Assessment of genotoxicity induced by subchronic exposure to graphene in HaCaT human skin cell line

Abstract

The applications of graphene-based materials (GBMs) and their processing involve prolonged contact with cellular barriers such as human skin. Even though the potential cytotoxicity of graphene has been studied in recent years, the impact of long-term graphene exposure has rarely been explored. We tested in the HaCaT epithelial cells, in vitro, the effect of subchronic treatments with sublethal doses of four different, well-characterized GBMs, two commercial graphene oxides (GO) and two few-layer graphenes (FLG). Cells were exposed weekly to low doses of the GBMs for 14 days, 30 days, 3 months, and 6 months. GBMs-cells uptake was assessed by confocal microscopy. Cell death and cell cycle were determined by fluorescence microscopy and cytometry. DNA damage was measured by comet assay and γ-H2AX staining, followed by the determination of p-p53 and p-ATR by immunolabeling. Subchronic exposure to different GBMs at noncytotoxic doses has potential genotoxic effects on HaCaT epithelial cells that can be recovered depending on the GBM and exposure time. Specifically, GO-induced genotoxicity can be detected after 14 and 30 days from treatment. At this time, FLG appears less genotoxic than GO, and cells can recover more quickly when genotoxic pressure disappears after some days of removal of the GBM. Long-term exposure, 3 and 6 months, to different GBMs induces permanent, nonreversible, genotoxic damage comparable to the exerted by arsenite. This should be considered for the production and future applications of GBMs in scenarios where low concentrations of the material interact chronically with epithelial barriers.

Keywords:

• Graphene
• subchronic
• skin cells
• genotoxiciy
• DDR

Author contributions

J.F.R., E.V., and M.D.P. are responsible for the study design. J.F.R. and V.J.G. performed the experiments. J.F.R. and V.J.G. are responsible for data and analysis. J.F.R. wrote the manuscript and conceived the figures. All coauthors contributed to critical revisions of the manuscript. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by author(s).

Data availability statement

The data sets during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

The financial support from the 785219-Graphene Core 2 and 881603-Graphene Core 3 European Union (Flagship project) and the Spanish Ministerio de Economía y Competitividad (project CTQ2017-88158-R) are gratefully acknowledged.