Few-Layer Graphene Induces Both Primary and Secondary Genotoxicity in in Vitro Alveolar Barrier Models

Abstract Background: Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). Results: In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p<0.05) cytotoxic response with each ENM following 24-hour exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p<0.05) micronuclei induction at 8µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress. Conclusions: Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.