An in vitro approach to assess the cyto-genotoxic potential of graphene

Graphene-family nanomaterials (GFNs) due to their unique properties have numerous promising applications in several fields as electronics, optics, biotechnology and medicine. The widespread applications of GFNs is arising concerns about their impact on human health due to occupational, consumer and environmental exposures. Inhalation exposure is particularly relevant for GFNs since some of these nanomaterials have aerodynamic sizes and can deposit in the human respiratory tract. Research towards the toxicity of GFNs is limited/controversial, and proper toxicological risk assessment is needed. Within the scope of the PLATOX project, funded by the FP7-SIINN ERANET on Nanosafety, commercially available GFNs were selected and their in vitro toxicity was assessed in human lung fibroblasts MRC-5 cells and RAW 264.7 macrophages (0-50 µg/cm2). Cell viability was assessed by LDH release and AlamarBlue assays. Graphene’s cellular uptake, effects on cell-cycle dynamics and inflammatory responses were analyzed by flow cytometry. Results showed that viability of MRC-5 cells was not affected by any of the GFNs, contrarily to the cell viability of RAW 264.7 cells that decrease upon exposure to all GFNs. Carboxyl graphene and single layer graphene markedly impaired metabolic activity of macrophages. Carboxyl graphene, graphene nanoplatelets and single layer graphene oxide were highly internalized by RAW 264.7 cells. Also, GFNs affected differently cell cycle dynamics of macrophages. Upregulation of pro-inflammatory TNF-α and MCP-1, was observed for single layer graphene oxide and graphene oxide. These findings highlight the importance of a comparative in vitro toxicity screening of GFNs with the ultimate goal of establishing a toxicological ranking.