Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment

Carcinogenesis, Oct 2000

Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 × 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H2O2) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects of γ-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA or an inducible change in the levels of free transition metals. We now demonstrate increased levels of heme oxygenase-1 (HO-1) in lymphocytes 24 h after HBO treatment of volunteers. Under the same conditions, superoxide dismutase, catalase and the DNA repair enzymes apurinic endonuclease and DNA polymerase β were not enhanced in expression. We also show that protection against the induction of DNA damage by H2O2 in lymphocytes even occurs with a shortened HBO treatment which did not induce significant DNA damage by itself. Our results suggest that increased sequestration of iron as a consequence of induced HO-1 might be involved in the adaptive protection after HBO treatment and that the induction of DNA damage is not the trigger for adaptive protection.

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Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment

Gu nter Speit 1 Claudia Dennog 1 Uta Eichhorn 0 1 Andreas Rothfu 1 Bernd Kaina 0 1 0 Universita t Mainz, Abteilung fu r Angewandte Toxikologie , D-55131 Mainz , Germany 1 Universita tsklinikum Ulm, Abteilung Humangenetik D-89070 Ulm 2To whom correspondence should be addressed Email: Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H2O2) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects of -irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA or an inducible change in the levels of free transition metals. We now demonstrate increased levels of heme oxygenase-1 (HO-1) in lymphocytes 24 h after HBO treatment of volunteers. Under the same conditions, superoxide dismutase, catalase and the DNA repair enzymes apurinic endonuclease and DNA polymerase were not enhanced in expression. We also show that protection against the induction of DNA damage by H2O2 in lymphocytes even occurs with a shortened HBO treatment which did not induce significant DNA damage by itself. Our results suggest that increased sequestration of iron as a consequence of induced HO-1 might be involved in the adaptive protection after HBO treatment and that the induction of DNA damage is not the trigger for adaptive protection. - We have been studying the biological consequences of hyperbaric oxygen (HBO) treatment as a model for the investigation of oxidative stress in humans (15). We have shown that HBO as used therapeutically (i.e. the inhalation of 100% oxygen under a pressure of 2.5 ATA in a hyperbaric chamber for a total of three 20 min periods, interspersed with 5 min of air breathing) caused clear and reproducible DNA defects (DNA strand breaks and oxidative base damage) as determined in the comet assay (single cell gel electrophoresis) with leukocytes (1). The DNA damaging effect of HBO was only seen immediately after a single HBO exposure of individuals. DNA damage was not detected after further treatments under the Abbreviations: APE, apurinic endonuclease; HBO, hyperbaric oxygen; HO-1, heme oxygenase; Pol , DNA polymerase ; ROS, reactive oxygen species; SOD, superoxide dismutase. same conditions, indicating increase in cellular defense against oxidative stress. DNA damage was also not detectable when HBO treatment was started with a reduced treatment time (1 20 min) which was then increased stepwise. Furthermore, blood taken 24 h after HBO treatment was well protected against the in vitro induction of DNA damage by hydrogen peroxide (H2O2). Treatment of isolated lymphocytes with H2O2 caused significant induction of DNA damage in the comet assay before HBO exposure of the volunteers, whereas the same treatment was ineffective in eliciting genotoxicity 24 h after HBO treatment (3). The cells were not comparably protected against the genotoxic effect of -irradiation suggesting, as the basis of the adaptive response, an increase in scavenging of reactive oxygen species (ROS) distant from nuclear DNA or an inducible change in the level of free transition metals. Resistance to H2O2, but not to ionizing radiation, could occur when iron sequestration is elevated as a result of adaptation to the initial HBO treatment. Heme oxygenase-1 (HO-1) is a protein which is highly inducible by a variety of agents causing oxidative stress and which seems to play a vital function in maintaining cellular homeostasis (6). HO-1 activity leads to degradation of the pro-oxidant heme and to accumulation of the antioxidant bilirubin. Various in vivo studies with animals and in vitro studies with mammalian cell lines have indicated involvement of HO-1 in the resistance to oxygen toxicity (79). We therefore measured the levels of HO-1 before and after HBO, to see whether this stress responsive protein is involved in the adaptive protection against the induction of DNA damage after HBO treatment in humans. We also measured several other putative oxidative stress response proteins such as catalase and superoxide dismutase (SOD), two main cellular antioxidant enzymes (10) and the DNA repair enzymes apurinic endonuclease (APE) and DNA polymerase (Pol ), that are involved in the repair of oxidative DNA damage and have been shown to be inducible (1114). The antioxidant adaptive response induced by HBO in human subjects was determined by treating lymphocytes from volunteers before and after various HBO exposures with H2O2 and evaluating the geno (...truncated)


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Günter Speit, Claudia Dennog, Uta Eichhorn, Andreas Rothfuβ, Bernd Kaina. Induction of heme oxygenase-1 and adaptive protection against the induction of DNA damage after hyperbaric oxygen treatment, Carcinogenesis, 2000, pp. 1795-1799, 21/10, DOI: 10.1093/carcin/21.10.1795