Fullerenols are attractive molecules for clinical drug delivery, because their hollow caged structure allows for both encapsulation of therapeutic and/or diagnostic loads within the fullerene cage, and attachment to the scaffolding of the fullerene backbone (Bolskar, 2008
). Additionally, derivatization of fullerene to fullerenol enhances its solubility and has been reported to dramatically decrease the toxicity of fullerene in some in vitro
systems (Sayes, et al., 2004
). Thorough evaluation of the biocompatibility and safety of nanotechnology platforms destined for clinical use is imperative (Stern and McNeil, 2008
; Hall, et al., 2007
). Ideally, characterization of these platforms should include initial screens utilizing in vitro
systems to identify possible adverse effects and mechanisms of toxicity. Fullerenol toxicity has been demonstrated in numerous animal and human cell lines (Gelderman, et al., 2008
, Yamawaki and Iwai, 2006
, Sayes et al., 2004
, Su et al.
, 2009). There are, however, no reports in the literature on the cytotoxic effects of fullerenol on kidney cells, and few reports on plausible intracellular targets of this nanomaterial.
Fullerenol nanoparticles used in this present study were purchased commercially. Elemental analysis of fullerenol was conducted by two independent laboratories for molecular formula determination, and fullerenol nanoparticles were tested for metal impurities in our laboratory by ICP-MS (See Supplemental Data
). The empirical molecular formula for fullerenol was concluded to be C60
and served as the basis for molecular weight and sample concentration determinations in this study. All fullerenol preparations were virtually free of metal contaminants that could potentially contribute to the biologic and toxic responses observed in this present study (See Supplemental Data
). In particular, brominated iron was used as a catalyst during commercial preparation of fullerenol. Quantitative ICP-MS analysis of fullerenol used in this study was determined to contain less than 0.01% of metal iron. Fullerenol preparations were also virtually free of bromine (See Supplemental Data
Renal cell responses to fullerenol exposure were evaluated in the porcine proximal tubule cell model, LLC-PK1. Carbon based nanomaterials have been documented to interfere with assay markers and cause variable and/or inconclusive assay results in classical toxicology assays (Monteiro-Riviere et al., 2009
). Thus, care must be taken to insure that nanoparticles do not cause assay interference. The use of multiple complementary in vitro
toxicology assays is also advised to confirm nanoparticle effects. In this study, possible fullerenol assay interference was evaluated in all experiments conducted, and when applicable, an orthogonal assay was utilized to confirm study results. Since fullerene derivatives, including fullerenol, can reduce tetrazolium based salts, the traditional MTT and XTT cytotoxicity assays were not used to evaluate cell viability effects in this study.
In this study, treatment of LLC-PK1 cells for 24 and 48 hours with fullerenol in the low millimolar range was cytotoxic, decreasing cell density and compromising the membrane integrity of LLC-PK1 cells, as determined by the SRB assay and Trypan Blue assay, respectively. Interestingly, in a study by Qingnuan, et al.
, administration of a 1 mg dose of technetium labeled fullerenol (99m
) to mice resulted in retention of approximately 5.25% of the injected dose in the kidney, or a concentration of ~15 mM, at 24 h post fullerenol exposure (Qingnuan et al., 2002
). Given these data, the cytotoxic fullerenol concentrations determined here, 6.0 – 60.0 mM, may be relevant to kidney exposures expected in vivo
Fullerenol’s mechanism of cell death appears to be cell type specific, and both apoptotic and non-apoptotic mechanisms have been reported in the literature (Yamawaki and Iwai, 2006
, Gelderman et al., 2008
). Previous studies by other research groups have identified oxidative stress as a primary mechanism of cytotoxicity for underivatized fullerene and nanomaterials in general. Mitochondrial dysfunction induced by fullerenol may be expected to result in ROS production, and oxidative stress. However, fullerenol treatment resulted in only limited oxidative stress in this study, as determined by lipid peroxidation and total glutathione measurement data of fullerenol treated cells. The minimal oxidative stress observed confirms other previous reports that fully hydroxylated fullerenes produce minimal oxygen radicals and lipid peroxidation products in culture (Sayes et al., 2004
, Xia et al., 2006
). It is certainly plausible, that in this study, fullerenol attenuated any oxidative stress response resulting from mitochondrial dysfunction by the reported free radical scavenging properties of this nanomaterial.
