Metal oxide nanoparticles (NPs), such as ZnO, ZnFe₂O₄, and Fe₂O₃, are widely used in industry. However, little is known about the cellular pathways involved in their potential toxicity. Here, we particularly investigated the key molecular pathways that are switched on after exposure to sub-toxic doses of ZnO, ZnFe₂O₄, and Fe₂O₃ in the in vitro rat alveolar macrophages (NR8383). As in our model, the calculated IC₅₀ were respectively 16, 68, and more than 200 μg/mL for ZnO, ZnFe₂O₄, and Fe₂O₃; global gene and protein expression profiles were only analyzed after exposure to ZnO and ZnFe₂O₄ NPs. Using a rat genome microarray technology, we found that 985 and 1209 genes were significantly differentially expressed in NR8383 upon 4 h exposure to ¼ IC₅₀ of ZnO and ZnFe₂O₄ NPs, respectively. It is noteworthy that metallothioneins were overexpressed genes following exposure to both NPs. Moreover, Ingenuity Pathway Analysis revealed that the top canonical pathway disturbed in NR8383 exposed to ZnO and ZnFe₂O₄ NPs was eIF2 signaling involved in protein homeostasis. Quantitative mass spectrometry approach performed from both NR8383 cell extracts and culture supernatant indicated that 348 and 795 proteins were differentially expressed upon 24 h exposure to ¼ IC₅₀ of ZnO and ZnFe₂O₄ NPs, respectively. Bioinformatics analysis revealed that the top canonical pathways disturbed in NR8383 were involved in protein homeostasis and cholesterol biosynthesis for both exposure conditions. While VEGF signaling was specific to ZnO exposure, iron homeostasis signaling pathway was specific to ZnFe₂O₄ NPs. Overall, the study provides resource of transcriptional and proteomic markers of response to ZnO and ZnFe₂O₄ NP-induced toxicity through combined transcriptomics, proteomics, and bioinformatics approaches.

There are many studies concerning titanium dioxide (TiO₂) nanoparticles (NP) toxicity. Nevertheless, there are few publications comparing in vitro and in vivo exposure, and even less comparing air–liquid interface exposure (ALI) with other in vitro and in vivo exposures. The identification and validation of common markers under different exposure conditions are relevant for the development of smart and quick nanotoxicity tests. In this work, cell viability was assessed in vitro by WST-1 and LDH assays after the exposure of NR8383 cells to TiO₂ NP sample. To evaluate in vitro gene expression profile, NR8383 cells were exposed to TiO₂ NP during 4 h at 3 cm² of TiO₂ NP/cm² of cells or 19 μg/mL, in two settings—submerged cultures and ALI. For the in vivo study, Fischer 344 rats were exposed by inhalation to a nanostructured aerosol at a concentration of 10 mg/m³, 6 h/day, 5 days/week for 4 weeks. This was followed immediately by gene expression analysis. The results showed a low cytotoxic potential of TiO₂ NP on NR8383 cells. Despite the absence of toxicity at the doses studied, the different exposures to TiO₂ NP induce 18 common differentially expressed genes (DEG) which are involved in mitosis regulation, cell proliferation and apoptosis and inflammation transport of membrane proteins. Among these genes, we noticed the upregulation of Ccl4, Osm, Ccl7 and Bcl3 genes which could be suggested as early response biomarkers after exposure to TiO₂ NP. On the other hand, the comparison of the three models helped us to validate the alternative ones, namely submerged and ALI approaches.