Nanoscale and fine zinc oxide particles: can in vitro assays accurately forecast lung hazards following inhalation exposures?

The development of accurate in vitro screening assays to assess lung hazard potential of nanomaterials is a highly desirable goal. However, some studies have noted little correlation between in vitro and in vivo results. Moreover, a recent National Academy of Sciences report predicts that future hazard testing will be conducted primarily using cell culture assays. The three major objectives of this study were to compare lung toxicity impacts of nanoscale (NZnO) vs fine zinc oxide (FZnO) particulates, assess predictability of in vitro cell culture systems, and compare effects of instillation vs inhalation exposures in rats. Physicochemical aspects of ZnO particle types were rigorously characterized and did not agree with specifications provided by the supplier; i.e., the ZnO particle types were closer in size than advertised. Rats were exposed in vivo either by intratracheal instillation to 1 or 5 mg/kg of nanoscale or fine size zinc oxide particle types or by inhalation to aerosols of 25 or 50 mg/m3 for 1 or 3 h. Lung inflammation, cytotoxicity, and histopathological endpoints were assessed at several time points postexposure. Three different in vitro culture conditions were utilized. Cultures of (1) rat lung epithelial cells, (2) primary alveolar macrophages, and (3) alveolar macrophages-L2 lung epithelial cell cocultures were incubated with fine or nano ZnO particles and evaluated for cytotoxicity biomarkers (LDH) and proinflammatory cytokines (MIP-2 and TNF-alpha). In vivo exposures to instilled or inhaled fine or nanoscale ZnO produced "metal fume fever" responses, characterized by transient short-term lung inflammatory or cytotoxic responses. Alternatively, in vitro exposures to fine or nanoscale ZnO particles produced minor cytotoxic responses at 4 and 24 h, only in cocultures and at the highest (particle overload) dose with little detectable proinflammatory cytokine generation (MIP-2, and TNF-alpha). To summarize, the comparisons of in vivo and in vitro toxicity measurements following nano or fine ZnO particle exposures demonstrated little convergence and few differences in potency.