Graphene and its derivatives are heralded as “miracle” materials with manifold applications in different sectors of society from electronics to energy storage to medicine. The increasing exploitation of graphene-based materials (GBMs) necessitates a comprehensive evaluation of the potential impact of these materials on human health and the environment. Here, we discuss synthesis and characterization of GBMs as well as human and environmental hazard assessment of GBMs using in vitro and in vivo model systems with the aim to understand the properties that underlie the biological effects of these materials; not all GBMs are alike, and it is essential that we disentangle the structure–activity relationships for this class of materials.

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.

Titanium dioxide nanoparticles (TiO2NPs) suspensions (concentration 1.0 g/L) in synthetic sweat solution were applied on Franz cells for 24 h using intact and needle-abraded human skin. Titanium content into skin and receiving phases was determined. Cytotoxicity (MTT, AlamarBlue® and propidium iodide, PI, uptake assays) was evaluated on HaCat keratinocytes after 24 h, 48 h, and seven days of exposure. After 24 h of exposure, no titanium was detectable in receiving solutions for both intact and damaged skin. Titanium was found in the epidermal layer after 24 h of exposure (0.47 ± 0.33 μg/cm2) while in the dermal layer, the concentration was below the limit of detection. Damaged skin, in its whole, has shown a similar concentration (0.53 ± 0.26 μg/cm2). Cytotoxicity studies on HaCaT cells demonstrated that TiO2NPs induced cytotoxic effects only at very high concentrations, reducing cell viability after seven days of exposure with EC50s of 8.8 × 10−4 M (MTT assay), 3.8 × 10−5 M (AlamarBlue® assay), and 7.6 × 10−4 M (PI uptake, index of a necrotic cell death). Our study demonstrated that TiO2NPs cannot permeate intact and damaged skin and can be found only in the stratum corneum and epidermis. Moreover, the low cytotoxic effect observed on human HaCaT keratinocytes suggests that these nano-compounds have a potential toxic effect at the skin level only after long-term exposure.

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 μg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS.

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Graphene related materials, if prepared with non-irritant exfoliation agents, do not induce skin irritation on a 3D model of human epidermis, following the OECD guideline 439.