Distinct Cytotoxic Mechanisms of Pristine versus Hydroxylated Fullerene

Abstract

The mechanisms underlying the cytotoxic action of pure fullerene suspension (nano-C 60 ) and water-soluble polyhydroxylated fullerene [C 60 (OH) n ] were investigated. Crystal violet assay for cell viability demonstrated that nano-C 60 was at least three orders of magnitude more toxic than C 60 (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-C 60 caused rapid (observable after few hours), reactive oxygen species (ROS)-associated necrosis characterized by cell membrane damage without DNA fragmentation. In contrast, C 60 (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-C 60 toxicity, but not C 60 (OH) n toxicity, while the pan-caspase inhibitor z-VAD-fmk blocked C 60 (OH) n -induced apoptosis, but not nano-C 60 -mediated necrosis. Finally, C 60 (OH) n antagomozed, while nano-C 60 synergized with, the cytotoxic action of oxidative stress-inducing agents hydrogen peroxide and peroxynitrite donor 3-morpholinosydnonimine. Therefore, unlike polyhydroxylated C 60 that exerts mainly antioxidant/cytoprotective and only mild ROS-independent pro-apoptotic activity, pure crystalline C 60 seems to be endowed with strong pro-oxidant capacity responsible for the rapid necrotic cell death.