Mutation Research 428 1999. 8389 www.elsevier.comrlocatermolmut Community address: www.elsevier.

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Antioxidant status in humans after exposure to hyperbaric oxygen

Claudia Dennog a , Peter Radermacher b, Yvonne A. Barnett c , Gunter Speit
b c

a,)

a Uniersitatsklinikum Ulm, Abteilung Medizinische Genetik, D-89070 Ulm, Germany Uniersitatsklinikum Ulm, Sektion Anasthesiologische Pathophysiologie und Verfahrensentwicklung, D-89070 Ulm, Germany Cancer and Ageing Research Group, School of Biomedical Sciences, Uniersity of Coleraine, Northern Ireland BT52 1SA, UK

Received 29 November 1998; accepted 9 January 1999

Abstract Hyperbaric oxygen HBO. treatment i.e., exposure to 100% oxygen at a pressure of 2.5 atmosphere absolute ATA. for a total of 3 = 20 min periods. of human subjects induced DNA damage in the alkaline comet assay with leukocytes and protected against the DNA damaging effects of subsequent in vivo HBO exposures. Furthermore, blood taken 24 h after the first HBO was well protected against the in vitro induction of genotoxic effects by hydrogen peroxide. To investigate the mechanisms which led to this apparent adaptive response, we determined the antioxidant status of blood from subjects before and after HBO. We did not find differences in the plasma concentrations of the antioxidant vitamins A, C and E after HBO treatment. HBO had also no effect on the antioxidant power of the plasma as measured with the FRAP-assay or on the concentration of reduced glutathione determined in the plasma or in lymphocytes. Red cell concentrate activities of superoxide dismutase, catalase, glutathione peroxidase were not influenced by HBO. In contrast, synthesis of the heat shock protein HSP70 which has been implicated to play an important role in cellular protection against oxidative stress, was significantly induced in lymphocytes after a single HBO treatment. To investigate whether intake of antioxidants may protect against HBO-induced DNA damage, we supplemented subjects with vitamin E 800 mg for 7 days. or with N-acetylcysteine 400 mg, 1 h before the HBO treatment.. However, these supplementations did not influence the induction of DNA damage by HBO. q 1999 Elsevier Science B.V. All rights reserved.
Keywords: Comet assay; Hyperbaric oxygen treatment; Antioxidant enzyme; Vitamin; Glutathione; N-acetylcysteine; Heat shock protein

1. Introduction Hyperbaric oxygen HBO. treatment seems to be a well-suited model for the investigation of oxidative
Abbreiations: HBO, hyperbaric oxygen; ATA atmospheres absolute; GSH, reduced glutathione; CAT, catalase; GPx, glutathione peroxidase; SOD, superoxide dismutase; HSP, heat shock protein; NAC, N-acetylcysteine; FRAP, ferric reducing ability of plasma ) Corresponding author. Tel.: q49-731-502-3429; Fax: q49731-502-3438; E-mail: guenter.speit@medizin.uni-ulm.de

stress and its biological consequences w13x. HBO implies the inhalation of 100% oxygen under a pressure of 2.5 ATA atmospheres absolute. in a hyperbaric chamber for a total of 3 = 20 min periods, interspersed with 5 min of air breathing. Exposure to HBO leads to an increase in the amount of dissolved oxygen and therefore reactive oxygen species ROS. in the blood. Toxicity of hyperoxia seems to result from oxidative stress, i.e., the increased formation of ROS, including superoxide, singlet oxygen, hydrogen peroxide and lipid peroxides w4x. Combina-

0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 4 2 9 9 . 0 0 0 3 4 - 4

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tions of these substances, particularly in the presence of Fe 2q ions, can produce the highly reactive and damaging hydroxyl radical, which is generally assumed to be most important in causing biomolecule damage w5x. ROS arise in vivo as a consequence of a number of intrinsic processes e.g., redox respiration chain in the mitochondria, phase I xenobiotic metabolising enzyme systems, the respiratory burst of neutrophils, etc.., and extrinsic exposures e.g., cigarette smoke, ozone, ionizing radiation, etc... Excessive amounts of ROS can cause cellular damage. To counteract the potential for damage, complex antioxidant systems to control production and reduce damage from ROS have evolved w68x. Antioxidants can act by reducing the production of ROS, removing catalytic metal ions, or removing ROS once they have been formed. These defence systems are not perfect and ROS-induced biomolecule damage may still occur. In these cases DNA repair pathways and stress response proteins have an important role in the recognition and removal of such damage w6x. Although the chemical reactions involved in the generation and detoxification of ROS have been studied in great detail, little is known about the cellular responses to oxidative stress in the human body. It has been shown that alterations in gene expression of antioxidant enzymes and stress-response genes play an important role in the response of mammalian cells to oxidative stress w8x. Various animal experiments have also demonstrated increase in antioxidant enzymes after hyperoxia w9x, but only few studies investigated the effects of HBO. In experiments with rats and guinea pigs, exposure to HBO had marked effects on antioxidant enzymes in the brain and lungs w10x. In another study, where rats were exposed to HBO, an increase in reduced glutathione GSH. in lung tissue was observed w11x. To determine the in vivo antioxidant response to HBO, we measured the in vivo antioxidant status w12x including levels of important plasma and cellular antioxidants in blood samples of subjects before and 24 h after HBO. We analysed the plasma concentrations of the antioxidant vitamins A, C and E, the antioxidant power of the plasma as indicated by the FRAP-assay and the concentration of reduced glutathione GSH. in the plasma and in lymphocytes. On the cellular level, we measured the activities of catalase CAT., glutathione peroxidase GPx. and superoxide dismutase

