Screening the micronucleus assay for reliable estimation of the genotoxicity of graphene and other 2D materials

• Published in: Carbon (New York) Vol. 215; p. 118426
• Main Authors: García-Carpintero, S., Jehová González, V., Frontiñán-Rubio, J., Esteban-Arranz, A., Vázquez, E., Durán-Prado, M.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01-11-2023
• Subjects: 2D nanomaterials; Genotoxicity; Graphene; Micronucleus assay; 2D nanomaterials; Graphene; Micronucleus assay; Genotoxicity
• ISSN: 0008-6223; 1873-3891
• DOI: 10.1016/j.carbon.2023.118426

In recent years, the genotoxicity of graphene-related materials (GRMs) and other 2D materials has been evaluated using different models to ensure their safety. The OECD TG 487 in vitro micronucleus (MNu) test is one of the most widely used methods to assess the genotoxicity of nanomaterials (NMs). However, the nature of NMs interferes with the standard MNu process, thus requiring an adaptation to assess this kind of compound. Improvements have been incorporated into the standard method to solve the interferences and build a modified, reliable protocol that can be used to test NMs further. The main interference observed was NM agglomeration, which hinders the correct visualization of micronuclei. Addition of 10% dextran to the culture medium reduced agglomeration but interfered with toxicity by reducing NM-induced cell death. Mechanical shaking during cell incubation with NMs minimized the size and number of agglomerates and avoided their rapid sedimentation and deposition onto cells, thus allowing accurate visualization of the micronucleate cells. Once set up, the proposed method was tested with graphene oxide (GO), few-layer graphene (FLG), molybdenum disulfide (MoS2), and boron nitride (hBN). The novel in vitro MNu test for NMs showed that GO (0.05–5 μg/ml) and hBN (0.05 μg/ml) were genotoxic at sublethal doses, inducing irreparable chromosomal damage. With these adaptations, the modified in vitro MNu test is strongly recommended to test the genotoxicity of NMs. The results obtained with this protocol indicate that GO and hBN represent a risk that should be considered.
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•Cyto-b interferes with the MNu assay with graphene-related materials but not with other 2D materials.•NM agglomeration interferes with the assay but it can be solved by introducing mechanical shaking during cell incubation.•Shaking improves the effective concentration of NMs delivered to the cells.•GO and hBN are genotoxic at low concentrations and cause irreparable DNA damage.