Investigating the immunomodulatory nature of zinc oxide nanoparticles at sub-cytotoxic levels in vitro and after intranasal instillation in vivo

BACKGROUND This study evaluates the time-dependent pro-inflammatory response of the model human lung epithelial cells (A549) to industrially relevant zinc oxide nanoparticles (ZnO NPs). In terms of toxicity, ZnO-NPs are categorised into the group of high toxicity nanomaterials. However information on pro-inflammatory potential of these NPs at sub-toxic concentrations is limited. Understanding how cellular defense mechanisms function in the presence of sub-cytotoxic concentrations of these NPs is vital. Moreover, there is an urgent need for additional in vivo studies addressing pulmonary toxicity due to accidental inhalation of ZnO NPs. RESULTS Exposure to sub-cytotoxic ZnO NP concentrations (20 μg/mL) induced significant up-regulation of mRNA for the pro-inflammatory cytokine IL-8 and redox stress marker heme oxygenase-1, along with increased release of IL-8. The highest pro-inflammatory response was recorded after 4 to 6 hr exposure to ZnO NPs over a 24 hr period. Pre-treatment of A549 cells with the sulfhydryl antioxidant N-acetyl cysteine (at 5 mM) resulted in significant reduction of the up-regulation of inflammatory markers, confirming the role of reactive oxygen species in the observed immunomodulatory effects, independent of cytotoxicity. Furthermore, we report for the first time that, intranasal instillation of a single dose (5 mg/kg) of pristine or surfactant-dispersed ZnO NPs can cause pulmonary inflammation, already after 24 hr in a murine model. This was confirmed by up-regulation of eotaxin mRNA in the lung tissue and release of pro-inflammatory cytokines in the sera of mice exposed to ZnO NPs. CONCLUSION Our study highlights that even at sub-cytotoxic doses ZnO NPs can stimulate a strong inflammatory and antioxidant response in A549 cells. ZnO NP mediated cytotoxicity may be the outcome of failure of cellular redox machinery to contain excessive ROS formation. Moreover exposure to a single but relatively high dose of ZnO NPs via intranasal instillation may provoke acute pulmonary inflammatory reactions in vivo.