SOD.. As the synthesis of heat shock proteins HSPs. may also play a significant role in cellular protection against oxidative injury w13,14x, we assessed levels of the inducible form of HSP70 in lymphocytes of subjects before and after HBO. In a further approach we investigated whether supplementation with vitamin E or with N-acetylcysteine NAC. afforded protection against HBO-induced DNA damage.

2. Materials and methods 2.1. Test subjects and treatment protocols Healthy volunteers non-smokers, 2039 years. gave informed consent to participate in this study. All subjects were on a normal diet without vitamin supplementation. They were exposed to HBO according to a routine therapy protocol: 100% oxygen at a pressure of 2.5 ATA in a hyperbaric chamber for a total of 3 = 20 min periods, interspersed with 5 min periods of air breathing. Venous heparinized blood samples were taken before HBO exposure, immediately on exit from the chamber and 24 h later. The blood samples were kept on ice and processed within 1 h. To assess any potential protective effects of antioxidant supplementation against HBO-induced DNA damage, three subjects were supplemented with a daily dose of 800 mg vitamin E for 7 days prior to a HBO and another four subjects received a supplement of 400 mg N-acetylcysteine p.o. 1 h before HBO. Subjects without supplementation took part in these tests the same HBO treatment. as internal controls. 2.2. Comet assay The alkaline comet assay was performed on whole blood, as described earlier w2x. The cells were exposed to alkali pH 13. for 25 min to permit DNA unwinding and expression of alkali-labile sites. Electrophoresis was performed for 25 min at 25 V 0.86 Vrcm. and 300 mA. Measurements were made by image analysis Comet Assay II, Perceptive Instruments, Haverhill, UK., determining the mean tailmoment of 50 cells per slide. The presence of oxida-

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tive DNA base damage was determined with a modified protocol w2x using the bacterial formamidopyrimidine-DNA glycosylase FPG protein. or endonuclease III Endo III.. After lysis, slides were washed three times in an enzyme buffer, drained and the agarose covered with 200 ml of either buffer or enzyme 1 mgrml. in buffer, sealed with a coverslip and incubated for 30 min at 378C. 2.3. Assessment of in io antioxidant status From blood samples before and after HBO, plasma and cell fractions were prepared by centrifugation and lymphocytes were obtained by density gradient centrifugation. All samples were frozen immediately. Plasma levels of vitamin A, C and E, of GSH and of the antioxidant power FRAP-assay. as well as red cell concentrate activities of the antioxidant enzymes SOD, CAT, GPx, and levels of the inducible form of HSP70 in lymphocytes were performed according to the manual and automated methods described in detail elsewhere w1519x.

Table 2 Determination of antioxidant power with the FRAP assay in of subjects before and one day after HBO Subject FRAP mmolrl. Before HBO PH IJ KK RK Mean 891.2 735.7 805.5 895.8 832.1 Day 1 1233.0 863.3 834.4 1158.9 1022.4

3. Results Table 1 shows that HBO does not have any significant effect on plasma levels of the antioxidant vitamins A, C and E. Neither individual antioxidant vitamin values nor the mean of the four subjects tested are altered after HBO. The ferric reducing ability of plasma FRAP. as a measure of antioxidant power of the plasma did also not give a clear indication for an adaptive response. Although the

values seen after HBO are higher than before HBO in all of the four subjects, this difference was not significant and clearly within the range of normal controls Table 2.. Table 3 shows the concentration of reduced glutathione GSH. in plasma and in lymphocytes before and after HBO. Obviously, there is considerable variation among subjects as well as before and after HBO. However, no clear effect of HBO can be derived from these data. Table 4 summarizes the effects of a single HBO on the red cell concentrate activities of the antioxidant enzymes. It can be seen that there was no difference in the individual activities of SOD, CAT and GPx before and 24 h after HBO. All values measured are within the range of normal controls w12x. A significant effect p - 0.01, paired t-test. was found for the induction
Table 3 Concentration of reduced glutathione GSH. in plasma and lymphocytes of subjects before and one day after HBO Subject Plasma GSH mM. Before HBO A JB CD KK OM Mean B TH PR WS AV Mean 8.8 41.9 51.0 149.5 62.8 Day 1 22.6 23.2 73.2 48.5 41.9 Cellular GSH mM. Before HBO 37.7 37.1 60.5 36.5 43.0 Day 1 64.8 47.9 54.5 16.7 46.0

