms for cell killing. For example, inhibition of the apoptotic cascade–initiating enzymes caspases usually blocks apoptosis, while caspase-independent necrosis in some cases can be forestalled by treatment with antioxidants (Edinger and Thompson, 2004). The toxicity of fullerenes is an important characteristic for defining and constraining their possible biomedical applications, and a complete knowledge of the mechanisms underlying fullerene-induced cell death is necessary for designing an efficient therapeutic strategy for its alleviation. In the present study, we examined the hypothesis that pristine and hydroxylated fullerenes might use distinct mechanisms for the induction of cell death. To that effect, various tests have been employed for analyzing the ability of pure and polyhydroxylated C60 to induce apoptotic or necrotic cell death, and to assess the involvement of oxidative stress in the cytotoxicity of the two fullerene-based agents. MATERIALS AND METHODS Preparation and characterization of colloid and hydroxylated fullerene. For preparation of fullerene colloid in water, we used a C60/70 extract of carbon soot (79% C60, 20% C70, 1% higher-order fullerenes) produced by arc discharge (Markovic et al., 2003). C60/70 colloid (referred to as nano-C60 for reasons of convenience) was produced by evaporating tetrahydrofuran (THF) from a mixture of water and molecularly dispersed C60/70 in THF (Sigma, St. Louis, MO), using the procedure first described by Deguchi et al. (2001) and modified by Fortner et al. (2005). The concentration of nano-C60 suspension in water was adjusted by evaporation to 10 μg/ml, as determined from the absorption spectrum and using a gravimetric procedure. Polyhydroxylated fullerene, referred to as C60(OH)n, was prepared as previously described (Zhao et al., 2004) from the same C60/70 extract of carbon soot used for the nano-C60 preparation. Immediately after preparation, both nano-C60 and C60(OH)n were transferred to light-protected glass bottles and stored at 4°C until used for experiments. Images of nano-C60 were obtained upon evaporation of diluted C60 colloid suspension on a 400-mesh carbon-coated copper grid and imaging with a Phillips (FEI Europe B. V., The Netherland) EN 401 transmission electron microscope at 120 kV. The particle size distribution of fullerene colloid was obtained using a Brookhaven Instruments (Holtsville, NY) light-scattering system equipped with a BI-200SM goniometer, a BI-9000AT correlator, a temperature controller, and a Coherent INNOVA 70C argon-ion laser. Dynamic light-scattering measurements were performed using 135 mW laser excitation on 514.5 nm at a 90° detection angle, and particle size distribution was calculated using a Brookhaven Instruments particle-sizing software. For the Fourier transform infrared (FTIR) spectroscopy analysis, nano-C60 suspension and C60(OH)n solution were dried on silicon wafers until thin films were formed. FTIR spectra were measured at room temperature in the spectral range from 400 to 4000 cm−1, on a BOMEM spectrometer. Cell Lines. The mouse fibrosarcoma cell line L929 was obtained from the European Collection of Animal Cell Cultures (Salisbury, UK), while the rat glioma cell line C6 and the human glioma cell line U251 were kindly donated by Dr Pedro Tranque (Universidad de Castilla-La Mancha, Albacete, Spain). The cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2, in a HEPES (20mM)–buffered RPMI 1640 cell culture medium (Sigma) supplemented with 5% fetal calf serum, 2mM L-glutamine, 50μM 2-mercaptoethanol, 10mM sodium pyruvate, and 100 IU/ml penicillin and streptomycin (all from Sigma). Experimental design. The cells were prepared for experiments using the conventional trypsinization procedure with trypsin/EDTA and incubated in flat-bottom 96-well or 6-well cell culture plates (Sarstedt, Newton, NC) for the cell viability assessment or flow cytometry/lipid peroxidation analysis, respectively. Cells (>95% viable, as determined by trypan blue staining) were seeded at a rate of 1 × 104 per well (96-well plates) and 5 × 105 per well (6-well plates) for the short-term treatment (0.5–4 h), or at 0.5 × 104 per well (96-well plates) and 2.5 × 105 per well (6-well plates) for the long-term treatment (18–24 h). After being rested for 18 h, cell cultures were washed to remove the small number of nonadherent dead cells (<5%) and incubated in 200 μl (96-well plates) or 4 ml (6-well plates) of cell culture medium. Cells were incubated alone (control), or treated with fullerenes, antioxidant N-acetylcysteine (NAC), pro-oxidants hydrogen peroxide and 3-morpholinosydnonimine (SIN-1), or pan-caspase inhibitor z-VAD-fmk (all from Sigma), as described in detail in Figures 2–5. To avoid photoexcitation of fullerenes, we tried to minimize their exposure to ambient light, while all cell incubations were performed in the dark. Cells were cultivated under conditions described in the