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

Günter Speit2, Claudia Dennog, Uta Eichhorn1, Andreas Rothfuß and Bernd Kaina1

Universitätsklinikum Ulm, Abteilung Humangenetik D-89070 Ulm and
1 Universität Mainz, Abteilung für Angewandte Toxikologie, D-55131 Mainz, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 x 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 {gamma}-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 ß 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.

Abbreviations: APE, apurinic endonuclease; HBO, hyperbaric oxygen; HO-1, heme oxygenase; Pol ß, DNA polymerase ß; ROS, reactive oxygen species; SOD, superoxide dismutase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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 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 (1x20 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 {gamma}-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 ß (Pol ß), 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 genotoxic effects using the comet assay.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test persons and HBO treatment
Fourteen healthy volunteers (non-smokers, aged 24–30 years) gave informed consent to participate in this study. This research was approved by the University Human Subjects Committee. The test persons were exposed to 100% oxygen at a pressure of 2.5 ATA in a hyperbaric chamber either for a total of three 20 min periods, interspersed with 5 min periods of air breathing (standard protocol) or for one or two 20 min periods, respectively. Venous blood samples were taken before HBO, immediately on exit from the chamber or 1 day later.

Western blot analysis
Protein extracts were obtained by sonication of lymphocytes in buffer containing 20 mM Tris–HCl pH 8.5, 1 mM EDTA, 1 mM ß-mercaptoethanol, 5% glycerol and protease inhibitor PMSF. The cell extract was centrifuged (10 min, 10 000 g) and the supernatant shock-frozen in liquid nitrogen. The protein content in the extracts were quantified as described (15). Prior to analysis, the frozen samples were thawed on ice and 40 µg of protein were separated onto a 12% SDS–polyacrylamide gel and electroblotted onto a nitrocellulose membrane (Schleicher & Schuell, Germany). Amounts of protein fixed on the membrane corresponding to individual lanes of the gel were checked by staining the protein with Ponceau S-red. The membrane was treated against unspecific binding of antibodies by blocking overnight with 5% non-fat dry milk in PBS/0.1% Tween-20. Rabbit anti-HO-1 antibody (PA3-019, ABR) diluted 1:1000 in blocking solution was added. After 2 h of incubation, membranes were extensively rinsed with PBS/0.1% Tween-20. Thereafter, membranes were incubated with peroxidase-conjugated secondary antibody diluted in blocking solution (1:5000) for 1 h. Finally, membranes were again washed extensively with PBS/0.1% Tween-20 and membrane-bound antibodies were visualized by enhanced chemiluminescence (Amersham-Pharmacia) according to the manufacturer's protocol. For re-incubation with another antibody, the membranes were rinsed for 15 min in 100 mM glycine pH 2.8, followed by incubation in PBS (15 min). Western blot analysis with catalase, SOD, APE and Pol ß occurred in the same way. Immunoblotting with ERK2 (sc154, Santa Cruz) was performed as a loading control. Quantification of expression levels was performed by densitometric measurements.

Mutagen treatment
H2O2, obtained from Sigma (München, Germany), was diluted in distilled water. For the in vitro-tests, peripheral blood lymphocytes were separated from whole blood on Histopaque gradients (Sigma, Germany), washed twice in 1x PBS and resuspended in PBS. Freshly isolated lymphocytes were treated with H2O2 by adding 100 µl H2O2 (final concentration: 10 and 20 µM) to 200 µl of lymphocyte suspension for 5 min on ice.

Comet assay (single cell gel electrophoresis)
Freshly taken blood samples were kept on ice and used for the comet assay within 1 h. Aliquots of 5 µl treated or untreated heparinized whole blood or 10 µl of isolated lymphocytes were mixed with 120 µl low melting agarose (LMA; 0.5% in PBS) and added to microscope slides (with frosted ends), which had been covered with a bottom layer of 1.5% agarose and dried. After solidifying the LMA, slides were processed as described previously (1). The time of alkali denaturation (pH 13) and electrophoresis (0.86 V/cm) was 25 min each. Images of 50 randomly selected cells stained with ethidium bromide were analysed from each coded slide. Measurements were made by image analysis (Comet Assay II, V1.02; Perceptive Instruments, Haverhill, UK) determining the median tail moment (DNA migrationxtail intensity) of the 50 cells.

