Impact of physico-chemical properties on the toxicological potential of reduced graphene oxide in human bronchial epithelial cells

The increasing use of graphene-based materials (GBM) requires their safety evaluation, especially in occupational settings. The same physico-chemical (PC) properties that confer GBM extraordinary functionalities may affect the potential toxic response. Most toxicity assessments mainly focus on graphene oxide and rarely investigate GBMs varying only by one property. As a novelty, the present study assessed the in vitro cytotoxicity and genotoxicity of six reduced graphene oxides (rGOs) with different PC properties in the human bronchial epithelial 16HBE14o - cell line. Of the six materials, rGO1-rGO4 only differed in the carbon-to-oxygen (C/O) content, whereas rGO5 and rGO6 were characterized by different lateral size and number of layers, respectively, but similar C/O content compared with rGO1. The materials were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, laser diffraction and dynamic light scattering, and Brunauer-Emmett-Teller analysis. Cytotoxicity (Luminescent Cell Viability and WST-8 assays), the induction of reactive oxygen species (ROS; 2 ',7 '-dichlorofluorescin diacetate-based assay), the production of cytokines (enzyme-linked immunosorbent assays) and genotoxicity (comet and micronucleus assays) were evaluated. Furthermore, the internalization of the materials in the cells was confirmed by laser confocal microscopy. No relationships were found between the C/O ratio or the lateral size and any of the rGO-induced biological effects. However, rGO of higher oxygen content showed higher cytotoxic and early ROS-inducing potential, whereas genotoxic effects were observed with the rGO of the lowest density of oxygen groups. On the other hand, a higher number of layers seems to be associated with a decreased potential for inducing cytotoxicity and ROS production.