Dna Repair

Besides its ability to detect DNA damage, the comet assay also allows to assess DNA repair following induced DNA effects. DNA repair can be determined by monitoring the time-dependent removal of induced lesions, i.e. the decrease in DNA migration. Follow-up experiments using the comet assay showed that HBO-induced DNA effects are rapidly repaired27, leading to a reduction in induced DNA migration of >50% during the first hour after exposure. Using a modified comet assay protocol in conjunction with the FPG protein, a similar repair kinetic for the induced oxidative damage was observed. Furthermore, blood taken 6 or 24 h after HBO did not show any effect, indicating complete repair of the induced DNA damage27.

It has to be mentioned, however, that the comet assay only measures the kinetic of strand break rejoining but not the accuracy of DNA repair. Incorrectly rejoined DNA breaks do no longer contribute to DNA migration in the comet assay, but nevertheless may lead to a disruption of genetic information with potentially functional consequences.

To further evaluate the biological significance of the comet assay effects in humans, the micronucleus test (MNT) with human lymphocytes was performed, which is a well established and sensitive test for the detection of chromosome breakage in humans exposed to mutagens. The same blood samples that exhibited a significant increase in DNA migration in the comet assay did not show increased micronucleus frequencies27. Although an effect in the MNT is confined to proliferating lymphocytes, the comet assay results clearly demonstrated that genotoxic effects occur in the whole population of white blood cells, so it can be assumed that under therapeutical exposure conditions the primary DNA damage is repaired before the cells enter the mitotic S-phase and chromosome aberrations can be produced. In lymphocytes of healthy human volunteers, no induction of chromosome mutations was found after a single HBO. Therefore, it can be assumed that under therapeutical exposure conditions the primary DNA damage is repaired before they can lead to chromosome aberrations in mitotic cells.

Further studies gave additional evidence that HBO does not induce gene mutations in healthy volunteers either. No increase in mutant frequencies was observed at the HPRT locus in peripheral lymphocytes after a single HBO28. The in vivo HPRT test is a unique system for studies on spontaneous and induced mutant frequencies in humans and the mechanisms of mutagenesis. The assay detects a broad spectrum of gene mutations from single base substitutions to larger deletions and should detect mutations caused by oxidatively damaged DNA bases. The negative result in combination with the comet assay experiments suggests that the induced DNA lesions are completely repaired before lymphocytes are stimulated to replicate. However, the detection of mutations with the in vivo HPRT gene mutation test is known to be rather insensitive, and in vitro HPRT tests with cultured mammalian cells indicated that HBO seems to have a low potential for the induction of gen mutations (see below). Taken together, the in vivo mutagenicity studies with healthy human subjects exposed to HBO indicated that neither induction of chromosome aberrations nor induction of gene mutations can be detected in peripheral blood lymphocytes despite the clear DNA-damaging effect in the comet assay.

In contrast to these negative results, increased frequencies of chromosome aberrations after HBO exposure were reported in an earlier study. However, this effect was found in patients with various diseases and drug treatments after repeated HBO exposures29. As the comet assay results suggest that repeated HBO does not lead to further DNA damage due to induced antioxidant protection, it is likely that the observed elevated chromosome aberration frequencies are not directly related to HBO exposure. However, it cannot be excluded that individuals with specific diseases or genetic susceptibility exhibit higher vulnerability.

lt seems that at least in healthy individuals, efficient antioxidant defences together with DNA repair maintain a steady-state level of damage with minimal risk to the cell or the organism30. However, under conditions where antioxidant defense is deficient or overcharged, a significant mutational burden cannot be excluded29. In vitro experiments with cultured mammalian and human cells enable to increase HBO exposure and to study a possible mutagenic potential of HBO-induced DNA damage under conditions where antioxidant capacities are overwhelmed30. The main difference in these studies in comparison to in vivo exposure of human subjects was a permanent oxygen exposure, i.e. the absence of interspersed normal air exposures. Using the comet assay, a genotoxic effect of HBO could be demonstrated in various cell types30.

Increased HBO exposures (3bar) clearly caused mutagenic effects in cultured mammalian cells. A dose-related induction of chromosome damage was measured in V79 cells (a permanent Chinese hamster cell line) with the micronucleus test (MNT) after treatment with HBO with increasing exposure time (0.5 to 3 hr)31 32. The clastogenic (chromosome-breaking) effect of HBO in V79 cells correlated very well with the increase in DNA damage obtained with the comet assay in the same cell population. The same exposure conditions also increased mutant frequencies in a mammalian cell gene mutation assay at the tk-locus of mouse lymphoma cells (the so called mouse lymphoma assay, MLA), but failed to induce mutations in the in vitro HPRT test with V79 cells31, 32. The HPRT test predominantly detects point mutations and the negative result suggests that HBO does not significantly lead to point mutations even under high exposure conditions. In accordance with this finding there is increasing evidence that oxidative DNA modifications generated by oxygen radicals are not a significant cause of gene mutations due to efficient repair of these lesions. It is more likely that oxygen radicals are mutagenic through other kinds of DNA lesions (e. g. DNA strand breaks) leading to large rearrangements and gross deletions after high exposures. The mutagenic effect in the MLA was solely based on the induction of small colony mutants, which have in fact been shown to arise as a consequence of gross genomic alterations such as deletions and recombinations. Molecular analysis of HBO-induced mutations in the MLA revealed that all investigated mutants had deletions of the tk-gene. These results also demonstrate that the mutagenic effect of HBO is based on a clastogenic mechanism. Such a mutagenic mechanism has also been proposed for normobaric hyperoxia which induced a similar pattern of mutagenic effects in vitro. Taken together, the in vitro mutagenicity studies clearly demonstrate that HBO under high pressure conditions has the potential to induce mutations via a clastogenic mechanism. The negative result in the in vivo MNT in HBO-exposed human subjects is obviously due to the lower exposure and/or a better protection of the peripheral blood cells.

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