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Assays that measure DNA damage and repair are critical in evaluating the extent to which therapeutic agents damage DNA and in identifying whether DNA repair can limit the toxicity of chemotherapy. The COMET assays described in this guide should help readers evaluate single and double-strand breaks cause by chemotherapeutic agents and also monitor the ability of the cells to repair such damage. The EJDR assay described is a valuable tool to assess the ability of drugs and DNA repair proteins to modulate DNA repair capacity. Finally, the immunofluorescence assay described should allow accurate assessments of DNA damage and the kinetics of repair as measured by Ɣ-H2AX foci. This procedure can also be used to mechanistically investigate the recruitment of specific DNA damage and repair proteins in CLL cells.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Human biomonitoring studies aim to identify potential exposures to environmental, occupational, or lifestyle toxicants in human populations and are commonly used by public health decision makers to predict disease risk. The Comet assay measures changes in genomic stability and is one of the most reliable biomarkers to indicate early biological effects and therefore accepted by various governmental regulatory agencies. The appeal of the Comet assay lies in its relative simplicity, rapidity, sensitivity, and economic efficiency. Furthermore, the assay is known for its broad versatility, as it can be applied to virtually any human cell and easily adapted in order to detect particular biomarkers of interest, such as DNA repair capacity or single and double-strand breaks. In a standard experiment, isolated single cells are first embedded in agarose, and then lysed in high-salt solutions in order to remove all cellular contents except the DNA attached to a nuclear scaffold. Subsequent electrophoresis results in accumulation of undamaged DNA sequences at the proximity of the nuclear scaffold, while damaged sequences migrate toward the anode. When visualized with fluorochromes, these migrated DNA fragments resemble a Comet tail and can be quantified for their intensity and shape according to internationally drafted guidelines.

The comet assay (single cell gel electrophoresis) is a sensitive, versatile method for detecting DNA damage in eukaryotic cells. The traditional comet assay format has 1 or 2 gels on a microscope slide, 1 sample per slide, and there is a limit of 40 gels per experiment given the size of a typical electrophoresis tank. To increase throughput, we have designed and tested a system with 12 minigels on one slide, allowing analysis of up to 12 times more samples in one electrophoresis run. The novel comet assay format compares well with the traditional technology. The various steps are suitable for further automation, and the formats can be adapted to fully automated scoring. The new procedures save time at all stages as fewer slides are handled, and the amounts of reagents needed are reduced significantly. This format is particularly useful for testing of numerous genotoxic agents and nanomaterials at different concentrations and on different types of cells; simultaneous analysis of different lesions using a range of enzymes; and analysis of cell extracts for DNA repair activity.