Carcinogenesis vol.22 no.12 pp.1979–1985, 2001
Involvement of heme oxygenase-1 (HO-1) in the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO) treatment
Andreas Rothfuss1, Peter Radermacher2 and Gu¨nter Speit1,3 1Abteilung Humangenetik and 2Sektion Ana ¨ sthesiologie und Pathophysiologische Verfahrensentwicklung, Universita¨tsklinikum Ulm, D-89070 Ulm, Germany 3To
whom correspondence should be addressed Email:
[email protected]
Accumulating evidence suggests that HO-1 plays an important role in cellular protection against oxidant-mediated cell injury. Our previous studies on hyperbaric oxygen (HBO; i.e. exposure to pure oxygen under high ambient pressure) indicated clearly increased levels of HO-1 in lymphocytes of volunteers 24 h after HBO treatment (1 h at 1.5 bar). Experiments with the comet assay (alkaline single cell gel electrophoresis) revealed that the same cells were almost completely protected against the induction of DNA damage by a repeated exposure or in vitro treatment with H2O2 24 h after the first HBO. In order to further investigate the role of HO-1 in HBO-induced adaptive response, we now performed experiments with isolated human lymphocytes exposed to HBO in vitro (2 h at 3 bar). Our results show that also under cell culture conditions, lymphocytes exhibit an adaptive protection similar to that observed in our previous work with healthy human subjects. The timecourse of HO-1 induction proceeds in parallel to the development of an adaptive protection against the induction of oxidative DNA damage. A comparable protection was not seen in V79 cells, indicating a specific difference between the two investigated cell systems. Treatment with the specific HO-1 inhibitor tin-mesoporphyrin IX (SnMP) led to a complete abrogation of HBO-induced adaptive protection in human lymphocytes. Our results indicate a functional involvement of HO-1 in the adaptive protection of human lymphocytes against the induction of oxidative DNA damage. The exact mechanism by which HO-1 contributes to an adaptive response remains to be elucidated. Introduction Adaptive protection generally implies enhanced protection of cells induced by an initial treatment which renders cells resistant to a subsequent treatment. Adaptive responses have been shown to be induced by a wide range of exposures and may therefore represent an important cellular defense mechanism. We have been studying the biological consequences of hyperbaric oxygen (HBO) treatment (i.e. exposure to pure oxygen under high pressures) as model for oxidative stress (1–5). Our previous investigations indicated that HBO treatment of human subjects leads to the induction of an adaptive response which protects blood cells against the induction of DNA damage by a second HBO or in vitro Abbreviations: FPG, formamidopyrimadine DNA-glycosylase; HBO, hyperbaric oxygen; HO-1, heme oxygenase-1; SnMP, tin-mesoporphyrin. © Oxford University Press
treatment with H2O2 (3,6). Using the comet assay, we could show that DNA damage was only induced immediately after a single HBO, while a repeated exposure under the same conditions 24 h later had no genotoxic effect (1). Furthermore, ex vivo experiments showed that blood cells taken from human subjects exposed to HBO were completely protected against the induction of (oxidative) DNA damage by hydrogen peroxide (3). Measurements of the antioxidant status of subjects after HBO and western blot experiments in adapted lymphocytes revealed that the observed adaptational effect could not be explained by enhanced activity of well-known antioxidant enzymes (6,7). Increased DNA repair activity as a cause for adaptation was unlikely because the difference in the amount of DNA damage was measured immediately after exposure, and the levels of apurinic endonuclease (APE) and Polβ, two inducible proteins involved in the repair of oxidative DNA damage, were not changed after HBO exposure (3,6). In contrast, a clear induction of human heme oxygenase-1 (HO-1; EC 1.14.99.3) was obvious 24 h after HBO treatment in all subjects (6). HO-1 catalyzes the rate-limiting step in the degradation of heme, producing bilirubin, ferrous ion and carbon monoxide. Various studies have shown that HO-1 is highly inducible by agents