The in vitro and in vivo toxicity of copper oxide nanoparticles (CuO NPs) is attributed to both particle and dissolved copper ion species. However, a clear understanding of (1) the specific cellular responses that are modulated by the two species and (2) the temporal dynamics in toxicity, as the proportional amount of particulate and ionic forms change over time, is lacking. In the current study, in vitro responses to microparticulate CuO (CuO MPs), CuO NPs, and dissolved Cu2+ were characterized in order to elucidate particle and ion-induced kinetic effects. Particle dissolution experiments were carried out in a relevant cell culture medium, using CuO NPs and MPs. Mouse lung epithelial cells were exposed for 2-48 h with 1-25 µg/mL CuO MPs, CuO NPs, or 7 and 54 µg/mL CuCl2. Cellular viability and genome-wide transcriptional responses were assessed. Dose and time-dependent cytotoxicity were observed in CuO NP exposed cells, which was delayed and subtle in CuCl2 and not observed in CuO MPs treated cells. Analyses of differentially expressed genes and associated pathway perturbations showed that dissolved ions released by CuO NPs in the extracellular medium are insufficient to account for the observed potency and cytotoxicity. Further organization of gene expression results in an Adverse Outcome Pathway (AOP) framework revealed a series of key events potentially involved in CuO NPs toxicity. The AOP is applicable to toxicity induced by metal oxide nanoparticles of varying solubility, and thus, can facilitate the development of in vitro alternative strategies to screen their toxicity.
Keywords: Soluble metal oxides; adverse outcome pathway; gene expression; in vitro; nanotoxicity.