EPR spin trapping evaluation of ROS production in human fibroblasts exposed to cerium oxide nanoparticles: Evidence for NADPH oxidase and mitochondrial stimulation

International audienceTo better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxicproperties of the industrially used cerium oxide nanoparticles (nano-CeO2), we investigated theireffects on reactive oxygen species formation and changes in the antioxidant pool of human dermaland murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagneticresonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cellswith 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 theparticles from the medium and paralleled with an increase of cell lipid peroxidation. These effects ofinternalized nano-CeO2 on spin adduct formation were then investigated at the cellular level by usingspecific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant.When micromolar doses of nano-CeO2 were used, weak DEPMPO–OH levels but no loss of cell viabilitywere observed, suggesting that cell signaling mechanisms through protein synthesis and membraneNADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a25–60-fold higher DEPMPO–OH formation together with a decrease in cell viability, early apoptosisinduction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrialsource and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanismsof nano-CeO2-induced free radical formation in cells, in vitro EPR and spectrophotometricstudies suggest that, contrary to Fe2+ ions, the Ce3+ redox state at the surface of the particles is probablynot an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo