The Detection Of Dnadamage

DNA-damage can be evaluated with the so called "Comet-assay", which is a well-established, highly sensitive genotoxicity test that detects DNA damage on the single cell level21-23. In this microgel electrophoresis technique, a small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed and stained with a fluorescent DNA binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus towards the anode, which resembles the shape of a comet. In its alkaline version (pH >13) DNA strand breaks, alkaline labile sites and incisions during DNA repair become apparent, and the amount of DNA migration22,24, expressed by characteristic differences in the DNA migration. Using image analysis, various parameters can be measured for determining the length and/or the amount of DNA migration. The so called "tail-moment" is frequently used because it represents a product of both length and intensity and sensitively indicates the amount of DNA damage in the cell.

A genotoxic effect in the comet assay is not directly related to mutagenesis and carcinogenesis. DNA effects in the comet assay can be based on various types of DNA damage with different mutagenic potential. Neither the shape of the comet nor the extent of DNA migration directly reflects the mutagenic risk. The DNA lesions leading to DNA migration can represent repairable DNA single strand breaks (which even can be produced during the process of excision repair itself) but might also include highly toxic and mutagenic lesions (e.g. DNA double strand breaks). Furthermore, the comet assay is very sensitive but not specific, so different types of genotoxic effects can lead to increased DNA migration, and, hence, additional information is needed to definitely evaluate the biological significance of the results.

Such additional information on the presence of a specific class of DNA damage can be obtained using the comet assay in combination with lesion-specific endonucleases26. Such tools are for instance formamido-pyrimidin-glycosylase (FPG) and endonuclease III (Endo III), which specifically detect oxidized DNA bases. Both proteins remove oxidized purines and pyrimidines, respectively, with the resulting abasic sites being converted into DNA single strand breaks by the associated endonuclease activity. This additional induced DNA damage leads to additional DNA migration in the comet assay and therefore provides an indirect measure for the presence of oxidative DNA damage.

Using this technique, Speit and co-workers26 showed that a DNA-damaging effect in the comet assay was seen in healthy young male volunteers who had been exposed according to a therapeutically used HBO treatment protocol, i.e. 100% oxygen exposure at a hyperbaric pressure of 2.5 bar abs in a hyperbaric chamber for a total of 3x20 min periods, interspersed with 5 min of air breathing. The presence of oxidative base damage after HBO therapy assay suggested that at least some of the HBO-induced DNA modifications might have some relevance for mutagenicity and carcinogenicity.

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