Abstract
The comet assay is a valuable experimental tool aimed at mapping DNA damage in human cells in vivo for environmental and occupational monitoring, as well as for therapeutic purposes, such as storage prior to transplant, during tissue engineering, and in experimental ex vivo assays. Furthermore, due to its great versatility, the comet assay allows to explore the use of alternative cell types to assess DNA damage, such as epithelial cells. Epithelial cells, as specialized components of many organs, have the potential to serve as biomatrices that can be used to evaluate genotoxicity and may also serve as early effect biomarkers. Furthermore, 80% of solid cancers are of epithelial origin, which points to the importance of studying DNA damage in these tissues. Indeed, studies including comet assay in epithelial cells have either clear clinical applications (lens and corneal epithelial cells) or examine genotoxicity within human biomonitoring and in vitro studies. We here review improvements in determining DNA damage using the comet assay by employing lens, corneal, tear duct, buccal, and nasal epithelial cells. For some of these tissues invasive sampling procedures are needed. Desquamated epithelial cells must be obtained and dissociated prior to examination using the comet assay, and such procedures may induce varying amounts of DNA damage. Buccal epithelial cells require lysis enriched with proteinase K to obtain free nucleosomes. Over a 30 year period, the comet assay in epithelial cells has been little employed, however its use indicates that it could be an extraordinary tool not only for risk assessment, but also for diagnosis, prognosis of treatments and diseases.
Highlights
In this special issue, we review the use of the comet assay to map DNA damage in different human cells since the inception of this field nearly 30 years ago by Ostling and Johanson (1984)
The study conducted by Rojas et al (1996) employed enzymatic lysis enrichment to compare DNA damage between buccal epithelial cells that were derived from smokers and non-smokers; in spite of the fact that the study employed a small number of subjects; the comet assay was found to be suitable for use in this cell type
In the study conducted by Valverde et al (1997), DNA damage induced by air pollution in Mexico City was compared in three different cell types, demonstrating that lysed buccal epithelial cells with proteinase K enrichment are suitable for use in comet analyses; differences between the exposure groups were not detected
Summary
We review the use of the comet assay to map DNA damage in different human cells since the inception of this field nearly 30 years ago by Ostling and Johanson (1984). The expert panel reached a consensus that the optimal version of the assay for identifying genotoxic activity was the alkaline (pH > 13) version of the assay that was developed by Singh et al (1988). This version of the comet assay is capable of detecting DNA single-strand breaks (SSB), alkali labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. The advantages of the comet assay relative to other genotoxicity tests include its sensitivity in detecting low levels of DNA damage; the requirement of a small number of cells per sample; and its flexibility, ease of application, and short duration. The expert panel identified the minimal experimental and methodological standards required to ensure that the results of comet studies would be accepted as valid by knowledgeable scientists and regulatory agencies (Tice et al, 2000)
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