previous section, and cell culture conditions were identical for all cell lines. All treatments in each experiment were performed and analyzed in triplicates, except for the flow cytometry analysis in which single cultures were analyzed. Each experiment was done at least three times. Determination of cell viability and lactate dehydrogenase release. For the assessment of cell viability, we used the crystal violet assay, which is based on the inability of dead cells to remain adherent to cell culture plastic (Flick and Gifford, 1984). After incubation, cells were washed with PBS to remove dead, nonadherent cells. The remaining adherent, viable cells were fixed with methanol and stained with 1% crystal violet solution at room temperature for 10 min. The plates were thoroughly washed with water, and crystal violet was dissolved in 33% acetic acid. The absorbance of dissolved dye, corresponding to the number of viable cells was measured in an automated microplate reader at 570 nm. The release of intracellular enzyme lactate dehydrogenase (LDH), as a marker of cell membrane damage, was determined exactly as previously described (Kaludjerovic et al., 2005). The results of both crystal violet and LDH release assays were presented as a percent of the control value obtained in untreated cells. Detection of apoptosis and necrosis. Apoptotic cell death and necrotic cell death were analyzed by double staining with annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI), in which annexin V bound to the apoptotic cells with exposed phosphatidylserine, while PI labeled the necrotic cells with a membrane damage. Staining was performed according to the instructions from the manufacturer (BD Pharmingen, San Diego, CA), and flow cytometry was conducted on a FACSCalibur flow cytometer (BD Pharmingen). DNA fragmentation, another apoptotic marker that is not characteristic for necrotic cells, was assessed by a flow cytometric analysis of ethanol-fixed cells stained with PI as previously described (Kaludjerovic et al., 2005). The percentage of apoptotic (annexin+/PI− or PIlow hypodiploid cells in sub-G1 fraction) and necrotic (annexin+/PI+) cells was determined using CellQuest Pro software. Measurement of ROS generation and lipid peroxidation. The production of oxygen radicals was determined by measuring the intensity of green fluorescence emitted by the redox-sensitive dye dihydrorhodamine 123 (DHR) upon excitation at 488 nm. DHR (Sigma) was added to cell cultures 10 min prior to fullerene treatment at a concentration of 1μM. At the end of incubation, cells were detached by trypsinization and washed in PBS, and the fluorescence intensity in treated cells was analyzed using a FACSCalibur flow cytometer with a 488-nm argon laser. Alternatively, DHR fluorescences in nano-C60–treated cell cultures and the cell-free suspension of nano-C60 in the cell culture medium were compared using a fluorescence microplate reader (Chameleon, Hidex, Finland) equipped with a 488-nm excitation filter and a 535-nm emission filter. Lipid peroxidation was measured using the colorimetric thiobarbituric acid assay for malondialdehyde (MDA), as previously described (Losa, 2003). Analysis of synergism and antagonism in cytotoxic interactions. To analyze the type (additive, synergistic, or antagonistic) of fullerene interaction with the pro-oxidant agents H2O2 and SIN-1, or the interaction between nano-C60 and C60(OH)n in inducing tumor cell death, cells were treated with each agent alone and in combination. Six twofold dilutions were made from the starting concentrations of 0.5 μg/ml nano-C60, 800 μg/ml C60(OH)n, 1mM H2O2, and 1mM SIN-1, and the cytotoxic effect of each dose alone, as well as its combination with the corresponding dose of appropriate agent, was tested using the crystal violet assay. The results were expressed as a percent of cytotoxic efficiency, and a combination index (CI) for mutually exclusive or mutually nonexclusive interactions was calculated according to the method designed by Chou and Talalay (1984). CI values = 1, <1, or >1 indicate additive, synergistic, or antagonistic interactions, respectively. Statistical analysis. The statistical significance of the observed differences was analyzed by t-test or ANOVA followed by the Student-Newman-Keuls test. A p value < 0.05 was considered significant. RESULTS Characterization of Nano-C60 and C60(OH)n The images obtained with the transmission electron microscope revealed that C60 in water formed nanocrystalline aggregates of approximately 100 nm (Fig. 1A). This is consistent with the results of dynamic light-scattering measurements, which determined the average particle size to be 96.3 nm (Fig. 1B). The dynamic light-scattering analysis of C60(OH)n showed that all particles were <5 nm (the detection limit), thus confirming the absence of C60(OH)n aggregation (data not shown). The UV/Vis absorbance spectra of nano-C60 and C60(OH)n in Figure 