causing oxidative stress (8,9), and accumulating evidence suggests that HO-1 induction is often connected with an increased resistance against oxidant-mediated cell injury (10–12). Enhanced cellular protection is commonly explained by an increased production of bilirubin, a well-known cellular antioxidant, and a subsequent increased sequestration of redox-active iron due to increased ferritin levels (8,12). In our previous studies however, it remained unclear whether the observed increase of HO-1 levels is directly related to the adaptive protection against the induction of DNA damage, or if it is only part of a general cellular stress response. Since we could show that an adaptive response is expressed in lymphocytes (3) and in order to facilitate mechanistic investigations, we established an in vitro HBO test system with isolated human lymphocytes (4). Our previous studies indicated that HBO treatment of cultured cells is well-suited for studying cellular consequences of oxidative stress (4,5). Using this model, we now investigated whether an adaptive protection can also be observed under in vitro conditions and whether HO-1 is directly involved in the adaptive response. Materials and methods Cell cultures and chemical treatment V79 Chinese hamster cells (V79-UL) were cultivated in RPMI 1640 (Dutch modification), supplemented with 10% fetal calf serum (FCS), 2 mM glutamine and antibiotics (complete RPMI 1640). Cells were maintained in a humidified incubator at 37°C with 5% CO2 and harvested with 0.15% trypsin and 0.08% EDTA. Heparinized blood samples were obtained by venepuncture of healthy subjects, which were not previously subjected to HBO therapy. Peripheral blood lymphocytes were separated from freshly collected whole blood using Ficoll gradient (Pharmacia Biotech, Sweden), washed in 1⫻ phosphatebuffered saline (PBS) and resuspended in RPMI 1640. For exposure to HBO, V79 cells (106 – 5 ⫻ 106) or freshly isolated lymphocytes (~.3 ⫻ 106 cells)
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A.Rothfuss, P.Radermacher and G.Speit were kept in 3 ml serum-free RPMI 1640 in 25 cm2 cell culture flasks. After exposure to HBO, cells were washed, and the medium was replaced with fresh complete RPMI 1640 medium. In the case of repeated exposures to HBO, lymphocytes from the same sample were exposed on different days. H2O2, obtained from Sigma (Munich, Germany), was diluted in distilled water. For the comet assay, V79 cells and isolated human lymphocytes were treated by adding 100 µl of H2O2 to 200 µl of cell suspension for 5 min on ice. For studies with the specific HO-1-inhibitor tin-mesoporphyrin IX (SnMP, Porphyrin Products, Logan), lymphocyte cultures were treated with 10 µM SnMP 10 min before HBO exposure. As SnMP is sensitive to light, the metalloporphyrin solution was prepared in a darkened room, and the cell culture flasks were wrapped with aluminium paper. Treatment with HBO HBO exposure was performed in a small, temperature-controlled (37°C) hyperbaric chamber as described previously (4). The gas composition was 98% O2 and 2% CO2 in order to maintain physiological pH. Before compression, the chamber was flushed with gas. In all experiments, a pressure of 3 bar was applied with a compression rate of 0.2 bar/min and maintained for 2 h. After exposure, the chamber was decompressed (0.2 bar/min) to atmospheric pressure. Control experiments with hyperbaric air (21% O2) revealed no induction of DNA damage under these conditions. Comet assay The comet assay was performed as described earlier (13). The time of alkali denaturation and electrophoresis (0.86 V/cm) was 25 min each for V79 cells. The standard comet assay protocol for human lymphocytes was 30 min denaturation followed by a 40 min electrophoresis. For determination of oxidative DNA base damage, the comet assay was combined with bacterial FPG protein (5). Briefly, slides were washed three times (for 5 min each) in enzyme buffer (40 mM Hepes; 100 mM KCl; 0.5 mM EDTA; 0.2 mg/ml BSA; pH 8.0) and covered with 100 µl of either buffer or FPG protein in buffer, sealed with a coverslip and incubated for 30 min at 37°C. Slides with and without FPG-post-treatment were denaturated for 25 min and electrophoresed for