Development of in vitro DNA damage assays to properly assess the genotoxicity of graphene and other 2D materials.

Abstract

The increasing production and application of graphene-related materials (GRMs) and other 2D nanomaterials (2D materials) raise concerns about their potential genotoxicity. This study aims to assess the genotoxic effects of these materials using a modified in vitro comet assay and double-strand break (DSB) detection via the γH2AX assay, which is crucial for determining the safety of these compounds. Chinese hamster ovary cells (CHO–K1) were exposed to graphene oxide (GO), few-layer graphene (FLG), monolayer GO (mlGO), molybdenum disulfide (MoS2), and hexagonal boron nitride (hBN) at concentrations of 0, 0.05, 0.5, 5, 20, and 50 μg/mL for 24 h. Adaptations to the standard comet assay were implemented to address interference caused by 2D material agglomeration, which can hinder accurate DNA damage detection. Controlled, gentle mechanical shaking during incubation minimized agglomeration, improving the visualization of DNA damage. Both the modified in vitro comet assay and γH2AX DSB assay indicated that all materials tested, except mlGO, exhibited genotoxic effects at sublethal concentrations. These findings suggest that certain GRMs should be classified as genotoxic, warranting careful consideration of their potential risks, and recommending the modified comet assay as a reliable tool for 2D material genotoxicity assessment.