Nano-C60 cytotoxicity is due to lipid peroxidation

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C₆₀, a fullerene aggregate that readily forms when pristine C₆₀ is added to water. Nano-C₆₀ was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses 50 ppb (LC₅₀=2–50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C₆₀ colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C₆₀ inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C₆₀ was completely prevented.