Graphene and graphene oxide induce ROS production in human HaCaT skin keratinocytes: the role of xanthine oxidase and NADH dehydrogenase

Abstract

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.

Skin irritation potential of graphene-based materials using a non-animal test

“…On these cells, GBMs exerted a significant cytotoxicity 11 mediated by ROS-dependent mitochondrial damage. 12 However, despite their wide use, HaCaT cells are a simplified in vitro model, useful as a firstround screening, also to investigate the mechanism of skin toxicity, but unable to completely predict the relevant toxicity after cutaneous exposure. Indeed, being a proliferating cell line, HaCaT cells fail to recapitulate in vitro one of the essential functions of the skin: the barrier function.…”

“…In addition, the inability of GBMs to reach the stratum basale may explain the apparent discrepancy between these results and those previously obtained on HaCaT skin keratinocytes, at least for FLG and GO, that induced significant cytotoxicity at high concentrations. [11][12][13] Indeed, HaCaT cells are a simplified model of proliferating keratinocytes more compliant with those typical of the stratum basale.…”

“…8 However, despite the importance of skin exposure and the dermotoxic potential of GBMs, very limited data on the cutaneous toxicity of these materials are currently available. Only few in vitro data regarding the effects on skin fibroblasts 9 and keratinocytes [10][11][12][13] are available, so far. In HaCaT skin keratinocytes, we previously demonstrated that FLG and GO reduced cell viability after interaction with plasma membrane.…”

“…11 The effect was significant after exposure times as long as 72 hours at concentrations higher than 1-10 µg mL −1 , involving a reactive oxygen species (ROS)-dependent mitochondrial dysfunction, driven by the activation of flavon-based oxidative enzymes. 12 Hence, considering the potential toxicity of GBMs at the cutaneous level, this study investigates skin irritation properties of a panel of different materials, including FLG, obtained by different procedures, disks of CVD-graphene monolayers, obtained by chemical vapor deposition, GO and its chemically reduced form rGO. Since no specific guidelines for skin testing of nanomaterials are currently available, a specific guideline given by the Organisation for Economic Co-operation and Development (OECD) for in vitro testing of chemicals at the skin level was followed.…”

Graphene-based materials do not impair physiology, gene expression and growth dynamics of the aeroterrestrial microalga Trebouxia gelatinosa

“…Conversely, in other green algae such as Chlamydomonas reinhardtii and Raphidocelis subcapitata, GBM treatments caused membrane damage due to the overproduction of ROS (Nogueira et al 2015;Mart ın-de-Luc ıa et al 2018). These differences may be explained considering that (i) in other organisms the ROS unbalance was caused by decoupling of the electron transport chain in mitochondrial lamellae induced by internalized GBMs (Pelin et al 2018), and (ii) Trebouxias are known to be tolerant to oxidative stress, having a strong constitutive antioxidant machinery able to scavenge an 'oxidative burst' within minutes after its insurgence (Kranner et al 2008;Candotto Carniel et al 2015). This was further confirmed by our preliminary tests implemented to define H 2 O 2 concentrations sufficient to affect the algae physiology, which revealed the presence of an extracellular catalase whose activity can remove low concentrations of H 2 O 2 before it can exert any negative effect.…”