Dataset Information

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.

SUBMITTER: Garcia-Rodriguez A

PROVIDER: S-EPMC6956333 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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

García-Rodríguez Alba A Kazantseva Liliya L Vila Laura L Rubio Laura L Velázquez Antonia A Ramírez María José MJ Marcos Ricard R Hernández Alba A

Nanomaterials (Basel, Switzerland) 20191124 12

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 robu ...[more]

PMID: 31771274

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Project description:Mitochondria are highly dynamic organelles whose fusion and fission play an increasingly important role in a number of both normal and pathological cellular functions. Despite the increased interest in mitochondrial dynamics, robust, and quantitative methods to analyze mitochondrial fusion and fission activity in intact cells have not been developed. The current state-of-the art method to measure mitochondrial fusion activity is the polyethylene glycol (PEG) fusion assay in which cells expressing distinct mitochondrially-targeted fluorescent proteins (FPs) are fused together and mitochondrial fusion activity is determined by the rate at which color mixing occurs. Although this assay is useful, cell-cell fusion events are rare, and finding the number of fused cells required to generate statistically rigorous data is both tedious and time-consuming. Furthermore, the data-collection methods available for fluorescence microscopy lead to inherent selection biases that are difficult to control for. To that end, we have developed an unbiased and high-throughput method to detect, image, and analyze fused cells using the Amnis ImagestreamX™ MKII. With IDEAS™ software, we developed algorithms for identifying the fused cells (two nuclei within a single cell), distinguishing them from cell aggregates. Additionally, using the fluorescence localization of the mitochondrially-targeted fluorescent proteins (YFP and DsRed), we applied a modified co-localization algorithm to identify those cells that had a high co-localization score indicating mitochondrial fusion activity. These algorithms were tested using negative controls (FPs associated with fusion deficient mitochondria) and positive controls (cells expressing both FPs in the same mitochondria). Once validated these algorithms could be applied to test samples to evaluate the degree of mitochondrial fusion in cells with various genetic mutations. Ultimately, this new method is the first robust, high-throughput way to directly measure mitochondrial fusion in intact cells. Given how many cellular processes are being linked mitochondrial dynamics, this technique will provide a powerful new tool in the study of this important organelle. © 2016 International Society for Advancement of Cytometry.

Dataset Information

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

Publications

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

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