Background
A key aspect of any new material safety assessment is the evaluation of their in vivo genotoxicity. Graphene oxide (GO) has been studied for many promising applications, but there are remaining concerns about its safety profile, especially after inhalation. Herein we tested whether GO lateral dimension, comparing micrometric (LGO) and nanometric (USGO) GO sheets, has a role in the formation of DNA double strand breaks in mouse lungs. We used spatial resolution and differential cell type analysis to measure DNA damages in both epithelial and immune cells, after either single or repeated exposure.

Results
GO induced DNA damages were size and dose dependent, in both exposure scenario. After single exposure to a high dose, both USGO and LGO induced significant DNA damage in the lung parenchyma, but only during the acute phase response (P<0.05 for USGO; P<0.01 for LGO). This was followed by a fast lung recovery at day 7 and 28 for both GOs. When evaluating the chronic impact of GO after repeated exposure, only a high dose of LGO induced long-term DNA damages in lung alveolar epithelia (at 84 days, P<0.05). Regardless of size, low dose GO did not induce any significant DNA damage after repeated exposure. A multiparametric correlation analysis of our repeated exposure data revealed that transient or persistent inflammation and oxidative stress were associated to either recovery or persistent DNA damages. For USGO, recovery from DNA damage was correlated to efficient recovery from acute inflammation (i.e., significant secretion of SAA3, P<0.001; infiltration of neutrophils, P<0.01). In contrast, the persistence of LGO in lungs was associated to a long-lasting presence of multinucleated macrophages (up to 84 days, P<0.05), an underlying inflammation (IL-1α secretion up to 28 days, P<0.05) and the presence of persistent DNA damages at 84 days.

Conclusions
Overall these results highlight the importance of the exposure scenario used. We showed that LGO was more genotoxic after repeated exposure than single exposure due to persistent lung inflammation. These findings are important in the context of human health risk assessment and toward establishing recommendations for a safe use of graphene based materials in the workplace.
Keywords: lungs; graphene oxide; genotoxicity; γ-H2AX; inflammation; toxicology.