1C indicate that fullerol does not have pronounced absorption bands in the UV region like nano-C60. Importantly, the spectra of our fullerene preparations are identical to the previously published spectra of similarly prepared commercial C60 and C60(OH)n (Fortner et al., 2005; Vileno et al., 2005). The FTIR spectra of nano-C60 and C60(OH)n show characteristic vibrational modes of C60 at 526, 575, and 1182 cm−1 (Kuzmany et al., 1995), as indicated by the arrows in Figure 1D. Expectedly, C60(OH)n has two broad absorption bands at about 1070 and 1608 cm−1, which reflect the presence of C–O and O–H covalent bonds, respectively. However, both bands were also found in the nano-C60 spectrum, which is consistent with the data that some degree of derivatization apparently occurs at the surface of nano-C60 crystals (Sayes et al., 2005). A strong absorption band at 860 cm−1 in the FTIR spectrum of C60(OH)n is another indication of a C–O bond, while the peaks observed in the region between 1200 and 1550 cm−1 originate from the silicon substrate. FIG. 1. Open in new tabDownload slide Characterization of nano-C60 and C60(OH)n. (A) Images of nano-C60 crystals obtained by transmission electron microscopy. (B) Particle size distribution of nano-C60 aggregates in water. Five thousand particles have been analyzed, and the highest number of particles (corresponding to a diameter of 96.3 nm) was normalized to 100. (C) UV/Vis spectra of nano-C60 (10 μg/ml) and C60(OH)n (1000 μg/ml). (D) FTIR spectra of nano-C60 and C60(OH)n. The arrows indicate characteristic peaks at 526, 575, 1070, 1182, and 1608 cm-1. FIG. 1. Open in new tabDownload slide Characterization of nano-C60 and C60(OH)n. (A) Images of nano-C60 crystals obtained by transmission electron microscopy. (B) Particle size distribution of nano-C60 aggregates in water. Five thousand particles have been analyzed, and the highest number of particles (corresponding to a diameter of 96.3 nm) was normalized to 100. (C) UV/Vis spectra of nano-C60 (10 μg/ml) and C60(OH)n (1000 μg/ml). (D) FTIR spectra of nano-C60 and C60(OH)n. The arrows indicate characteristic peaks at 526, 575, 1070, 1182, and 1608 cm-1. Nano-C60 and C60(OH)n Differ in the Efficacy and Kinetics of Their Cytotoxic Action In the preliminary experiments, we used cells of different types and origin (rat primary astrocytes and fibroblasts, rat/mouse peritoneal macrophages, C6 rat astrocytoma, U251 human glioma, and L929 mouse fibrosarcoma cell line) to investigate if fullerenes might exert cell-selective toxicity. However, repeated experiments in which the cell viability after 24-h incubation was evaluated by the crystal violet assay clearly demonstrated that the toxicity of nano-C60 and C60(OH)n was neither species/cell type specific, nor selective for primary or transformed cells (data not shown). Having established that, for reasons of convenience and reproducibility, we used well-defined transformed cell lines (C6, U251, and L929) to delineate the cytotoxic mechanisms of the two different fullerene preparations. To compare the cytotoxic efficiencies of nano-C60 and C60(OH)n, different doses of each agent were incubated with the L929, C6, or U251 cell line, and the number of adherent, live cells was assessed by crystal violet staining after 24 h. In accordance with the results of Sayes et al. (2005), the data presented in Figure 2A clearly show that pristine fullerene was at least three orders of magnitude more toxic to the tested cell lines than its hydroxylated counterpart [LC50 values were 0.25 μg/ml and 800–1000 μg/ml for C60 and C60(OH)n, respectively]. Microscopic examination revealed a dramatic change in the morphology of nano-C60–treated cells. In contrast to control cells, nano-C60–treated cells lost their processes and became round and detached from the culture well surface after only 6 h of treatment (Fig. 2B). The observed morphological changes followed by cell detachment were indicative of rapid cell death. Accordingly, the crystal violet analysis of cell viability at various time points following nano-C60 addition revealed an extremely fast kinetics of its cytotoxic action, reaching almost maximal efficiency after 6 h of cultivation (Fig. 2C, line). In the same time range, C60(OH)n failed to cause any discernible changes in cellular morphology (data not shown) or to induce cell death (Fig. 2C, bars). Therefore, pristine C60 was a much faster and more efficient cytotoxic agent than its hydroxylated derivative. Importantly, the cytotoxic activity of crystalline C60 was completely unrelated to the residual amount (<10% wt/wt) of organic solvent intercalated into its lattice (Sayes et al., 2005), as THF was without any toxic effect (data not shown) even at concentrations 100-fold higher (10 μg/ml) than its estimated residual presence (1 μg/ml nano-C60 contains <0.1 μg/ml THF). FIG. 2. Open in new tabDownload slide Different cytotoxic efficiency and kinetics