another 25 min. Images of 50 randomly selected cells stained with ethidium bromide were analyzed by image analysis (Perceptive Instruments, Haverhill, UK). The mean tail moment (percentage of DNA in the tail ⫻ tail length) of the individual cells was determined according to the image analysis software (Comet assay II V1.02). Results are expressed as induced tail moment values with the tail moment value of the untreated control being subtracted from the value obtained for HBO or H2O2-exposed cells. In case of repeated exposure, a control value was established by determining the actual degree of DNA damage immediately before a subsequent exposure with a second HBO or with H2O2, so that the induced tail moment value of repeated exposures indicates the extent of DNA damage induced by the second treatment. RNA extraction and northern blot analysis For northern blot experiments, lymphocytes were isolated from peripheral blood and resuspended in RPMI 1640 without serum. After treatment with a single HBO, cells were washed and incubated in RPMI 1640 containing 10% FCS. RNA was isolated before and at various time points after HBO exposure as indicated. Total RNA was isolated using the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA samples (30 µg/lane) were loaded on a MOPS/formaldehyde agarose gel (1%). After electrophoresis, RNA was transferred by capillary action in 50 mM sodiumphosphate buffer (pH 6.5) to Hybond-N nylon filters (Amersham International, Uppsala, Sweden). After crosslinking, the membrane was prehybridized in hybridization buffer (0.5 M phosphate buffer, pH 7.2; 7% SDS; 1 mM EDTA) at 60°C for 4 h followed by incubation in hybridization buffer containing 32Plabeled human HO1-cDNA probe at 65°C for 24 h. To control the amount of RNA in different samples, blots were hybridized with 32P-labeled human βactin probe. The filter was washed and autoradiographed using Kodak Super RX films. Autoradiograms were scanned by densitometry (ImageQuant, Molecular Dynamics, Krefeld, Germany), and the results were normalized to the β-actin signal. The HO-1 mRNA level is expressed as fold induction compared with untreated control cells. Western blot analysis Protein extracts were obtained from human lymphocytes isolated from 20 ml peripheral blood before and at various time points after a single HBO by sonication of lymphocytes in RIPA-buffer containing 50 mM Tris-HCl, pH 8; 150 mM NaCl; 1% NP-40, 0.1% SDS and protease inhibitor PMSF. The cell extract was centrifuged (10 min, 15 000 rpm) and the supernatant was shockfrozen in liquid nitrogen. Protein content of the samples was determined as described (14). Prior to analysis, 20 µg of protein were boiled for 5 min in an equal volume of 2⫻ SDS sample buffer (0.125 M Tris-HCl, pH 7.4; 4% SDS, 2% β-mercaptoethanol and 20% glycerol). Samples were separated onto
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Fig. 1. DNA migration (induced tail moment) in human lymphocytes after repeated HBO exposure in the standard comet assay (A) and after posttreatment with FPG (B). DNA damage was evaluated in cells which were exposed to a single HBO (HBO1, HBO2) or in cells which were exposed for a second time (HBO 1 ⫹ 2) 4 h or 24 h after the initial HBO. Note that the time of alkaline unwinding/electrophoresis in (A) was 30 min/40 min and in (B) was 25 min/25 min. Mean ⫾ SEM of three experiments. without FPG post-treatment, j with FPG post-treatment
a 12% SDS-polyacrylamide gel for 2 h at 25 mA. The proteins were then electroblotted onto a PVDF-membrane (Immobilon-P; Millipore, Bedford, USA) and incubated for 2 h in phosphate-buffered saline–0.1% Tween20 buffer (PBST) containing 5% non-fat dry milk. Rabbit anti-human HO-1 antibody (StressGen, Victoria, Canada) diluted 1:500 in blocking solution was added. After incubation for 2 h, the membrane was washed extensively with PBST and incubated with goat anti-rabbit IgG antibody (StressGen, Victoria, Canada) diluted 1:5000 for 1 h. The membrane was washed again and membrane-bound antibodies were visualized by enhanced chemiluminescence (Amersham-Pharmacia, Freiburg, Germany) according to the manufacturer’s protocol. Immunoblotting with actin (C-11; Santa Cruz) was performed as a loading control.
