Understanding the cellular uptake and genotoxic potential of industrial relevant nanomaterials utilizing electron microscopy and the ToxTracker assay <i>in vitro</i>

Evaluating the genotoxic potential of nanomaterials (NMs) presents unique challenges not associated with traditional toxicological assessment. A key question in any NM focused toxicity study is whether the material has reached the target cell and what its subsequent subcellular localization is. This current study aimed to assess the potential of a panel of industrially relevant NMs; TiO2-NM102, TiO2-NM105, TiO2-E171, silica, polyethylene, polystyrene, carbon black, gold nanorods, tungsten carbide/cobalt, and tungsten carbide, to undergo cellular uptake in mouse embryonic stem cells, which are applied in the ToxTracker genotoxicity assay. Ultrastructural cellular analysis by transmission electron microscopy was undertaken following 100 μg/ml treatment with the test NMs for 24 h; any observed uptake was confirmed by energy-dispersive X-ray spectroscopy. Induction of DNA damage, cytotoxicity, p53 activation, protein stress, and oxidative stress was evaluated by the ToxTracker assay following 24-h treatment with the test NMs (0-100 μg/ml) in the absence of S9. TiO2-NM105, silica, polystyrene, carbon black, and tungsten carbide were all shown to undergo cellular uptake, localized in membrane-bound vesicles within the cytoplasm. None of the internalized NMs promoted a genotoxic response in ToxTracker, and similarly, no DNA damage was observed by the materials not internalized. Interestingly, of the internalized NMs, only polystyrene caused a slight cytotoxic response at 100 μg/ml treatment (10% loss in cell viability). Of the NMs not internalized, cytotoxicity was observed in mES cells treated with 100 μg/ml TiO2-NM102 (15%), polyethylene (15%), gold nanorods (35%), and tungsten carbide/cobalt (45%). In summary, this study demonstrated that TiO2-NM105, silica, polystyrene, carbon black, and tungsten carbide are non-genotoxic in vitro despite undergoing cell uptake in the ToxTracker cells. A continued focus is needed to supplement NM genotoxicity studies with cellular uptake analysis.