of nano-C60 and C60(OH)n. (A) The cells were incubated for 24 h in the absence (control) or presence of nano-C60 and C60(OH)n at different concentrations, and the cell viability was assessed by crystal violet staining. (B) Light micrographs of normal L929 cells (control) and L929 cells exposed to 1 μg/ml nano-C60 for 6 h. (C) L929 cells were treated with 1 μg/ml nano-C60 (line) or 1000 μg/ml C60(OH)n (bars), and the cell viability was analyzed by crystal violet assay at the indicated time points (nano-C60) or after 6 h [C60(OH)n] (B). The values in A are means from four independent experiments (SD values were <15% of the corresponding means), while the values in C are mean ± SD of triplicate measurements and are representative of three experiments (*p < 0.05). FIG. 2. Open in new tabDownload slide Different cytotoxic efficiency and kinetics of nano-C60 and C60(OH)n. (A) The cells were incubated for 24 h in the absence (control) or presence of nano-C60 and C60(OH)n at different concentrations, and the cell viability was assessed by crystal violet staining. (B) Light micrographs of normal L929 cells (control) and L929 cells exposed to 1 μg/ml nano-C60 for 6 h. (C) L929 cells were treated with 1 μg/ml nano-C60 (line) or 1000 μg/ml C60(OH)n (bars), and the cell viability was analyzed by crystal violet assay at the indicated time points (nano-C60) or after 6 h [C60(OH)n] (B). The values in A are means from four independent experiments (SD values were <15% of the corresponding means), while the values in C are mean ± SD of triplicate measurements and are representative of three experiments (*p < 0.05). Nano-C60 and C60(OH)n Induce Distinct Types of Cell Death In the following experiments, we compared the ability of nano-C60 and C60(OH)n to induce apoptotic or necrotic cell death. To discriminate between the two distinct types of cell death, we used double staining with annexin V–FITC and PI. Annexin V binds to the phosphatidylserine that is typically exposed at the outer side of cell membrane during apoptosis, while PI only enters the cells with membrane damage that occurs in necrotic cell death. Therefore, normal, healthy cells are annexin−/PI− (Fig. 3A, 3B, lower left quadrant), apoptotic cells express phosphatidylserine, but have preserved membrane integrity (annexin+/PI−; lower right quadrant), while necrotic cells with damaged membrane stain for both annexin V and PI (annexin+/PI+; upper right quadrant). The results presented in Figures 3A and 3B clearly show that the treatment of U251 cells with C60(OH)n led to a significant increase in the number of apoptotic, but not necrotic, cells (Fig. 3A), while nano-C60 caused a massive increase in the number of necrotic, but not apoptotic, cells (Fig. 3B). The analysis of cellular DNA content, which is reduced in apoptosis due to DNA fragmentation, was consistent with the data from annexin/PI staining. Namely, the number of hypodiploid, presumably apoptotic, cells with fragmented DNA (sub-G1 fraction of PI-stained cells) markedly increased upon the treatment of U251 cells with fullerol (Fig. 3C), while pristine fullerene was without effect (Fig. 3D). Similar results were obtained with C6 and L929 cells (data not shown). The ability of nano-C60 to cause cell membrane damage indicative of necrosis was also confirmed by a significant increase in LDH release (159 ± 15%, n = 3, p < 0.05), which was not observed after C60(OH)n treatment. Due to a difference in the kinetics of cytotoxic action of C60(OH)n and nano-C60 , the assessment of apoptosis/necrosis presented in Figures 3A and 3C and Figures 3B and 3D was performed after 24 and 6 h, respectively. It should be noted, however, that the appearance of annexin+ cells in nano-C60–treated cultures could not be observed even at earlier time points (0.5–4 h; data not shown), which excluded the possibility that some early apoptotic events were overlooked. Finally, we used z-VAD-fmk, a pharmacological inhibitor of caspase activation and subsequent apoptotic cell death, to evaluate the role of apoptosis in fullerene-mediated cytotoxicity. In accordance with the data presented in Figures 3A–3D, z-VAD-fmk exerted a significant protective effect in C60(OH)n-treated U251 cultures, but completely failed to rescue U251 cells from the toxicity of nano-C60 (Fig. 3E). Therefore, pure C60 and C60(OH)n apparently employ distinct cytotoxic mechanisms resulting in the preferential induction of caspase-independent necrosis and caspase-dependent apoptosis, respectively. FIG. 3. Open in new tabDownload slide Pure and hydroxylated C60 induce different types of cell death. (A–D) U251 cells were incubated without (control) or with 1000 μg/ml C60(OH)n for 24 h (A, C) or with 1 μg/ml nano-C60 for 6 h (B, D). The number of cells staining with annexin V–FITC and/or PI (A, B), or those with fragmented DNA (PIlow sub-G1 fraction) (C, D), was assessed by flow cytometry. (E) The cytotoxicity of nano-C60 (1 μg/ml) and C60(OH)n (1000 μg/ml) toward U251 cells was investigated after 24-h incubation in the absence or presence of the pan-caspase inhibitor z-VAD-fmk (0.5μM). The values in A–D are means ± SD from three separate experiments, while those in E are mean ± SD of triplicates and are representative of three experiments (*p < 0.05). FIG. 3. Open in new tabDownload slide Pure and hydroxylated C60 induce different types of cell death. (A–D) U251 cells were incubated without (control) or with 1000 μg/ml C60(OH)n for 24 h (A, C) or with 1 μg/ml nano-C60 for 6 h (B, D). The number of cells staining with annexin V–FITC and/or PI (A, B), or those with fragmented DNA (PIlow sub-G1 fraction) (C, D), was assessed by flow cytometry. (E) The cytotoxicity of nano-C60 (1 μg/ml) and C60(OH)n (1000 μg/ml) toward U251 cells was investigated after 24-h incubation in the absence or presence of the pan-caspase inhibitor z-VAD-fmk (0.5μM). The values in A–D are means ± SD from three separate experiments, while those in E are mean ± SD of triplicates and are representative of three experiments (*p < 0.05). Cytotoxicity of Nano-C60, but Not C60(OH)n, Depends on ROS Generation We next investigated the involvement of oxidative stress in the observed cytotoxic action of nano-C60 and C60(OH)n. The treatment of C6 cells with C60(OH)n for 18 h failed to induce detectable intracellular production of ROS, as judged by unaltered intracellular DHR fluorescence (Fig. 4A, lower panel). On the other hand, a considerable increase in DHR fluorescence was observed in C6 cells after 3 h of incubation with pristine C60 (Fig. 4A, upper panel), indicating a significant intracellular generation of ROS. It should be noted that a shorter time course was chosen for nano-C60 because of the promptness of its cytotoxic action. Nevertheless, the inability of C60(OH)n to generate ROS was confirmed in the experiments in which the incubation time was reduced from 18 h to 3 or 6 h (data not shown). We used a powerful antioxidant agent, NAC (Zafarullah et al., 2003), to examine the role of ROS production in fullerene-mediated cytotoxicity. While NAC was completely unable to prevent C60(OH)n-mediated tumor cell killing (Fig. 4B), it markedly downregulated the production of oxygen radicals and completely prevented viability loss, induction of necrosis (measured as number of annexin+/PI+ cells), and LDH release associated with nano-C60 treatment (Fig. 4C). Moreover, an increase in MDA levels in C6 cells, reflecting membrane lipid peroxidation, was readily observed upon addition of nano-C60 and efficiently blocked by NAC treatment (Fig. 4C). The protective effect of NAC was also observed in U251 and L929 cells, and lasted for at least 24 h, as demonstrated by crystal violet staining (data not shown) and the completely preserved morphology of cells treated with nano-C60 (Fig. 4D). Because fluorescence-activated cell sorting analysis could not distinguish cell-derived from nano-C60–produced oxygen radicals, we used a fluorescence microplate reader to detect DHR fluorescence in cell-free nano-C60 suspension. However, the results presented in Figure 4E show that the time-dependent increase in DHR fluorescence in nano-C60–treated C6 cultures was slightly less pronounced than that observed in the absence of cells. Therefore, it appears that intracellular increase in ROS production was not a cellular response to nano-C60 treatment, but rather depended on the ability of pristine fullerene to directly generate oxygen radicals. Collectively, these results suggest that nano-C60–produced oxygen radicals are involved in lipid peroxidation and the consequent necrotic cell death, while C60(OH)n-triggered apoptosis seems to be ROS independent. FIG. 4. Open in new tabDownload slide The involvement of ROS in cytotoxic action of nano-C60. (A) C6 cells were incubated in the absence (control) or presence of nano-C60 (1 μg/ml) or C60(OH)n (1000 μg/ml). The production of ROS was measured by flow cytometric analysis of DHR fluorescence after 3 h (nano-C60) or 18 h [C60(OH)n]. (B) The viability of C6 cells incubated for 24 h without (control) or with C60(OH)n (1000 μg/ml), in the presence or absence of the antioxidant NAC (2mM). (C) The influence of NAC (2mM) on various parameters determined after exposure of C6 cells to nano-C60 (1 μg/ml) for 3 h (DHR, MDA) or 6 h (viability, number of annexin+/PI+ cells, LDH release). (D) Light micrographs of L929 cells incubated for 24 h without (control) or with nano-C60 (1 μg/ml), in the presence or absence of NAC (2mM). (E) The ability of nano-C60 (1 μg/ml) to generate ROS in C6 cultures and cell-free suspension in cell culture medium was assessed by measuring DHR fluorescence in a microplate reader. The values in B, C, and E are presented as mean ± SD of triplicates and are representative of at least three experiments, with the exception of values for DHR and annexin+/PI+ cells in C, which are means ± SD from three separate experiments (*p < 0.05 refers to nano-C60 treatment without NAC [C] or the corresponding DHR value obtained in cell-free conditions). FIG. 4. Open in new tabDownload slide The involvement of ROS in cytotoxic action of nano-C60. (A) C6 cells were incubated in the absence (control) or presence of nano-C60 (1 μg/ml) or C60(OH)n (1000 μg/ml). The production of ROS was measured by flow cytometric analysis of DHR fluorescence after 3 h (nano-C60) or 18 h [C60(OH)n]. (B) The viability of C6 cells incubated for 24 h without (control) or with C60(OH)n (1000 μg/ml), in the presence or absence of the antioxidant NAC (2mM). (C) The influence of NAC (2mM) on various parameters determined after exposure of C6 cells to nano-C60 (1 μg/ml) for 3 h (DHR, MDA) or 6 h (viability, number of annexin+/PI+ cells, LDH release). (D) Light micrographs of L929 cells incubated for 24 h without (control) or with nano-C60 (1 μg/ml), in the presence or absence of NAC (2mM). (E) The ability of nano-C60 (1 μg/ml) to generate ROS in C6 cultures and cell-free suspension in cell culture medium was assessed by measuring DHR fluorescence in a microplate reader. The values in B, C, and E are presented as mean ± SD of triplicates and are representative of at least three experiments, with the exception of values for DHR and annexin+/PI+ cells in C, which are means ± SD from three separate experiments (*p < 0.05 refers to nano-C60 treatment without NAC [C] or the corresponding DHR value obtained in cell-free conditions). Nano-C60 and C60(OH)n Differently Cooperate with the Cytotoxic Action of Oxidative Stress–Inducing Agents Having demonstrated that the cytotoxicity of nano-C60, but not C60(OH)n, depends on production of oxygen radicals, we wanted to examine if these fullerene preparations could influence the toxicity of other ROS-generating agents. To that effect, we assessed the cytotoxic effects of simultaneous application of nano-C60 or C60(OH)n with hydrogen peroxide and peroxynitrite donor SIN-1, the agents well known for their ability to induce oxidative stress–mediated cell death (Bauer et al., 1998; Wang and Joseph, 1999). In contrast to pristine C60, C60(OH)n significantly improved the viability of L929 cells treated with H2O2 or SIN-1 (Figs. 5A and 5B). We next sought to confirm these observations by using mathematical analysis of drug interaction based on the approach by Chou and Talalay (1984). As shown in Figures 5D and 5E, the CI for combination of C60(OH)n with H2O2 or SIN-1 was > 1 throughout the 0.1–0.99 efficiency range, thus confirming the putative ability of hydroxylated fullerene to antagonize the cytotoxic action of both agents. On the other hand, the CI for combination of nano-C60 with H2O2 or SIN-1 was around or less than 1 (Figs. 5D and 5E), indicating additive/synergistic cooperation in killing L929 cells. Similar results were obtained with U251 and C6 cells (data not shown). These data suggest that pure C60 cooperates with, while its hydroxylated derivative antagonizes, the cytotoxic action of oxidative stress–inducing agents. Interestingly, C60(OH)n readily antagonized the oxidative stress–dependent cytotoxicity of C60 nanocrystals (Figs. 5C and 5F), thus further reinforcing the concept of cell-damaging/pro-oxidant versus cytoprotective/antioxidant activity of pristine fullerene and its hydroxylated counterpart, respectively. FIG. 5. Open in new tabDownload slide Hydroxylated C60 antagonizes, and nano-C60 cooperates with, the cytotoxic action of H2O2 and SIN-1. (A, B) Viability of L929 cells exposed for 24 h to various doses of H2O2 (A) or SIN-1 (B), in the presence or absence of nano-C60 (0.25 μg/ml) or C60(OH)n (250 μg/ml). (C) Viability of L929 cells treated for 24 h with different concentrations of nano-C60, in the presence or absence of C60(OH)n (250 μg/ml). (D–F) The interaction of nano-C60 and C60(OH)n with H2O2 (D) or SIN-1 (E), as well as the interaction of nano-C60 and C60(OH)n (F) in causing L929 cell death was assessed by Chou-Talalay analysis as described in the “Materials and Methods” section. The results in A–C are presented as mean ± SD of triplicate observations and are representative of at least three experiments, while the CI values in D–F are means ± SD from four separate experiments (*p < 0.05 in A–C refers to the corresponding H2O2-, SIN-1-, or nano-C60–treated cultures, while *p < 0.05 in D–F indicates significant antagonism [CI > 1] or synergism [CI < 1]). FIG. 5. Open in new tabDownload slide Hydroxylated C60 antagonizes, and nano-C60 cooperates with, the cytotoxic action of H2O2 and SIN-1. (A, B) Viability of L929 cells exposed for 24 h to various doses of H2O2 (A) or SIN-1 (B), in the presence or absence of nano-C60 (0.25 μg/ml) or C60(OH)n (250 μg/ml). (C) Viability of L929 cells treated for 24 h with different concentrations of nano-C60, in the presence or absence of C60(OH)n (250 μg/ml). (D–F) The interaction of nano-C60 