EPR Spin Trapping Evaluation of ROS Production in Human Fibroblasts Exposed to Cerium Oxide Nanoparticles: Evidence for NADPH Oxidase and Mitochondrial Stimulation

To better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxic properties of the industrially used cerium oxide nanoparticles (nano-CeO(2)), we investigated their effects on reactive oxygen species formation and changes in the antioxidant pool of human dermal and murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagnetic resonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cells with the nanoparticles dose-dependently triggered the release in the culture medium of superoxide dismutase- and catalase-inhibitable DEPMPO/hydroxyl radical adducts (DEPMPO-OH) and ascorbyl radical, a marker of ascorbate depletion. This DEPMPO-OH formation occurred 2 to 24 h following removal of the particles from the medium and paralleled with an increase of cell lipid peroxidation. These effects of internalized nano-CeO(2) on spin adduct formation were then investigated at the cellular level by using specific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant. When micromolar doses of nano-CeO(2) were used, weak DEPMPO-OH levels but no loss of cell viability were observed, suggesting that cell signaling mechanisms through protein synthesis and membrane NADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a 25-60-fold higher DEPMPO-OH formation together with a decrease in cell viability, early apoptosis induction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrial source and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanisms of nano-CeO(2)-induced free radical formation in cells, in vitro EPR and spectrophotometric studies suggest that, contrary to Fe(2+) ions, the Ce(3+) redox state at the surface of the particles is probably not an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo.