The differences between the subjects reflect normal individual variations, as has been described earlier. Immediately after HBO exposure, a clear increase in DNA migration was found for all subjects.Three of the subjects were tested repeatedly and the same effect was seen in all trials. No increase in DNA migration was observed hafter HBO.In a few tests, DNA migration was determined hafter HBO and there was no increase as well. Figure indicates even lower values for the tail moment hafter treatment compared with the values before HBO treatment, an observation that was constantly made during the whole study.It can be seen that after HBO exposure, the reasch Butylated hydroxyanisole majority of cells exhibit increased DNA migration in comparison with the control.Figure also indicates that a normal size distribution for the tail moment was not observed and, consequently, the median was used to compare individual effects. All values obtained for the four subjects studied were in the range of controls. A clear and reproducible genotoxic effect was seen with the comet assay immediately after HBO exposure, as used therapeutically.This effect was found in all subjects tested and was due to increased DNA damage in the majority of leukocytes.The comet assay is a well established genotoxicity test that detects DNA damage on the single cell level with high sensitivity. In its alkaline version as used in the present study, DNA strand breaks and alkalilabile sites lead to increased DNA migration and provides a measure of DNA damage. Using the FPG protein to convert oxidative DNA base damage into strand breaks resulted in a small but significant increase in DNA migration of the control values before HBO exposure.This effect might be due to the presence of spontaneous oxidative DNA base damage in human blood, as previously shown for damage detected by endonuclease HI. Immediately after HBO treatment, the increase in DNA migration is strongly enhanced by addition of FPG protein.Furthermore, hafter HBO treatment, when the DNA damaging effect cannot be seen any more, there is also no enhancing effect of the FPG protein.Experiments performed with endonuclease IE instead of FPG protein resulted in comparable effects, indicating a broad spectrum of induced oxidative damage. To test whether an accumulation of DNA damage occurs after repeated HBO treatments, we investigated four subjects undergoing daily HBO treatment over a period of days. Comet assays were performed on the first, die third and the fifth day of the HBO therapy.While after the first HBO treatment the comet assay reveals a clear genotoxic effect, no such effect is seen after the third and the fifth day.DNA migration at day and is in the range of the pretreatment control or even smaller.A further time protocol was used to consider the typical weekend pause under standard treatment conditions.These experiments demonstrate that after the genotoxic effect on day, no induction of DNA damage occured on day after a day pause. This finding suggests that an increased antioxidant protection lasts for more dian days.To see whether the generally applied high partial pressure of oxygen can be used in HBO therapy without the observed genotoxic side effect, we changed the standard protocol by increasing the duration of HBO stepwise from X min to x min and X min during the first days.