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April 01, 2009 (Vol. 29, No. 7)

High-Content Methods to Assess Toxicity

Extensions to Thermo Scientific Cellomics Platform Address Comet and Zebrafish Applications

  • Comet Assay

    Click Image To Enlarge +
    Figure 1. 20x image of CHO-K1 cells treated with 150 µM 9-aminoacridine.

    The Comet (more correctly known as single-cell gel electrophoresis) assay is a simple-to-perform, sensitive technique for the detection of DNA damage in single cells. In the assay, cells having been exposed to a potential agent of interest are embedded in a thin layer of agarose on a microscope slide. The DNA is allowed to unwind under neutral/alkaline conditions.

    The sample then undergoes electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus. After staining, the resulting image looks like a comet with a distinctive head and tail (Figure 1). The image is then evaluated for the amount of fluorescence in the head and tail and the length of the tail. The length of the tail is directly proportional to the amount of DNA damage.

    The comet assay is already being adopted by regulatory agencies as approved for regulatory submissions, and it is being widely adopted for genetic toxicology for screening and regulatory testing of industrial chemicals, pharmaceuticals, biocides, and cosmetics, as well as for ecogenotoxicology.

    There is also a move to replace some traditional assays (e.g., liver UDS assay) with an in vivo Comet assay. Comet assays are therefore gaining popularity; they are  limited in scalability by the manual scoring, a subjective assessment. Automating and quantifying the assay would overcome the limitations and allow it to be used more widely.

    To address the need for improved genotoxicity testing and the study of DNA damage using Comet, the Thermo Scientific Cellomics HCS Platform recently added the capability to measure Comet assays.

    The high-content approach to evaluating Comet assays offers a number of advantages in terms of speed, robustness, reproducibility, and objectivity. The Comet BioApplication allows scoring of Comets in seconds, removing tedious manual scoring as well as providing a wealth of objective measurements such as olive tail moment, tail length, tail extent moment, and percent DNA in tail.

    Comets can be classified into normal/ abnormal, increasing statistical robustness, and the available measurements provide much more information on DNA damage than manual scoring by eye. When combined with suitable assay systems (e.g., Trevigen Comet slide; dnadamage/cometslides.php) many compounds and many Comets can be analyzed, offering a step-change in productivity and the ability to easily scale the assessment of DNA damage.

    Comet assays are seen as key in defining new reliable and validated in vitro assays to test for genotoxic and cytotoxic effects of chemicals (as in the REACH program) without resorting to animal experiments. The Thermo Scientific automated high-content approach will certainly go a long way to aiding that initiative.

    The Cellomics Comet BioApplication also allows the analysis of Comet-FISH  assays, which extends its utility into determining sequence or gene-specific damage and repair as well as possible diagnostic use.

    The focus on Comet addresses one aspect of the innovation need, in that it allows a tedious manual assay to be automated and made more robust. The search for more relevant, predictive models of toxicity is ongoing, however, and again high content has a role to play here.

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