and C60(OH)n with H2O2 (D) or SIN-1 (E), as well as the interaction of nano-C60 and C60(OH)n (F) in causing L929 cell death was assessed by Chou-Talalay analysis as described in the “Materials and Methods” section. The results in A–C are presented as mean ± SD of triplicate observations and are representative of at least three experiments, while the CI values in D–F are means ± SD from four separate experiments (*p < 0.05 in A–C refers to the corresponding H2O2-, SIN-1-, or nano-C60–treated cultures, while *p < 0.05 in D–F indicates significant antagonism [CI > 1] or synergism [CI < 1]). DISCUSSION The present study describes the rapid ROS-dependent, caspase-independent necrotic cell death that occurs within only few hours of exposure to pure buckminsterfullerene aggregates (nano-C60). On the other hand, polyhydroxylated C60 caused delayed, ROS-independent, caspase-mediated apoptosis. It should be noted that the observed toxic effects were not restricted to the transformed cell lines, as similar results were obtained in primary rat astrocytes and fibroblasts, as well as in rat and mouse peritoneal macrophages (Isakovic et al., unpublished observation). While cytotoxic/antiproliferative effects of pristine C60 and its various water-soluble derivatives, including C60(OH)n, have been observed in different experimental systems (Bogdanovic et al., 2004; Bosi et al., 2004; Chen et al., 2004; Lu et al., 1998; Mashino et al., 2003; Sayes et al., 2004, 2005; Tsuchiya et al., 1996; Yang et al., 2002), their capacity for preferential induction of apoptosis or necrosis has not been compared. Although the release of LDH observed after 48-h incubation of human dermal fibroblasts, astrocytes, or liver carcinoma cells with nano-C60 (Sayes et al., 2005) is consistent with necrotic cell death, secondary necrosis following an initially apoptotic death could not be excluded. A line of evidence from the present study, including the results of cell membrane asymmetry/integrity assessment by annexin V/PI staining or LDH release test, as well as the data from DNA fragmentation analysis, clearly demonstrate that cell death induced by nano-C60 and C60(OH)n proceeds exclusively through the necrotic and apoptotic pathways, respectively. Since aspartate-specific cysteine proteases, termed caspases, are apparently required for apoptotic, but not necrotic, cell death (Ferrari et al., 1999; Los et al., 2002), the protective effect of caspase inhibition in C60(OH)n-treated cells, but not C60-treated cells, further supports the view that pristine fullerene induces necrotic cell death, as opposed to the mainly pro-apoptotic activity of its hydroxylated derivative. The mode of cell death might profoundly influence the healing of the surrounding tissue, since necrotic cell products could initiate additional injury by promoting inflammation (Jaeschke et al., 2002). On the other hand, in view of the immunostimulatory properties of necrotic cells and resistance of tumor cells to apoptosis, it has been proposed that necrosis might be more efficient than apoptosis in inducing tumor regression (Edinger and Thompson, 2004; Galluci et al., 1999; Reiter et al., 1999). Therefore, the ability to rapidly induce necrotic, rather than apoptotic, cell death could pose a difficulty for counteracting the toxicity of nanocrystalline C60, as well as potentially increase its efficiency in cancer therapy. By using the redox-sensitive fluorescent dye DHR, MDA measurement, and cell incubation with the well-known antioxidant agent NAC, we have shown that nano-C60–induced necrosis was exclusively a consequence of oxidative stress that probably resulted in lipid peroxidation–mediated damage of the cell membrane. The putative involvement of oxygen radicals in nano-C60–induced necrosis is consistent with the ability of another antioxidant, L-ascorbic acid, to completely prevent lipid peroxidation and ensuing cell death in nano-C60–treated cultures of human dermal fibroblasts, described recently by Sayes et al. (2005). Based on their observation that C60 nanocrystals generate superoxide anions in a cell-free system (Sayes et al., 2004), the authors assumed that this feature of nano-C60 could be solely responsible for the induction of oxidative stress and subsequent cell death. However, it was not possible to conclude from these studies whether the target cells contributed to production of oxygen radicals in response to treatment with pure C60. By comparing the nano-C60–evoked increase in DHR fluorescence in tumor cell cultures and cell-free solution, we have been able to provide some direct evidence in support of the hypothesis by Sayes et al. (2004), as the cells themselves apparently did not contribute to ROS generation in our experiments. Moreover, we have demonstrated a slight, but significant, decrease in the ability of nano-C60 to generate oxygen radicals in the presence of cells, which might be a consequence of the