Results Isolated human lymphocytes and V79 Chinese hamster cells were exposed to HBO (2 h; 3 bar) in vitro and, using the comet assay, analyzed for the presence of an adaptive protection against further induction of DNA damage. Figure 1 summarizes the effects of repeated HBO exposure in the comet assay with isolated lymphocytes. Lymphocytes treated in serum-free RPMI 1640 medium showed a clear and reproducible induction of DNA damage (induced tail moment) after an initial HBO (HBO1). The absolute mean tail moment value increased about 3-fold from 0.90 to 2.95 under these experimental conditions (30 min alkaline unwinding and 40 min electrophoresis). This corresponds to an increase in tail intensity from 4.6 ⫾ 1.2 to 14.0 ⫾ 1.5. After incubation at 37°C for 4 h, about 75% of the induced DNA effects were repaired (i.e. induced tail moment values between 0.44 and 0.87), and control values
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Fig. 2. Effects of H2O2 treatment (10 µM and 20 µM) on DNA migration (induced tail moment) in freshly isolated human lymphocytes without previous exposure to HBO or in lymphocytes 4 h and 24 h after an initial HBO treatment (HBO ⫹ 10 µM, HBO ⫹ 20 µM). Mean ⫾ SEM of three experiments.
Fig. 3. DNA migration (induced tail moment) in V79 cells after repeated HBO exposure in the comet assay. DNA damage was evaluated in cells which were exposed to a single HBO treatment (HBO1, HBO2) or in cells which were exposed for a second time (HBO 1 ⫹ 2) 4 h or 24 h after the initial exposure. Mean ⫾ SEM of three experiments.
were measured after 24 h incubation (data not shown). At these time points, cells were exposed for a second time under the same conditions (HBO 1 ⫹ 2). Analysis of DNA migration in these cells revealed a clear protection against the induction of DNA damage by the second HBO exposure. No difference in the extent of protection was obvious at both time points. Control experiments using untreated lymphocytes confirmed that the exposure at the second time point (HBO2) caused an induction of DNA damage comparable to the initial HBO (HBO1). Using FPG protein, we can show that exposure to a single HBO (HBO1; HBO2) led to a clear induction of FPGsensitive sites in the comet assay (Figure 1B). In these experiments the time of alkali denaturation and electrophoresis was 25 min each. Tail moment values for controls were 0.33 in the absence of FPG postincubation and 1.04 in the presence of FPG postincubation, respectively. Repeated exposure to HBO did not significantly induce oxidative DNA damage in human lymphocytes, as DNA migration was not enhanced by FPG post-treatment. Again, a comparable degree of protection was obvious both 4 h and 24 h after the initial HBO. To test whether the observed adaptive response also protects cells against the induction of oxidative DNA damage by other agents than HBO, we treated human lymphocytes with 10 µM and 20 µM H2O2 (Figure 2). Treatment with H2O2 alone induced a clear and dose-dependent effect in the comet assay. It is interesting to note that the incubation time of lymphocytes before exposure to H2O2 obviously influenced the extent of DNA damage, as cells which were treated 24 h after isolation always had somewhat lower effects in the comet assay compared with cells which were treated earlier (4 h). Anyway, pre-exposure with HBO efficiently diminished H2O2-induced DNA effects in the comet assay. This effect was seen when cells were treated with H2O2 both 4 h or 24 h after HBO, but
