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

Michael J Burgum,Stephen J Evans,Gareth J Jenkins,Nicole Hondow,Martin J D Clift,Shareen H Doak,Afshin Tarat

doi:10.1186/s12951-021-00769-9

Michael J Burgum, Stephen J Evans + Show 5 more

Open Access

https://doi.org/10.1186/s12951-021-00769-9

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Abstract

BackgroundToxicological 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)).ResultsIn 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.ConclusionsTherefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

Highlights

engineered nanomaterials (ENMs) have unique physico-chemical features which provide the basis for novel applications in medicine, engineering and other such disciplines [26]
Neutral-few-layer graphene (FLG), amine-FLG, carboxyl-FLG particle and agglomerate sizes were investigated with several techniques including,plunge-freeze scanning electron microscopy (SEM), dynamic light scattering (DLS), atomic force microscopy (AFM) and Raman spectroscopy
FLG induces cytotoxicity and primary‐indirect DNA damage in TT1 cells This study aimed to evaluate the genotoxic potential of FLG and functionalised variants upon an epithelial barrier model of the lung

Summary

Introduction

ENMs have unique physico-chemical features which provide the basis for novel applications in medicine, engineering and other such disciplines [26]. Secondary genotoxicity is typically observed in vivo and is the result of DNA damage induced through a (sub)-chronic immune response, involving immune cell activation and recruitment, heightened inflammatory activity and subsequent oxidative stress, promoting genotoxicity in the surrounding epithelial cells [7]. Given both primary and secondary genotoxicity are important mechanisms for ENM induced DNA damage, it is important that they are both evaluated to develop a comprehensive understanding of a materials’ genotoxic potential. 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))

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