Engineered Nanoparticles Induce DNA Damage in Primary Human Skin Cells, Even at Low Doses

It is well documented that various particulate matter — either incidental or engineered — are known to generate reactive oxygen species (ROS) in living cells. In circumstances where these reactive species are generated, antioxidant production is often increased. This balance in the biological reduction/oxidation (a.k.a. redox) state within the cell has not been thoroughly studied in exposures involving engineered nanoparticles. However, nanoparticle exposure has been postulated to induce a DNA damage cascade. In this study, we examined primary human dermal ̄broblasts (HDF) exposed to three di®erent, but commonly used engineered nanoparticles (i.e., ceriumdioxide (CeO2), titanium dioxide (TiO2) and zinc oxide (ZnO)) in an attempt to determine the potential DNA damaging e®ects through the analysis of ROS generation, relevant protein upregulation response and single and double DNA strand breaks. Cell death was most elevated with exposure to ZnO, followed by TiO2 and CeO2. ROS generation was measured at 1 h, 6 h and 24 h after exposure to particles via a cell-based DCFH-DA (2 0; 7 0-dichlor°uorescein-diacetate) assay and indicated that ZnO generated themost signi ̄cant amount ofROS. ZnO also caused upregulation of oxidative stress protein, heme oxygenase-1 and phosphorylation of p38; whereas CeO2 caused upregulation of superoxide dismutase. Results from the comet assay indicated that ZnO triggered signi ̄cant DNA damage in cells at relatively low dosing concentrations (20 ppm). Immunocytochemistry with ZnOtreated cells revealed notable DNA double strand breaks evidenced by a marked increase in the presence of -H2AX foci. This ̄nding was also indicated by western blot, as well as cell cycle arrest by the phosphorylation of cyclin-dependent kinase 1. These data suggest that the three particletypes induce di®erent degrees of DNA damage. And, of the three particle-types tested, exposure to ZnO nanoparticles may cause the most signi ̄cant DNA damage.