was more pronounced at the later time point. We also performed experiments with FPG post-treatment indicating that pretreatment with HBO led to a reduced induction of oxidative DNA damage by H2O2 (data not shown). In contrast, cells were not comparably protected against the genotoxic effects induced by γ-irradiation. Irradiation with 1 and 2 Gy caused a similar induction of DNA damage in cells independent of pretreatment with HBO, thus confirming our earlier results with human subjects (data not shown). In opposition to the results obtained with human lymphocytes, repeated exposure of V79 cells to HBO did not indicate the presence of an adaptive protection. Figure 3 shows that a second treatment of V79 cells with HBO (HBO 1 ⫹ 2) induced a genotoxic effect comparable with that of cells treated only with a single HBO (HBO1 or HBO2). This effect was seen both with the second HBO treatment 4 h or 24 h after the initial treatment, respectively. Untreated control V79 cells had a mean tail moment of 0.40 under these experimental conditions. Furthermore, pretreatment of V79 cells with a single HBO did not protect against the induction of DNA damage by 10 µM and 20 µM H2O2 (Figure 4). In contrast, the results obtained 4 h after HBO indicate that V79 cells pretreated with HBO were even more sensitive to the induction of DNA damage by 20 µM H2O2. As our previous studies had revealed an induction of HO-1 in lymphocytes of human subjects 24 h after exposure to HBO, we wanted to know if this is also the case in our cell culture model. Furthermore, using both northern blot and western blot analysis, we tried to gain insight in the kinetics of a possible HO-1 induction in human lymphocytes. Figure 5 shows the results of the northern blot experiments. A rapid induction of the HO-1 mRNA, leading to an almost doubling of HO-1 mRNA levels compared with untreated control cells, was noticed already, 1 h after HBO. The highest mRNA-induction 1981
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Fig. 4. Effects of H2O2 treatment (10 µM and 20 µM) on DNA migration (induced tail moment) in V79 cells without previous exposure to HBO or 4 h and 24 h after an initial HBO treatment (HBO ⫹ 10 µM, HBO ⫹ 20 µM). Mean ⫾ SEM of three experiments.
Fig. 6. Time-dependent increase in heme oxygenase-1 (HO-1) protein levels by HBO in human lymphocytes (A) and V79 cells (B). Cells were exposed to a single HBO and protein was isolated before exposure and at various time points after HBO. Equal loading was verified by immunoblotting with actin.
was found at 8 h after treatment, when about a three-fold increase in HO-1 mRNA level was obvious. HO-1 mRNA levels subsequently declined, and 24 h after treatment they reproducibly fall below the level observed before HBO exposure. Figure 6A shows that the induction of HO-1 mRNA was succeeded by enhanced HO-1 protein levels. HO-1 protein was clearly induced after 4 h, peaked at 8 h and remained induced at least until 24 h after HBO treatment. In contrast to the results obtained in human lymphocytes, no comparable induction of HO-1 was observed in V79 cells exposed to HBO (Figure 6B). In order to further corroborate a possible involvement of HO-1 in adaptive protection of human lymphocytes, we performed experiments with tin-mesoporphyrin IX (SnMP), a specific inhibitor of HO-1 activity (24). In preceding experiments, we had observed that treatment of human lymphocytes with 10 µM SnMP for 24 h did not lead to enhanced DNA migration in the comet assay (data not shown). Figure 7 shows that treatment with 10 µM SnMP completely abrogated the adaptive protection of human lymphocytes towards a repeated HBO exposure 4 h or 24 h after the initial treatment. There was no clear effect of SnMP on the induction of DNA migration induced by a single exposure (HBO 0 h, 4 h, 24 h). Furthermore, treatment with SnMP also prevented the development of an adaptive protection against H2O2 treatment 24 h after HBO exposure (Figure 8). H2O2-induced DNA migration in cells pretreated with HBO and SnMP was comparable to that of cells not exposed to HBO but treated with H2O2. Again, SnMP itself had no consistent effect on DNA migration induced by H2O2 alone. Discussion Fig. 5. Time-dependent increase in heme oxygenase-1 (HO-1) mRNA by HBO in human lymphocytes. Cells were exposed to a single HBO and RNA was isolated before exposure and at various time points after HBO. Total RNA was analyzed for HO-1 and actin mRNA expression by northern blot analysis. (A) shows a representative northern blot. Results are presented as fold induction compared with untreated controls (mean ⫾ SEM of three experiments; B).