Fullerenol strongly induced conversion of LC3-I to the autophagy biomarker, LC3-II, in LLC-PK1 cells. LC3-II conversion correlated with lysosomal uptake of Lysotracker Red dye by fullerenol treated cells in both a dose-responsive and time-responsive manner. These results support the use of the Lysotracker Red assay as an initial screen for autophagy interaction following nanoparticle exposure, as reported by our group previously (Stern, et al., 2008
.). The robust autophagic response shown here for fullerenol builds upon previous reports of induction of this pathway by fullerene-based nanoparticles (Yamawaki and Iwai, 2006
, Zhang et al., 2009
, Harhaji et al., 2007
). The underlying mechanism(s) responsible for fullerene interaction with the autophagy pathway has not been elucidated. Given that the autophagy response seen here occurred at sub-lethal fullerenol concentrations, it is plausible that autophagy upregulation is a protective cell mechanism intended to remove fullerenol from the cell. With increasing fullerenol concentrations, this autophagic pathway could potentially be overwhelmed as autophagosomes and autophagolysosomes accumulate increasing amounts of fullerenol nanoparticles. To support this hypothesis, future works should include detection of fullerenol within autophagosomes and/or autophagolysosomes for definitive confirmation of uptake of this nanoparticle within autophagy machinery.
There are reports in the literature detailing the effects of carbon-based nanomaterials on actin cytoskeletal structure and organization (Tian, et al., 2006
, Walker, et al., 2009
). These studies demonstrated compromised actin filament integrity following administration of single- or multi- walled carbon nanotubes in culture. Cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, as there is evidence that cytoskeleton disruption can interfere with both autophagy processing and mitochondrial capacity.
Cytoskeleton proteins, more specifically, microtubules have been shown to assist in autophagosome formation, movement, and fusion with lysosome (Fass et al., 2006
, Kochl et al., 2006
). Studies in Saccharomyces cerevisiae (baker’s yeast) have identified actin-related protein complexes that target the autophagy transport machinery (Monastyrska, et al., 2008
). Recently, a study has suggested a role for actin in mammalian autophagy (Lee et al., 2010
). Lee et al.
have shown that histone deacetylase-6 is involved in autophagosome-lysosome fusion during basal autophagy in mammals, by promoting actin remodeling (Lee et al., 2010
Nocodazole was used as a positive control in our actin confocal studies. Nocodazole is more commonly used to elicit microtubule disruption, however, there is documented evidence in the literature that this compound also has disruptive effects on the actin cytoskeletal (Takenouchi et al., 2004
). Specific interaction and/or binding of fullerenol particles with actin protein was not determined in this study, however given the hydrodynamic size of fullerenol nanoparticles used here, it is expected that this compound can freely diffuse through the cell membrane and enter the cell. It is certainly plausible that fullerenol could bind to actin proteins, thereby potentially affecting actin polymerization and depolymerization states. Interestingly, concentrations of fullerenol that elicited actin filament effects also elicited mitochondrial dysfunction and ATP loss. Induction of mitochondrial dysfunction has also recently been documented for other carbon-based nanoparticles (Yang, et al., 2010
These data led us to postulate that fullerenol induced cytoskeletal disruption, subsequently disrupts homeostatic mitophagy (mitochondrial specific autophagy) which then leads to mitochondrial dysfunction and ATP depletion, and finally cell death. Elegant studies conducted in yeast have demonstrated a role for autophagy in mitochondrial maintenance (Zhang, et al., 2007a
). These studies showed that yeast strains with mutated autophagy genes had lower oxygen consumption rates, lower mitochondrial membrane potential, high levels of reactive oxygen species (ROS), and an accumulation of dysfunctional mitochondria compared to wild-type yeast strains. The current data suggest that autophagic maintenance of cellular mitochondria may also be important in mammalian cells. The apparent partial recovery of mitochondrial function and ATP levels resulting from 3-MA co-treatment supports this hypothesis. Co-treatment of fullerenol and 3-MA, however, was not sufficient for complete recovery of ATP beyond a maximum restorative value of 20% of control. Autophagy-independent fullerenol-induced cytoskeletal disruption, or direct effects of fullerenol on mitochondrial function, could account for the lack of complete recovery.
