Influence of TiO₂ nanoparticles on cellular antioxidant defense and its involvement in genotoxicity in HepG2 cells

We investigated the effects of two types of TiO₂ nanoparticles (<25 nm anatase, TiO₂-An; <100 nm rutile, TiO₂-Ru) on cellular antioxidant defense in HepG2 cells. We previously showed that in HepG2 cells, TiO₂ nanoparticles are not toxic, although they induce oxidative DNA damage, production of intracellular reactive oxygen species, and up-regulation of mRNA expression of DNA-damage-responsive genes (p53, p21, gadd45α and mdm2). In the present study, we measured changes in mRNA expression of several antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase, nitric oxide synthase, glutathione reductase and glutamate-cysteine ligase. As reduced glutathione has a central role in cellular antioxidant defense, we determined the effects of TiO₂ nanoparticles on changes in the intracellular glutathione content. To confirm a role for glutathione in protection against TiO₂-nanoparticle-induced DNA damage, we compared the extent of TiO₂-nanoparticle-induced DNA damage in HepG2 cells that were glutathione depleted with buthionine-(S,R)-sulfoximine pretreatment and in nonglutathione-depleted cells. Our data show that both types of TiO₂ nanoparticles up-regulate mRNA expression of oxidative-stress-related genes, with TiO₂-Ru being a stronger inducer than TiO₂-An. Both types of TiO₂ nanoparticles also induce dose-dependent increases in intracellular glutathione levels, and in glutathione-depleted cells, TiO₂-nanoparticle-induced DNA damage was significantly greater than in nonglutathione-depleted cells. Interestingly, the glutathione content and the extent of DNA damage were significantly higher in TiO₂-An- than TiO₂-Ru-exposed cells. Thus, we show that TiO₂ nanoparticles cause activation of cellular antioxidant processes, and that intracellular glutathione has a critical role in defense against this TiO₂-nanoparticle-induced DNA damage.