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Exposure to reactive oxygen species (ROS) is a common threat to mammalian cells, particularly to human lymphocytes, which participate in immune reactions at inflammatory sites. Cellular antioxidants normally help to protect against oxidant-mediated cell injury. Under circumstances where the amount of oxidants exceeds a certain threshold level however, oxidative stress
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Fig. 7. Effects of 10 µM tin-mesoporphyrine (SnMP) on HBO-induced DNA migration after a single (left part) or a repeated HBO exposure 4 h and 24 h after the initial treatment (right part). Mean ⫾ SEM of three experiments. without SnMP treatment, j 10 µM SnMP.
Fig. 8. Effects of 10 µM tin-mesoporphyrine (SnMP) on H2O2-induced (10 µM and 20 µM) DNA damage without previous HBO exposure (left part) or 24 h after the initial HBO treatment (right part). Mean ⫾ SEM of three experiments. without SnMP treatment, j 10 µM SnMP.
results, which may lead to the induction of oxidative damage. Using the comet assay (alkaline single cell gel electrophoresis), we have shown that hyperbaric oxygen (HBO) induces oxidative DNA damage in lymphocytes from subjects exposed to HBO therapy (1). Furthermore, isolated lymphocytes exposed to HBO in vitro gave similar results and therefore have been
suggested as a well-suited model for investigating cellular consequences of oxidative stress (3). One consequence of increased amounts of oxidants can be the development of adaptive responses (15). Our previous studies identified an adaptive protection in lymphocytes of human subjects exposed to HBO, as DNA damage could only be found after the first HBO exposure but not after further treatments under the same conditions (1). We can now show that the development of an adaptive response after HBO treatment also occurs under in vitro conditions: isolated lymphocytes showed resistance towards the induction of (oxidative) DNA damage by a second HBO exposure. This result indicates a solely cellular basis of the adaptive response and thus confirms the suitability of our HBO in vitro model for performing mechanistic investigations. Our results also reveal that pretreatment of isolated human lymphocytes with HBO efficiently protects the cells against the induction of (oxidative) DNA damage by H2O2. These observations suggest that the adaptive protection observed in isolated human lymphocytes is very similar to that found in our earlier studies with human subjects (1,3). It should however be noted that the exposure conditions required to induce oxidative stress (i.e. induction of DNA effects in the comet assay) were higher in vitro than in vivo (1,4). In our study using lymphocytes from HBO-exposed volunteers (3,6), the investigation of an adaptive protection was limited to one time point (i.e. 24 h) after HBO treatment. We now show that the adaptive protection in lymphocytes is already developed at a much earlier time point after the initial HBO (i.e. 4 h), indicating a quick and efficient induction of cellular response mechanisms. It should be noted however, that in particular the experiments with H2O2 indicate a trend towards a more efficient protection, when the second treatment occurs 24 h after an initial HBO exposure. In this context, it is noteworthy that isolated human lymphocytes obviously became more resistant to H2O2 exposure with increasing culture time, possibly as a consequence of adaptation to atmospheric oxygen pressures (21% O2). Therefore, the better protection of lymphocytes 24 h after HBO may be the result of a combined adaptive response towards HBO and atmospheric oxygen. Our previous work indicated increased levels of human heme oxygenase-1 (HO-1) in lymphocytes 24 h after exposure of human subjects to HBO. The question remained whether this upregulation is functionally linked to the observed adaptive protection. Time-dependent investigations of HO-1 induction now reveal that at the time points, when lymphocytes elicit an adaptive protection (i.e. 4 h and 24 h after an initial HBO), HO-1 protein is clearly induced. Northern blot experiments show that this increased protein level is due to increased transcription of the HO-1 gene and provide evidence for a typical stress response, as HO-1 mRNA levels are quickly induced and then subsequently decline. A similar response was observed in different mammalian cell lines exposed to agents causing oxidative stress (16–18). HO-1 protein levels have been shown to remain increased over hours (19). Particularly for human lymphocytes, it was demonstrated that exposure to strong oxidative stress (500 µM H2O2) led to a quick induction of a 32 kDa protein, probably HO-1, which stayed induced until 18 h after treatment (15). Several lines of evidence show that HO-1 functions as an efficient system for cellular protection against oxidative threat. Upregulation of HO-1 has been shown to confer increased resistance against oxidative stress (16,19,20). In addition, moderate overexpression of HO-1 in vitro and in vivo leads to enhanced protection 1983
