Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a research-grade GO1, and two commercial GOs, GO2 and GO3). Even though no significant effects were observed after 24 h, after 72 h the less oxidized compound (FLG) was the less cytotoxic, inducing mitochondrial and plasma-membrane damages with EC₅₀s of 62.8 μg/mL (WST-8 assay) and 45.5 μg/mL (propidium iodide uptake), respectively. By contrast, the largest and most oxidized compound, GO3, was the most cytotoxic, inducing mitochondrial and plasma-membrane damages with EC₅₀s of 5.4 and 2.9 μg/mL, respectively. These results suggest that only high concentrations and long exposure times to FLG and GOs could impair mitochondrial activity associated with plasma membrane damage, suggesting low cytotoxic effects at the skin level.

The extraordinary physicochemical properties of graphene-based nanomaterials (GBNs) make them promising tools in nanotechnology and biomedicine. Considering the skin contact as one of the most feasible exposure routes to GBNs, the mechanism of toxicity of two GBNs (few-layer-graphene, FLG, and graphene oxide, GO) towards human HaCaT skin keratinocytes was investigated. Both materials induced a significant mitochondrial membrane depolarization: 72 h cell exposure to 100 μg mL⁻¹ FLG or GO increased mitochondrial depolarization by 44% and 56%, respectively, while the positive control valinomycin (0.1 μg mL⁻¹) increased mitochondrial depolarization by 48%. Since the effect was not prevented by cyclosporine-A, it appears to be unrelated to mitochondrial transition pore opening. By contrast, it seems to be mediated by reactive oxygen species (ROS) production: FLG and GO induced time- and concentration-dependent cellular ROS production, significant already at the concentration of 0.4 μg mL⁻¹ after 24 h exposure. Among a panel of specific inhibitors of the major ROS-producing enzymes, diphenyliodonium, rotenone and allopurinol significantly reverted or even abolished FLG- or GO-induced ROS production. Intriguingly, the same inhibitors also significantly reduced FLG- or GO-induced mitochondrial depolarization and cytotoxicity. This study shows that FLG and GO induce a cytotoxic effect due to a sustained mitochondrial depolarization. This seems to be mediated by a significant cellular ROS production, caused by the activation of flavoprotein-based oxidative enzymes, such as NADH dehydrogenase and xanthine oxidase.

Graphene-based materials (GBMs) are a family of novel materials including graphene, few layer graphene (FLG), graphene oxide (GO), reduced graphene oxide (rGO) and graphene nanoplatelets (GNP). Currently, the risk posed by them to human health is associated mainly with the occupational exposure during their industrial and small-scale production or waste discharge. The most significant occupational exposure routes are inhalation, oral, cutaneous and ocular, inhalation being the majorly involved and most studied one. This manuscript presents a critical up-to-date review of the available in vivo toxicity data of the most significant GBMs, after using these exposure routes. The few in vivo inhalation toxicity studies (limited to 5-days of repeated exposure and only one to 5 days per week for 4 weeks) indicate inflammatory/fibrotic effects at the pulmonary level, not always reversible after 14/90 days. More limited in vivo data are available for the oral and ocular exposure routes, whereas the studies on cutaneous toxicity are at the initial stage. A long persistence of GBMs in rodents is recorded, while contradictory genotoxic data are reported. Data gap identification is also provided. Based on the available data, the occupational exposure limit cannot be determined. More experimental toxicity studies according to specific guidelines (tentatively validated for nanomaterials) and more information on the actual occupational exposure level to GBMs are needed. Furthermore, ADME (Absorption, Distribution, Metabolism, Excretion), genotoxicity, developmental and reproductive toxicity data related to the occupational exposure to GBMs have to be implemented. In addition, sub-chronic and/or chronic studies are still needed to completely exclude other toxic effects and/or carcinogenicity.