80 Advancing In Vitro Airway Models for Engineered Nanomaterial Genotoxicity Testing

Abstract A primary toxicological focus has been the development of cellular models and exposure systems representative of nanomaterial inhalation due to the inevitable risk of environmental or occupational exposure. This current study compared the genotoxic and immunogenic potential of two nanomaterials: copper oxide (CuO) and zinc oxide (ZnO) and a positive-particle control tungsten-carbide cobalt (WC-Co), in type-II alveolar epithelial cells (A549) utilising cell culture and exposure systems of increasing complexity. Firstly, treatment was undertaken in submerged-culture at a dose range of 1–100 μg/cm2 for all materials. This was repeated with A549s established at the air-liquid interface using a quasi-ALI exposure technique. Finally, complexity of the quasi-ALI exposure was increased with the addition of a dynamic-flow system (Kirkstall Quasi-Vivo,600) allowing media circulation on the basal side of A549 cultures. For all exposures, cytotoxicity, chromosomal damage, and immunogenicity were assessed by relative population doubling, cytokinesis blocked micronucleus assay and interleukin-8 quantification respectively. Compared to the untreated control, significant cytotoxicity (~50%;p<0.05)) was observed in A549s treated with CuO at 100 μg/cm2 following static quasi-ALI exposure. Moreover, both CuO and ZnO demonstrated a significantly increased micronucleus frequency and IL-8 response at 20 and 100 μg/cm2. WC-Co did not promote any (pro-)inflammatory response under any exposure scenario, however genotoxicity was observed in the static and dynamic-flow ALI cultures. Subsequently the variation between the applied exposure scenarios across the chosen test materials has highlighted the importance of considering the impact of the culture environment and exposure system applied when undertaking nanomaterial genotoxicity assessment in vitro.