Dose-time dependency of hyperbaric hyperoxia-induced DNA strand breaks in human immune cells visualized with the comet assay.

PURPOSE: Hyperbaric oxygen exposure may induce dose-dependent DNA damage in peripheral blood mononuclear cells (PBMCs), and repetitive exposures of man may have protective cellular effects. METHOD: PBMCs, freshly isolated from non-divers and pure oxygen divers, were exposed to ambient air (21kPa) and hyperoxia at different levels: 100kPa, 240kPa, 400kPa and 600kPa) for up to 6.5 hours in an experimental pressure chamber. DNA double-strand breaks were studied in the comet assay by calculating the "tail moment" and an alternative "Yes or No" method for damaged nuclei. Previously, the experimental procedure had been optimized for human cell experiments: Pre-tests assured that DNA damage could be considered to be oxygen-induced; and cell viability remained over 95% during exposure time. RESULTS: Visible DNA damage increased with the partial pressure of oxygen (pO2) and exposure time dose-dependently. Linear regressions revealed r2 between 0.61 and 0.98 with the Yes/No method, and significant differences in slopes from control. Tail moment showed similar results, but with less accuracy. The PBMCs of oxygen divers exposed to 400kPa pO2 (up to six hours) showed a significant lower slope in the linear regression. CONCLUSION: Oxygen induces dose-dependent DNA double-strand breaks, and the Yes/No discrimination is superior to the tail moment in linearity and accuracy. Oxygen diver PBMCs seem to be more resistant to hyperbaric oxygen.

Influence of hyperoxia and physical exercise on *OH-radical stress in humans as measured by dihydroxylated benzoates (DHB) in urine.

BACKGROUND: Hyperoxia and physical exercise are known to produce reactive oxygen species (ROS), and the *OH radical is the most aggressive among them. However, knowledge is limited about *OH stress during physical work under hyperoxic conditions. METHODS: This study monitored *OH stress in human volunteers before and after a total of 135 exposures to ambient air (control), different levels of hyperoxia at rest and challenging open-water closed-circuit dives by measurement of dihydroxylated benzoates (DHB) with HPLC by electrochemical detection in urine. RESULTS: Changes in DHB in urine after control were only 3.43 +/- 4.8% (n = 9). After exposures to 100 kPa oxygen (O2) for 110 minutes DHB revealed increases in urine of 23.14 +/- 5.12% (n = 9); exposures to 240 kPa O2 for 90 minutes increases of 22.38 +/- 8.91% (n = 8); and 280 kPa 02 for 30 minutes of 21.92 +/- 10.76% (n = 17). Closed-circuit dives in open water (45-54 minutes of 125-160 kPa O2) revealed DHB increases of 66.34 +/- 25.73% (n = 92). All results differed significantly from control (p < 0.001). The closed-circuit dives also differed significantly from all exposures to hyperoxia without exercise (p < 0.001). Standardization of "oxygen burden" during each exposure (pO2 x exposure time x VO2) allowed for comparison of different exposures vs. DHB changes and revealed goodness of linear fit of r2 = 0.432 (p < 0.0001). CONCLUSIONS: Increases in urine DHB after exposures to different levels of hyperoxia at rest and during exercise are consistent with *OH stress that is greater during exercise than at rest, although other interpretations are possible. Standardization of the individual "oxygen burden" for a given exposure may become useful in future for the estimation of *OH stress.