There are many other examples from animal and in vitro
models of human disease that also demonstrate the importance of autophagy in mitochondrial maintenance. For example, the ubiquitin ligase protein, Parkin is often mutated in familial forms of Parkinson disease and appears to play a role in recruitment of damaged mitochondria for autophagic degradation (Narendra et al., 2009
). Knockout of Parkin in mice results in loss of mitochondrial function (Palacino et al., 2004
). Excessive autophagy, resulting from either unregulated induction or blocked autophagosome cycling, can also have a detrimental effect on mitochondria. In a mouse model of the lysosomal storage disorder G(M1)-gangliosidosis, knockout of the lysosomal beta-galactosidase enzyme in mice resulted in autophagosome accumulation and loss of mitochondrial membrane potential, that were ameliorated by treatment with the autophagy inhibitor, 3-methyladenine (Takamura et al., 2008
). In an in vitro
model of neurodegeneration, nerve growth factor withdrawal from primary neurons in culture resulted in cytoskeleton disruption, autophagosome accumulation and loss of mitochondrial membrane potential (Yang et al., 2007
). The disruption of mitochondrial membrane potential by nerve growth factor withdrawal could be prevented by treatment with the autophagy inhibitor 3-methyladenine.
In addition to autophagy-mediated mitochondrial dysfunction, there is also ample evidence that actin cytoskeleton disruption itself can interfere with mitochondrial capacity directly (Anesti and Scorrano, 2006
). For example, yeast mutants with actin instability, displaying a clumped actin phenotype similar to that observed following treatment of LLC-PK1 cells with fullerenol, also had greatly reduced mitochondrial membrane potential (Gourlay et al., 2004
). A study in neuroblastoma cells demonstrated that disorganization of the actin cytoskeleton by overexpression of transgelin coincided with mitochondria depolarization (Ward et al., 2010
Lastly, it is important to note that a direct fullerenol mitochondrial mechanism may be involved in this study, with fullerenol-induced mitochondrial damage resulting in mitophagy induction, disruption of actin cytoskeleton, and apoptotic cell death. Indeed, there is evidence of direct inhibition of mitochondrial function by fullerenol (Ueng et al., 1997
). Mitochondria have been reported to serve as a switch between apoptosis and autophagy, with increasing levels of stress resulting in the initial induction of mitophagy, followed by caspase activation, apoptotic cell death, and finally necrotic cell death under the most extreme stress conditions (Nishida et al., 2008
). Initial induction of mitophagy by the cell to clear damaged mitochondria is consistent with the fact that in this study, there is evidence of autophagy induction at sub-lethal fullerenol concentrations ( and ) that are approximately one order of magnitude lower than fullerenol concentrations that induced ATP depletion () and mitochondrial dysfunction (). Furthermore, the TEM image of fullerenol treated cells shows the presence of damaged mitochondria (). With increasing fullerenol concentrations, possible direct fullerenol effects on cytoskeletal structure could serve as a negative feedback mechanism to stall stress-induced mitophagy (cause mitophagy dysfunction) and trigger cell death. Alternatively, direct fullerenol-induced mitochondrial damage could result in downstream disruption of actin cytoskeleton structure due to alteration in calcium homeostasis (Nicotera et al., 1992
) and/or diminished cellular bioenergetics (Molitoris et al., 1996
In summary, fullerenol cytotoxicity in the LLC-PK1 cells was associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by cotreatment with the autophagy inhibitor, 3-methyladenine. As there is evidence that cytoskeleton disruption can interfere with both autophagy processing and mitochondrial capacity, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction, and loss of mitochondrial capacity. While this proposed mechanism is consistent with the data presented, other mechanisms are certainly plausible, as discussed above. Nanoparticle-induced cytoskeleton disruption, as well as autophagy and mitochondrial dysfunctions, have been reported commonly in the literature, suggesting the proposed mechanism of fullerenol toxicity may be relevant for a variety of nanomaterials. It is important to note, however, that nanomaterials as a class include highly varied physicochemical characteristics, thus it would not be appropriate to attribute this mechanism of fullerenol toxicity to the entire class.