A common result of cellular stress is an increase in DNA damage. DNA damage may be manifest in the form of base alterations, adduct formation, strand breaks, and cross linkages (19). Strand breaks may be introduced in many ways, directly by genotoxic compounds, through the induction of apoptosis or necrosis, secondarily through the interaction with oxygen radicals or other reactive intermediates, or as a consequence of excision repair enzymes (20-22). In addition to a linkage with cancer, studies have demonstrated that increases in cellular DNA damage precede or correspond with reduced growth, abnormal development, and reduced survival of adults, embryos, and larvae (16, 23, 24).
The Comet assay can be easily utilized for collecting data on DNA strand breakage (9, 25, 26). It is a simple, rapid, and sensitive method that allows the comparison of DNA strand damage in different cell populations. As presented in this guide, the assay facilitates the detection of DNA single strand breaks and alkaline labile sites in individual cells, and can determine their abundance relative to control or reference cells (9, 16, 26). The assay offers a number of advantages; damage to the DNA in individual cells is measured, only extremely small numbers of cells need to be sampled to perform the assay (<10 000), the assay can be performed on practically any eukaryotic cell type, and it has been shown in comparative studies to be a very sensitive method for detecting DNA damage (2, 27).
These are general guidelines. There are numerous procedural variants of this assay. The variation used is dependent upon the type of cells being examined, the types of DNA damage of interest, and the imaging and analysis capabilities of the lab conducting the assay. To visualize the DNA, it is stained with a fluorescent dye, or for light microscope analysis the DNA can be silver stained (28). Only fluorescent staining methods will be described in this guide. The microscopic determination of DNA migration can be made either by eye using an ocular micrometer or with the use of image analysis software. Scoring by eye can be performed using a calibrated ocular micrometer or by categorizing cells into four to five classes based on the extent of migration (29, 30). Image analysis systems are comprised of a CCD camera attached to a fluorescent microscope and software and hardware designed specifically to capture and analyze images of fluorescently stained nuclei. Using such a system, it is possible to measure the fluorescence intensity and distribution of DNA in and away from the nucleus (8). Using different procedural variants, the assay can be utilized to measure specific types of DNA alterations and DNA repair activity (1, 3, 8, 10, 13, 14, 17, 18). Alkaline lysis and electrophoresis conditions are used for the detection of single-stranded DNA damage, whereas neutral pH conditions facilitate the detection of double-strand breaks (31). Various sample treatments can be used to express specific types of DNA damage, or as in one method, to preserve strand damage at sites of DNA repair (10). Nuclease digestion steps can be used to introduce strand breaks at specific lesion sites. Using this approach, oxidative base damage can be detected by the use of endonuclease III (18), as well as DNA modifications resulting from exposure to ultraviolet light (UV) through the use of T4 endonuclease V (3). Modifications of this type vastly expand the utility of this assay and are good examples of its versatility.
A sufficient knowledge of the biology of cel.......
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