cellular antioxidative defense. Indeed, it has been shown that cells increase their production of antioxidant glutathione in response to oxidative stress induced by exposure to pristine C60 (Sayes et al., 2005). However, it still remains to be established if ROS production and subsequent damage were limited to cell membrane, or nano-C60 could somehow gain access to the cell cytoplasm. In contrast to nano-C60–triggered necrosis, apoptosis induced by C60(OH)n proceeded entirely in a ROS-independent fashion. Furthermore, C60(OH)n significantly antagonized the cytotoxic action of the oxidative stress–inducing agents H2O2 and peroxynitrite donor SIN-1, as demonstrated by the Chou-Talalay approach. This is consistent with the previously reported ability of C60(OH)n to prevent oxidative stress and subsequent cell death in various experimental settings (Chen et al., 2004; Dugan et al., 1996; Jin et al., 2000; Murugan et al., 2002; Tsai et al., 1997), supporting a general view that C60(OH)n mainly acts as a “free radical sponge” with only mild cytotoxic activity (Bosi et al., 2003). On the other hand, the present study confirmed the recently described capacity of nanocrystalline C60 to generate oxygen radicals in cell-free systems (Sayes et al., 2004), while extending this observation by demonstrating ROS production in nano-C60–treated cells. This pro-oxidant activity might account for our novel finding that pristine C60, in contrast to its hydroxylated derivative, readily cooperated in a synergistic fashion with H2O2 and peroxynitrite in the induction of cell death. It seems conceivable to assume that the mechanisms underlying the pro- versus antioxidant effects of pristine and hydroxylated fullerene might be related to differences in their chemical structures. Namely, the transmission electron microscopy images and light-scattering measurements obtained in the present study confirm that a substantial fraction of pristine C60, unlike the fullerene core in intentionally water-solubilized derivatives, is contained in the interior of the nanocrystalline aggregates of approximately 90–100 nm in size (Fortner et al., 2005; Sayes et al., 2004). As a consequence, C60 in such conditions remains largely underivatized (>99%), and its high toxicity is therefore consistent with the finding that ROS generation and cytotoxicity decrease with increasing derivatization of the fullerene cage (Sayes et al., 2004). However, it remains to be revealed how this structural difference could lead to distinct oxidant and cytotoxic properties. To further emphasize the complexity of the oxidant behavior of fullerenes in biological systems, we note that even polyhydroxylated fullerenes could generate oxygen radicals under certain circumstances (e.g., upon photosensitization) (Kamat et al., 1998, 2000). On the other hand, the ability of nano-C60 to produce ROS does not seem to depend on the presence of light (Sayes et al., 2004), which is consistent with our results that exposure to visible light or UV did not affect the cytotoxicity of nano-C60 (unpublished data). In contrast to the mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity of polyhydroxylated C60, the present study clearly demonstrates the ability of pure crystalline C60 to induce rapid ROS-dependent necrosis and to synergistically enhance the cytotoxicity of other oxidative stress–inducing agents. This is consistent with a recent study in which the presence of pristine C60 in water caused lipid peroxidation–mediated damage to fish brain (Oberdorster, 2004), but the possible relevance of our findings for the in vivo toxicity of nano-C60 is still to be investigated. While the difference in the cytotoxic mechanisms of pure versus hydroxylated C60 suggests distinct remediation for their unwarranted biological effects, their divergent cytotoxic/cytoprotective properties also provide grounds for the development of fullerenes as either cytoprotective or anticancer therapeutics. This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia (Grant No. 145073). 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Author notes *Institute of Biochemistry, School of Medicine, University of Belgrade, 11000 Belgrade, Serbia and Montenegro; †Vinca Institute of Nuclear Sciences, 11000 Belgrade, Serbia and Montenegro; ‡Institute for Biological Research, 11000 Belgrade, Serbia and Montenegro; §Faculty of Physics, University of Belgrade, 11000 Belgrade, Serbia and Montenegro; and ¶Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, 11000 Belgrade, Serbia and Montenegro © The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
TI - Distinct Cytotoxic Mechanisms of Pristine versus Hydroxylated Fullerene
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfj127
DA - 2006-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/distinct-cytotoxic-mechanisms-of-pristine-versus-hydroxylated-jqbdlodqF2
SP - 173
EP - 183
VL - 91
IS - 1
DP - DeepDyve
ER -