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The fullerene C(60), due to the physicochemical properties of its spherical cage-like molecule build exclusively from carbon atoms, is able to both scavenge and generate reactive oxygen species. While this unique dual property could be exploited in biomedicine, the low water solubility of C(60) hampers the investigation of its behavior in biological systems. The C(60) can be brought into water by solvent extraction, by complexation with surfactants/polymers, or by long-term stirring, yielding pristine (unmodified) fullerene suspensions. On the other hand, a modification of the C(60) core by the attachment of various functional groups results in the formation of water-soluble fullerene derivatives. Assessment of toxicity associated with C(60) preparations is of pivotal importance for their biomedical application as cytoprotective (antioxidant), cytotoxic (anticancer), or drug delivery agents. Moreover, the widespread industrial utilization of fullerenes may also have implications for human health. However, the alterations in physicochemical properties imposed by the utilization of different methods for C(60) solubilization profoundly influence toxicological effects of fullerene preparations, thus making the analysis of their potential therapeutic and environmental toxicity difficult. This review provides a comprehensive evaluation of the in vitro and in vivo toxicity of fullerenes, focusing on the comparison between pristine and derivatized C(60) preparations and the mechanisms of their toxicity to mammalian cells and tissues.

The present study investigated the hemolytic properties of fullerene (C(60)) nanoparticles prepared by solvent exchange using tetrahydrofuran (nC(60)THF), or by mechanochemically assisted complexation with macrocyclic oligosaccharide gamma-cyclodextrin (nC(60)CDX) or the copolymer ethylene vinyl acetate-ethylene vinyl versatate (nC(60)EVA-EVV). The spectrophotometrical analysis of hemoglobin release revealed that only nC(60)THF, but not nC(60)CDX or nC(60)EVA-EVV, was able to cause lysis of human erythrocytes in a dose- and time-dependent manner. Atomic force microscopy revealed that nC(60)THF-mediated hemolysis was preceded by erythrocyte shrinkage and increase in cell surface roughness. A flow cytometric analysis confirmed a decrease in erythrocyte size and demonstrated a significant increase in reactive oxygen species production in red blood cells exposed to nC(60)THF. The nC(60)THF-triggered hemolytic activity was efficiently reduced by the antioxidants N-acetylcysteine and butylated hydroxyanisole, as well as by serum albumin, the most abundant protein in human blood plasma. These data indicate that nC(60)THF can cause serum albumin-preventable hemolysis through oxidative stress-mediated damage of the erythrocyte membrane.

Purpose: The fullerene (C60/C70 mixture-C60/70) nanocrystalline suspension prepared by solvent exchange method using tetrahydrofyran (THF/nC60/70) and polyhydroxylated C60/70 [C60/70(OH)n] were compared for their ability to modulate cytotoxicity of the proinflammatory cytokine tumor necrosis factor (TNF)., Materials and Methods: TNF-induced cytotoxicity was assessed in L929 fibrosarcoma cells by crystal violet assay. The type of cell death (apoptosis/necrosis), production of reactive oxygen species, mitochondrial depolarization and caspase activation were determined by flow cytometry using the appropriate reporter dyes., Results: THF/nC60/70 augmented, while C60/70(OH)n reduced the cytotoxicity of TNF. The numbers of cells undergoing apoptosis/necrosis, as well as of those displaying the activation of apoptosis-inducing enzymes of caspase family, were respectively increased or reduced by THF/nC60/70 or C60/70(OH)n. The antioxidant N-acetylcysteine and mitochondrial permeability transition inhibitor cyclosporin A each partly blocked the cytotoxic action of TNF, indicating the involvement of oxidative stress and mitochondrial dysfunction in the TNF cytotoxicity. Accordingly, THF/nC60/70 or C60/70(OH)n potentiated or suppressed, respectively, TNF-triggered oxidative stress and mitochondrial depolarization., Conclusion: The ability of different fullerene preparations to modulate TNF-induced oxidative stress and subsequent cell death suggests their potential value in the TNF-based cancer therapy or prevention of TNF-dependent tissue damage.

Because of the ability to induce cell death in certain conditions, the fullerenes (C(60)) are potential anticancer and toxic agents. The colloidal suspension of crystalline C(60) (nano-C(60), nC(60)) is extremely toxic, but the mechanisms of its cytotoxicity are not completely understood. By combining experimental analysis and mathematical modelling, we investigate the requirements for the reactive oxygen species (ROS)-mediated cytotoxicity of different nC(60) suspensions, prepared by solvent exchange method in tetrahydrofuran (THF/nC(60)) and ethanol (EtOH/nC(60)), or by extended mixing in water (aqu/nC(60)). With regard to their capacity to generate ROS and cause mitochondrial depolarization followed by necrotic cell death, the nC(60) suspensions are ranked in the following order: THF/nC(60)>EtOH/nC(60)>aqu/nC(60). Mathematical modelling of singlet oxygen ((1)O(2)) generation indicates that the (1)O(2)-quenching power (THF/nC(60)

Using the rat glioma cell line C6 and the human glioma cell line U251, we demonstrate the multiple mechanisms underlying the in vitro anticancer effects of the C(60) fullerene water suspension (nano-C(60) or nC(60)) produced by solvent exchange method. Nano-C(60) in a dose-dependent manner reduced the tumor cell numbers after 24 h of incubation. The observed antiglioma action of nC(60) at high concentration (1 microg/ml) was due to a reactive oxygen species-mediated necrotic cell damage that was partly dependent on oxidative stress-induced activation of extracellular signal-regulated kinase (ERK). On the other hand, low-dose nC(60) (0.25 microg/ml) did not induce either necrotic or apoptotic cell death, but caused oxidative stress/ERK-independent cell cycle block in G(2)/M phase and subsequent inhibition of tumor cell proliferation. Treatment with either high-dose or low-dose nC(60) caused the appearance of acidified intracytoplasmic vesicles indicative of autophagy, but only the antiglioma effect of low-dose nC(60) was significantly attenuated by inhibiting autophagy with bafilomycin A1. Importantly, primary rat astrocytes were less sensitive than their transformed counterparts to a cytostatic action of low-dose nC(60). These data provide grounds for further development of nC(60) as an anticancer agent.

We investigated the effect of gamma-irradiation on the cytotoxicity of pure C60 solubilized in water by using tetrahydrofuran (THF/n-C60 or THF/n-C60). In contrast to THF/n-C60, its gamma-irradiated counterpart failed to generate oxygen radicals and cause extracellular signal-regulated kinase (ERK)-dependent necrotic cell death in various types of mammalian cells. Moreover, gamma-irradiated THF/n-C60 protected cells from the oxidative stress induced by native THF/n-C60 or hydrogen peroxide. The observed biological effects were associated with gamma-irradiation-mediated decomposition of THF and subsequent derivatization of the n-C60 surface. These results for the first time demonstrate gamma-irradiation-mediated changes in the physico-chemical properties of THF-prepared nanocrystalline C60, resulting in a complete loss of its cytotoxic effect and its conversion to a cytoprotective agent.

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.