Micronuclei Detection by Flow Cytometry as a High-Throughput Approach for the Genotoxicity Testing of Nanomaterials

Abstract

Thousands of nanomaterials (NMs)-containing products are currently under development or incorporated in the consumer market, despite our very limited understanding of their genotoxic potential. Taking into account that the toxicity and genotoxicity of NMs strongly depend on their physicochemical characteristics, many variables must be considered in the safety evaluation of each given NM. In this scenario, the challenge is to establish high-throughput methodologies able to generate rapid and robust genotoxicity data that can be used to critically assess and/or predict the biological effects associated with those NMs being under development or already present in the market. In this study, we have evaluated the advantages of using a flow cytometry-based approach testing micronucleus (MNs) induction (FCMN assay). In the frame of the EU NANoREG project, we have tested six different NMs—namely NM100 and NM101 (TiO2NPs), NM110 (ZnONPs), NM212 (CeO2NPs), NM300K (AgNPs) and NM401 (multi-walled carbon nanotubes (MWCNTs)). The obtained results confirm the ability of AgNPs and MWCNTs to induce MN in the human bronchial epithelial BEAS-2B cell line, whereas the other tested NMs retrieved non-significant increases in the MN frequency. Based on the alignment of the results with the data reported in the literature and the performance of the FCMN assay, we strongly recommend this assay as a reference method to systematically evaluate the potential genotoxicity of NMs.

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…Cytochalasin B is an agent that is used to block cytokinesis, and thus to prevent separation of daughter cells after mitosis, leading to the formation of binucleated cells. This assay is often used for NM testing; however, exposure with NMs should occur before cytochalasin B is added to allow uptake of NMs by cells [ 15 , 31 , 121 ].…”

An Automated, Single Cell Quantitative Imaging Microscopy Approach to Assess Micronucleus Formation, Genotoxicity and Chromosome Instability

“…In general, these approaches require that cells are exposed to a test chemical and grown to provide sufficient time for chromosomal damage and/or cell cycle proliferation defects to manifest, which are required for micronucleus formation. Many of these approaches involve microscopy, flow cytometry, or image-based flow cytometry and include the use of DNA counterstains (Giemsa or fluorescent DNA dyes), fluorescence in situ hybridization (FISH) probes (chromosome enumeration probes), or kinetochore antibodies to identify micronuclei and/or their contents [48][49][50][51][52][53][54]. As an alternative, our scQuantIM approach does not depend on the efficient inhibition of cytokinesis by cytochalasin B, a compound that by itself has been shown to induce genome instability [34,55] and could conceivably synergize with test compounds or gene silencing to exacerbate micronucleus formation.…”

Assessment of the Carcinogenicity of Carbon Nanotubes in the Respiratory System

“…Chromosome damage and chromosome mis-segregation have also been described in airway epithelial cells chronically exposed to sub-toxic doses of short NM-400 and NM-403 MWCNTs [ 52 ], while no primary DNA damage or oxidized DNA bases have been observed in short-term experiments with NM-400, NM-401, and NM-403 [ 50 , 79 , 80 ]…”

“…With the cytokinesis-blocked micronucleus assay (CBMN), authors did not observe significant increases in the frequency of micronucleated binucleated cells or induction of DNA damage by the comet assay [ 81 ]. When analyzed by flow cytometry, NM-401 at 20 and 50 µg/mL were able to increase the MN formation [ 79 ]. No genotoxic effects with the CBMN assay were detected also for NM-400, NM-402, and NM-403 [ 81 ].…”