Lung recovery from DNA damage induced by graphene oxide is dependent on size, dose and inflammation profile

Abstract

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.

Functioning human lung organoids model pulmonary tissue response from carbon nanomaterial exposures

“…We selected two extensively studied carbon-based NMs, GO sheets 1,4,5,37 and MWCNT 8,24,26,27 , to validate our HLO exposure model. Thin GO sheets are known to induce minimal-to-transient pulmonary toxicity in vivo 37 , whilst MWCNT (Mitsui-7) have consistently been associated with adverse pulmonary effects 27,30,41,43 .…”

“…The current standard for pulmonary tissue response analysis combines mono/co-culture in vitro systems 1,2 , with time-intensive and ethically charged in vivo models [3][4][5] . Though mono/coculture systems allow for large-scale screening of nanomaterials (NMs), and have provided a better understanding of cellular effects with regards to physicochemical features of NMs 2 , such systems are often too simplistic.…”

“…GO has fast become one of the most studied two-dimensional NMs, mainly due to its ease of production, high surface area exploitable for drug loading, and oxygen-rich functional groups that enable its good colloidal dispersion in physiological milieu. Because of its similarity in chemical composition to other carbon nanostructures, namely CNTs, efforts are made to evaluate the impact of GO on lungs after inhalation 4,5,34,35 . Our group and others have previously shown that GO-induced toxicity is strongly dependent on the lateral dimension of the material 1,2,36 .…”

“…In contrast, nanometric-sized GO sheets are not associated with any adverse pulmonary effects 37,38 . More recently, it was shown that murine lungs can rapidly recover from repeated low dose exposures to micrometric GO sheets, despite initial signs of material-induced lung inflammation and genotoxicity 4,5 .…”

“…The impact of small (30-750nm, sGO) and large (11-67µm, lGO) GO sheets in lateral dimension was compared with long and rigid MWCNT on HLOs after 1d and 7d exposure. Key biological endpoints relevant to pulmonary tissue response of carbon-based NMs 4,5,37 were investigated, including cytotoxicity, material-cell interactions, tissue remodelling and pulmonary fibrosis.…”