Few-layer graphene induces both primary and secondary genotoxicity in epithelial barrier models in vitro

• Published in: Journal of nanobiotechnology Vol. 19; no. 1; pp. 24 - 15
• Main Authors: Burgum, Michael J, Clift, Martin J D, Evans, Stephen J, Hondow, Nicole, Tarat, Afshin, Jenkins, Gareth J, Doak, Shareen H
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 19-01-2021; BioMed Central; BMC
• Subjects: Alveolar Epithelial Cells; Analysis; Animals; Cell Differentiation; Cell Line; Cell Survival - drug effects; Coculture Techniques; DNA damage; DNA Damage - drug effects; Filaggrin Proteins; Graphene; Graphite - toxicity; Humans; Macrophages; Macrophages - drug effects; Management; Mutagenicity Tests - methods; Nanostructures - chemistry; Oxidative Stress - drug effects; THP-1 Cells; United Kingdom
• ISSN: 1477-3155; 1477-3155
• DOI: 10.1186/s12951-021-00769-9

Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)).
In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p < 0.05) cytotoxic response with each ENM following 24-h exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p < 0.05) micronuclei induction at 8 µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5 mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress.
Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.