Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

Abstract

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C60OHx), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

Nanocomposite and Nanostructured Carbon-based Films as Growth Substrates for Bone Cells

“…Thus, the fullerene C 60 layers in our study were colonized with human osteoblast-like MG 63 cells to a similar extent as standard cell culture polystyrene dishes. We considered this result rather surprising, because many studies have reported cytotoxic and even genotoxic effects of fullerenes (Xu et al, 2009;Johnson-Lyles et al, 2010; for a review, see Bacakova et al, 2008a). However, the cytotoxic action of fullerenes was usually associated with suspending fullerenes in the cell culture medium (Yamawaki et al, 2006;Gelderman et al, 2008) or UV-light irradiation (Prylutska et al, 2010), while our fullerene layers were resistant to be dissolved in a water environment, and the cells were cultured in the dark.…”

The mechanisms of graphene-based materials-induced programmed cell death: a review of apoptosis, autophagy, and programmed necrosis

“…81 It was elicited that lysosomal membrane permeabilization, lysosomal membrane rupture, and cytoskeleton disruption might be involved in lysosomal membrane destabilization, eventually resulting in autophagosome accumulation. 92 GO also leads to a decrease of p62, a protein that directly binds to LC3 and selectively incorporates into autophagosomes. 93 Moreover, p62 is preferentially degraded by autolysosomes; this degradation process is inversely correlated with autophagosomal maturation and degradation.…”

Analysis of zinc oxide nanoparticles binding proteins in rat blood and brain homogenate

“…In previous studies, it has been suggested that the relationship between mitochondria dysfunction and reactive oxygen species production could be the main pathway in causing nanotoxic effects in various cell lines via mitochondria dysfunction, linked with ATP production. [32][33][34][35][36] Based on these results, NPs might be capable of causing toxic effects induced by mitochondria dysfunction by interacting with proteins in ATP production, especially within electron transport, such as ATP synthase. In addition, NPs inside the vacuole or cell could induce cytotoxic effects through interactions with tubulin, such as disrupting cytoskeletons and causing cell death.…”

“…Previously, fullerene was reported to cause cell death from cytoskeleton disruption and autophagic vacuole accumulation. 35 Regulation of actin polymerization or depolymerization was also involved with ZnO SM20(−) and ZnO SM20(+) . Commonly, tubulin and heat shock protein were also found to bind tightly to all types of ZnO NPs.…”