The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials

Abstract

The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design—including treatment conditions, cell number, cell culture, format and volume of medium on the plate—is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential.

Rapid Identification of in Vitro Cell Toxicity Using an Electrochemical Membrane Screening Platform

“…It involves embedding cells (previously exposed to the test compound) in a low melting point (LMP) agarose gel, lysis of the cells in alkaline conditions and electrophoresis of the nuclei. The procedure of the alkaline comet assay has been published in Dusinska et al, [61], Balintová et al, [62] and El Yamani et al, [63] and most recently in Nature Protocols [64]. Median DNA tail intensity, proportional to the number of strand breaks (SBs), was calculated as a measure of DNA SBs.…”

“…Three independent experiments were performed. The positive control for SBs was H 2 O 2 exposure (100 µM for 5 min) as explained in detail in Dusinska et al, [61], Elje et al, [44] and El Yamani et al, [63].…”

Current status and future challenges of genotoxicity OECD Test Guidelines for nanomaterials: a workshop report

“…The work has been completed, and results have been made available publicly in the form of project deliverables, papers, and training materials. Furthermore, SOPs have been validated, and an SOP for the in vitro comet assay and colony forming ability will be proposed for inclusion in the OECD Test Guideline Programme for development of new Test Guidelines [ 11–14 ]. Additionally, the interference of nanomaterials with test components and/or damage detection systems of in vitro assays has been an issue.…”