Statistical analysis
Differences between comet assay mean values were tested for significance (P < 0.05 and P < 0.01) using Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Having shown that treatment of individuals with a single standard HBO induces adaptation of lymphocytes against the genotoxic effects of an oxidative agent such as H2O2 (3), we looked for a molecular basis of this adaptive response. Adaptation can be caused by increase in the level of DNA repair functions or enhancement of general defense against oxidative reactive species. The two DNA repair enzymes, APE and Pol ß, which are inducible and putatively involved in the oxidative stress response, were analysed by western blot analysis. In two independent series of treatments for a total of 10 individuals (nos 1–10) prior to and 24 h after a single HBO treatment, there was no increase in the expression of APE and Pol ß. Also, there was no clear difference in the level of catalase and SOD. However, the amount of HO-1 in lymphocytes was clearly enhanced in all individuals having received HBO therapy. Figure 1Go shows the western blot results of a representative experiment with six donors (nos 1–6). Quantitation of data obtained in western blots for all 10 subjects revealed that HO-1 was enhanced, on average, ~30-fold, as compared with lymphocytes from the same individual prior to HBO treatment (Figure 2Go).



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Fig. 1. Western blot analysis of lymphocytes of individuals prior to and after HBO treatment. Data of a representative experiment with six volunteers (nos 1–6) are shown. Per treatment level, 40 µg of protein (total cell extract) were subjected to analysis with antibodies against HO-1, SOD, Pol ß, APE and catalase. For each immunoblot, quantitation (loading control) was performed by reincubation with ERK2, which is not damage-inducible and constitutively expressed in the cells.

 


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Fig. 2. Level of induction of HO-1 in lymphocytes of 10 volunteers (nos 1–10) after HBO treatment. Data are from densitometric measurements of the corresponding blots (six of them are shown in Figure 1Go). Quantitation occurred in comparison with ERK2. Induction factor (IF) of HO-1 for each subject was set in relation to expression level prior to HBO treatment. –HBO, prior to treatment; +HBO, 24 h after treatment.

 
Adaptive protection occurred after a single standard HBO (3). This treatment also induced DNA damage in lymphocytes taken immediately on exit from the chamber (1). We therefore wanted to see whether a modified HBO protocol with reduced exposure is able to induce adaptive protection without inducing DNA damage. Four subjects (nos 11–14) were studied before and after a single HBO which lasted for only one 20 min period, as well as before and after HBO exposure for two 20 min periods the next day (Figure 3Go). In both cases, HBO-induced DNA damage could not be detected in any of the subjects. When blood from these subjects was taken and isolated lymphocytes were exposed in vitro to H2O2 (Figure 4Go), significant induction of DNA damage was found before HBO treatment (sample 1) for both H2O2 concentrations used. The mean tail moment value for the four subjects was increased from 0.05 (control) to 10.05 (10 µM H2O2) and to 16.08 (20 µM H2O2). This corresponds to an increase in the amount of DNA in the tail from 0.42 to 22.88 and 29.76%, respectively. However, 1 day after the first (one 20 min period) HBO exposure (sample 2) and 1 day after the second (two 20 min periods) exposure (sample 3), the DNA-damaging effect was much smaller than before HBO treatment. The difference between the samples 1 and 2 and 1 and 3 is statistically significant (P < 0.05 for 10 µM H2O2; P < 0.01 for 20 µM H2O2).



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Fig. 3. DNA migration (tail moment) in the comet assay with blood cells of four human subjects (nos 11–14) before and after a reduced HBO treatment (one 20 min period and two 20 min periods on successive days).

 


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Fig. 4. The effects of H2O2 (white bars, untreated control; gray bars, 10 µM; black bars, 20 µM) on DNA migration (tail moment) in isolated lymphocytes of four human subjects (nos 11–14) before and after HBO. The subjects were exposed to a reduced (one 20 min period and two 20 min periods) HBO treatment on two successive days. Blood samples were taken and treated with H2O2 before HBO exposure (sample 1), 24 h after the first exposure (sample 2) and 24 h after the second exposure (sample 3).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Oxidative stress is thought to underly the etiology of numerous human diseases, including cancer (16,17). Cellular antioxidants appear to be crucial for the prevention of damage induced by ROS which may lead to the respective diseases (10). HBO treatment has been shown to increase free radical levels in the blood of humans (18) and we have demonstrated previously that a single HBO treatment of human subjects causes DNA damage in leukocytes as indicated by the comet assay (1). DNA damage was not detected after further treatments under the same conditons, indicating an increase in antioxidant defence. Our present results contribute to a better understanding of the mechanisms possibly involved in the protection against oxidative stress of lymphocytes exhibiting an adaptive response. In accordance with our previous results (4), we show now that SOD and catalase, two of the main cellular antioxidant enzymes, were not enhanced in expression level. Although our comet assay experiments showed that adaptive increase in DNA repair could not be the sole cause for the induced protection, they did not exclude a contribution of enhanced repair to the protective effect (3). Two DNA repair enzymes involved in the repair of oxidative DNA damage have been shown to be inducible, namely APE (1113) and Pol ß (14). For APE, induction was shown to occur at the gene level by oxidative treatments, causing an adaptive response to the toxic and clastogenic effect of a challenge dose of oxidative treatment (11,12). In human lymphocytes adapted to HBO we could not find an increase in APE and Pol-ß protein levels, indicating that under these conditions both genes were not induced in vivo and that the corresponding proteins did not cause the adaptive effects observed. This is in line with the finding that blood taken 24 h after HBO was well protected against the in vitro induction of DNA damage by H2O2, but not to genotoxicity of {gamma}-irradiation, suggesting increased scavenging of ROS distant from nuclear DNA or increased sequestration of iron (3).

