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Few-layer graphene (FLG) has garnered much interest owing to applications in hydrogen storage and reinforced nanocomposites. Consequently, these engineered nanomaterials (ENMs) are in high demand, increasing occupational exposure. This investigation seeks to assess the inhalation hazard of industrially relevant FLG engineered with: (i) no surface functional groups (neutral), (ii) amine, and (iii) carboxyl group functionalization. A monoculture of human lung epithelial (16HBE14o-) cells is exposed to each material for 24-h, followed by cytotoxicity and genotoxicity evaluation using relative population doubling (RPD) and the cytokinesis-blocked micronucleus (CBMN) assay, respectively. Neutral-FLG induces the greatest (two-fold) significant increase (p < 0.05) in micronuclei, whereas carboxyl-FLG does not induce significant (p < 0.05) genotoxicity. These findings correlate to significant (p < 0.05) concentration-dependent increases in interleukin (IL)-8, depletion of intracellular glutathione (rGSH) and a depletion in mitochondrial ATP production. Uptake of FLG is evaluated by transmission electron microscopy, whereby FLG particles are observed within membrane-bound vesicles in the form of large agglomerates (>1 µm diameter). The findings of the present study have demonstrated the capability of neutral-FLG and amine-FLG to induce genotoxicity in 16HBE14o- cells through primary indirect mechanisms, suggesting a possible role for carboxyl groups in scavenging radicals produced via oxidative stress.

Background It is well established that toxicological evaluation of engineered nanomaterials (NMs) is vital to ensure the health and safety of those exposed to them. Further, there is a distinct need for the development of advanced physiologically relevant in vitro techniques for NM hazard prediction due to the limited predictive power of current in vitro models and the unsustainability of conducting nano-safety evaluations in vivo. Thus, the purpose of this study was to develop alternative in vitro approaches to assess the potential of NMs to induce genotoxicity by secondary mechanisms. Results This was first undertaken by a conditioned media-based technique, whereby cell culture media was transferred from differentiated THP-1 (dTHP-1) macrophages treated with γ-Fe2O3 or Fe3O4 superparamagnetic iron oxide nanoparticles (SPIONs) to the bronchial cell line 16HBE14o−. Secondly construction and SPION treatment of a co-culture model comprising of 16HBE14o− cells and dTHP-1 macrophages. For both of these approaches no cytotoxicity was detected and chromosomal damage was evaluated by the in vitro micronucleus assay. Genotoxicity assessment was also performed using 16HBE14o− monocultures, which demonstrated only γ-Fe2O3 nanoparticles to be capable of inducing chromosomal damage. In contrast, immune cell conditioned media and dual cell co-culture SPION treatments showed both SPION types to be genotoxic to 16HBE14o− cells due to secondary genotoxicity promoted by SPION-immune cell interaction. Conclusions The findings of the present study demonstrate that the approach of using single in vitro cell test systems precludes the ability to consider secondary genotoxic mechanisms. Consequently, the use of multi-cell type models is preferable as they better mimic the in vivo environment and thus offer the potential to enhance understanding and detection of a wider breadth of potential damage induced by NMs. Electronic supplementary material The online version of this article (10.1186/s12989-019-0291-7) contains supplementary material, which is available to authorized users.