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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility., The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive in vitro genotoxicity data. The CometChip® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip® assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter® and TempO-Seq®). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional in vitro tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT’s mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of in vitro test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip® results, which were negative for all three drugs except at overtly cytotoxic concentrations. This

Copyright © 2020 American Chemical Society. Environmental exposures have long been known to impact public health and safety. For example, exposures to airborne particulates, heavy metals in water, or certain industrial chemicals can contribute to aging and to risk of developing cancer and other diseases. Environmental factors can impact health in a variety of ways, but a key concern is DNA damage, which can lead to mutations that cause cancer. Cancer can take years to develop following chemical exposure; however, one way to predict carcinogenicity in a more practical time frame is by studying the chemical's ability to induce DNA damage. The comet assay (or single-cell gel electrophoresis assay) has been used successfully for genotoxicity testing. The comet assay allows for the detection of DNA strand breaks via analysis of DNA migration during electrophoresis. Previously, the Engelward laboratory, in collaboration with the Bhatia laboratory, developed the CometChip for measurements of DNA damage and repair. The CometChip is a high-throughput comet assay that improves user reproducibility and significantly shortens total assay time. Here, we describe how the high-throughput CometChip platform can be used to measure DNA damage in established cell lines, animal models, and human samples. We also discuss technical challenges associated with these studies and provide recommendations on how to achieve optimal results for researchers interested in adopting this assay.

Copyright © 2020 American Chemical Society. Environmental exposures have long been known to impact public health and safety. For example, exposures to airborne particulates, heavy metals in water, or certain industrial chemicals can contribute to aging and to risk of developing cancer and other diseases. Environmental factors can impact health in a variety of ways, but a key concern is DNA damage, which can lead to mutations that cause cancer. Cancer can take years to develop following chemical exposure; however, one way to predict carcinogenicity in a more practical time frame is by studying the chemical's ability to induce DNA damage. The comet assay (or single-cell gel electrophoresis assay) has been used successfully for genotoxicity testing. The comet assay allows for the detection of DNA strand breaks via analysis of DNA migration during electrophoresis. Previously, the Engelward laboratory, in collaboration with the Bhatia laboratory, developed the CometChip for measurements of DNA damage and repair. The CometChip is a high-throughput comet assay that improves user reproducibility and significantly shortens total assay time. Here, we describe how the high-throughput CometChip platform can be used to measure DNA damage in established cell lines, animal models, and human samples. We also discuss technical challenges associated with these studies and provide recommendations on how to achieve optimal results for researchers interested in adopting this assay.

Three-dimensional tissue culture models are emerging as effective alternatives to animal testing. They are especially beneficial for liver toxicity studies, enabling hepatocytes to display improved levels of liver-specific functions. One common model is hepatocyte spheroids, which are spontaneously formed cell aggregates. Techniques for spheroid formation include the use of ultralow attachment plates and the hanging drop method, both of which are technically challenging and relatively low throughput. Here, we describe a simple-to-use platform that improves spheroid production and is compatible with genotoxicity testing by the comet assay. To achieve this, we created a chip containing a microwell array where dozens of spheroids are contained within a single well of a 96-well plate. The microwells are made from agarose, a nontoxic material suitable for cell growth and spheroid formation. HepG2 cells loaded into customizable microwells formed spheroids through agarose-assisted aggregation within one to two days. In addition, the agarose matrix allows the same platform to be used in DNA damage analysis. Specifically, the comet assay enables quantification of DNA breaks based on the increased migration of damaged DNA through agarose during electrophoresis. Here, we developed a modified comet assay and show that intact HepG2 spheroids cultured in microwells can be electrophoresed to reveal the extent of DNA damage following exposure to inflammatory chemicals (H2O2 and SIN-1). With this SpheroidChip analysis method, we detected a dose-dependent increase in DNA damage and observed rapid repair of H2O2-induced DNA damage. In summary, we utilized an agarose microarray to condense what had required an entire 96-well plate into a single well, enabling analysis techniques that were cumbersome or impossible under conditions of a single spheroid per well of a 96-well plate., National Institute of Environmental Health Sciences (Grants T32-ES007020, P42-ES027707, P30-ES002109)