Most interesting in the present study is the finding of enhanced levels of HO-1 after HBO. To our knowledge this is the first demonstration of induction of HO-1 in human subjects after oxidative stress. HO-1 is one of two isoforms of heme oxygenase that catabolize cellular heme to biliverdin, carbon monoxide and free iron. It is up-regulated strongly during stress and is considered one of the most sensitive and reliable indicators of oxidative stress (6). Overexpression of HO-1 in human pulmonary epithelial cells stably transfected with the rat HO-1 cDNA resulted in increased resistance to hyperoxia (7). Suppression of HO-1 expression by transfection with HO-1 antisense oligonucleotides or treatment with specific inhibitors increased the sensitivity of a hamster fibroblast cell line to hyperoxia (8). In the HO-1 cDNA transfected cells, cellular heme was lowered compared with controls but cellular redox active iron levels were increased. Cells from knockout mice lacking functional HO-1 were hypersensitive to cytotoxicity caused by H2O2 (9), indicating that HO-1 serves as a mechanism to protect cells from oxidative stress.

Enhanced levels of HO-1 were demonstrated in blood samples 24 h after a single HBO. Under the same test conditions (i.e. 24 h after a single standard HBO) we also showed increased resistance of lymphocytes to H2O2 genotoxicity. The reduced induction of DNA damage was seen in the whole cell population and significant shifts in the blood cell population were not observed (3). Although we do not prove that HO-1 is directly responsible for the adaptive protection against the induction of DNA damage, the reactions catalysed by HO-1 suggest such a link. It is known that bilirubin, a metabolite of heme degradation is in itself a potent antioxidant (19). Furthermore, induction of ferritin synthesis as a result of iron removal from the degradation of heme by HO-1 may be involved (20). It is well known that the deleterious effects (e.g. genotoxicity) of ROS such as H2O2 are dependent on the presence of iron. Intracellular free iron can react with H2O2 and give rise to the toxic hydroxyl radical via the Fenton reaction. Due to the release of free iron during the catalysis of heme by HO-1, ferritin may be released and restrict iron from participation in the Fenton reaction. Thus, accumulation of ferritin is related to iron sequestration and protection against oxidative damage (20). The induction of HO-1 and increased sequestration of iron could explain why the cells are protected after HBO against the induction of DNA damage by H2O2 but not by ionizing radiation (3).

In vitro experiments with mammalian cell lines suggested that HO-1 gene expression is regulated by cellular oxygen tension (21). However, it is still unclear what kind of cellular effect triggers the upregulation of the antioxidant defense as a consequence of HBO exposure. Our results indicate that increased resistance to the in vitro induction of DNA damage already occurs after HBO conditions which did not cause detectable DNA damage by themselves. Obviously, oxidative stress induced DNA damage does not seem to be the trigger for the induction of the adaptive response to HBO. However, other deleterious effects of ROS, e.g. `early' effects like lipid peroxidation (22) may occur under these conditions and possibly trigger the response. Our study also supports the view that due to efficient protective mechanisms, relevant DNA damage is induced in humans only after acute exposures that are high enough to overcome the antioxidant defense (10). It would be an interesting issue of future research to elucidate whether induction of HO-1 is directly related to the adaptive protection against the induction of DNA damage, whether HO-1 is induced also under various other oxidative stress conditions and whether it elicits adaptation also in proliferating cells which are more vulnerable and a more critical target for fixation of DNA damage than lymphocytes.


    Notes
 
2 To whom correspondence should be addressed Email: guenter.speit{at}medizin.uni-ulm.de Back


    Acknowledgments
 
We would like to thank the volunteers for taking part in this study and Dr Lampl and Dr Ehrmisch of the Bundeswehrkrankenhaus Ulm for supervising the HBO treatments. This work was supported by the program `Environment and Health' (PUG) at the Forschungszentrum Karlsruhe with funds of the Department for Environment Baden-Württemberg and by the DFG (SFB 519/B4).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 2, 2000; revised February 2, 2000; accepted June 19, 2000.