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against oxidant-mediated cell injury (21–24). Finally, experiments with HO-1 knock-out mice revealed a reduced stress defense when exposed to oxidative stress (25). Our results indicate a close relationship between the induction of HO-1 and the phenotypic expression of an adaptive protection in human lymphocytes and point towards a direct involvement of HO-1 in HBO-induced adaptive protection. In contrast to the situation observed in human lymphocytes, V79 cells do not seem to be able to express an adaptive response. Interestingly, when V79 cells were tested for HO-1 induction after exposure to a single HBO (2 h; 3 bar), no increase in HO-1 levels was observed, indicating that the lack of adaptation in V79 cells may be due to the inability to induce HO-1. The underlying genetic defect causing this inability is not yet clear but it is known that permanent cell lines like V79 have various genetic alterations. Additionally, V79 cells in general show a higher sensitivity towards HBOinduced DNA damage compared to lymphocytes (4). Our experiments with the specific HO-1 inhibitor tinmesoporphyrin IX (SnMP) corroborate the involvement of HO-1 in HBO-induced adaptive response. We chose 10 µM SnMP for our investigations, because this concentration has been shown to significantly decrease HO activity (24,26) and did not induce DNA damage in human lymphocytes by itself. The finding that SnMP treatment led to a complete abrogation of cellular protection against the induction of DNA damage by HBO and H2O2 suggests a direct connection between the activity of HO-1 and the HBO-mediated adaptive protection. We can however still not rule out the possibility that the potentiation effects of SnMP are due to non-specific inhibition of other enzymes which also could account for the comet assay results. A direct involvement of HO-1 in the adaptive protection against membrane damage by UVA-irradiation was also described in human skin fibroblasts. Pretreatment of cells with HO-1 antisense oligonucleotide inhibited the induction of the HO-1 enzyme and abolished the protective effect of preirradiation (11). The mechanism(s) by which HO-1 exhibits its role in antioxidant protection is not yet clear. A protective role of HO-1 could be explained by the increased production of bilirubin, which is regarded as a very effective physiological antioxidant (27,28). Bilirubin acts as a radical scavenger, and increased formation of this antioxidant could therefore explain the observed adaptive protection against genotoxic effects induced by HBO and H2O2. Besides an enhanced bilirubin production, a subsequent induction of ferritin due to increases in free iron levels (10,11) could also contribute to the phenotypic expression of an adaptive response. Increased ferritin levels would restrict redox-active iron from participating in the Fenton reaction, and this increased sequestration of iron could explain why adapted lymphocytes are efficiently protected from the induction of oxidative DNA damage. Furthermore, increased HO-1 activity leads to enhanced formation of the gaseous molecule CO. Although the biological role of CO remains unclear, some studies described a protective effect of CO on hyperoxic lung injury (23,29). Up to now, the contribution of the products of HO-1 mediated heme degradation to the observed protective effect in human lymphocytes after HBO exposure is not understood and it is not clear whether the same reactions occur in vivo and in isolated cells. This has to be investigated in the future to elucidate the mechanism(s) of HBO-induced adaptive protection. 1984
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29. Otterbein,L., Bach,F., Alam,J., Soares,M., Lu,H., Wysk,M., Davis,R., Flavell,R. and Choi,A.M. (2000) Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat. Med., 6, 422–428. Received June 22, 2001; revised and accepted September 6, 2001
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