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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.
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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)