Table 1 Measurement of vitamins in plasma of subjects before and one day after HBO Subject Vitamin A mmolrl. Before HBO AH KD OM SP Mean 1.8 2.6 1.9 2.3 2.1 Day 1 2.0 2.4 2.1 2.2 2.2 Vitamin C mmolrl. Before HBO 33.8 32.2 36.4 31.4 33.5 Day 1 36.8 37.6 34.4 30.7 34.9 Vitamin E mmolrl. Before HBO 22.3 23.4 16.3 25.7 21.9 Day 1 25.0 23.3 18.2 22.7 22.3

7.1 16.7 17.3 35.9 19.2

10.1 21.5 26.3 14.9 18.2

25.1 33.5 61.1 55.1 43.7

39.5 36.5 21.5 46.7 36.0

A. without NAC-supplementation. B. subjects after oral supplementation of 400 mg NAC.

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Table 4 Measurement of antioxidative enzymes before and one day after HBO Subject Superoxide-dismutase UrgHb. Before HBO AH KD OM SP CD KK RG SH Mean Normal range 781.0 835.8 1455.1 1704.4 1250.8 1494.5 1312.5 1333.3 1270.9 5531660 Day 1 1053.5 852.8 1459.5 1928.2 1235.3 1477.9 1100.0 1184.1 1286.4 Catalase UrgHb. Before HBO 68.4 41.6 28.6 23.3 25.8 66.7 135.0 45.2 54.3 14191 Day 1 63.4 41.1 66.8 96.8 24.2 23.8 17.5 23.1 44.6 Glutathione-peroxidase UrgHb. Before HBO 8.6 18.9 32.2 37.9 52.8 50.4 42.5 55.8 37.4 2162 Day 1 14.7 13.7 30.4 52.7 52.8 48.1 38.2 59.6 38.8 Hemoglobin grl. Before HBO 341.5 301.5 267.0 326.5 319.0 274.0 304.0 315.0 306.1 132308 Day 1 299.0 295.5 259.0 299.5 332.0 279.0 315.0 302.0 297.6

of HSP70 synthesis by HBO Table 5.. In four subjects tested, a clear increase in HSP70 was seen one day after HBO. Heat treatment 20 min at 448C and recovery for 4 h at 378C. served as a positive control. Preliminary experiments with supplementation indicated that N-acetylcysteine NAC, 400 mg., given p.o. 1 h before HBO, did not significantly prevent HBO-induced DNA damage Fig. 1.. The mean tailmoment before and after HBO are somewhat lower in the group with supplementation because one of the four subjects PR. did not show increasd DNA damaging effects after HBO. One subject in the control group KK. had unusually high levels of DNA damge before and after HBO, when compared to the other subjects examined. In another test, two volunteers received 400 mg NAC intravenously 30 min before entering the HBO chamber. Again, there was no effect on the induction of DNA damage by

HBO compared to four subjects in the same HBO without supplementation data not shown.. Fig. 2 summarizes the comet assay effects of three subjects with and without vitamin E supplementation 800 mg daily for 1 week.. Vitamin E did not lead to reduced induction of DNA damage, as measured by the comet assay after HBO. Experiments with repair enzymes FPG protein and Endo III. specific for oxidative DNA base damage indicated that supple-

Table 5 Determination of the heat shock protein HSP70 in lymphocytes of subjects before and after HBO Subject HSP 70 a Before HBO BL OM TR PW
a

Day 1 128 96 149 123 Fig. 1. DNA migration tail-moment. in leukocytes of subjects before I. and after HBO B.. Subjects in A. were supplemented with an oral dose of 400 mg NAC one hour prior to HBO treatment. Subjects in B. were controls without supplementation in the same experiment.

26 31 41 58

Percentage %. of positive control.

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Fig. 2. DNA migration tail-moment. in leukocytes of subjects before I. and after HBO B.. The comet assay was performed without post-incubation with enzyme and with post-incubation with FPG or Endo III, respectively. A. subjects received 800 mg vitamin E daily for 7 days prior to the HBO. B. subjects without vitamin supplementation. Mean and SEM of 3 subjects.

mentation with vitamin E did not protect the cells against these types of ROS-induced lesions.

4. Discussion Our previous studies have indicated that a single exposure to HBO induces an adaptive response within lymphocytes in humans against subsequent HBO w1x. Furthermore, blood taken 24 h after HBO was shown to be well protected against the in vitro induction of DNA damage by hydrogen peroxide w3x. These findings could not be solely explained by increased DNA repair. An adaptive response to the genotoxic effects of ROS by the activation of a repair endonuclease has been demonstrated recently w20x, but we determined the effects in the comet assay immediately after a short hydrogen peroxide exposure so that repair effects could not occur w3x. Thus, the available data point to increased protection by antioxidants as the cause for the adaptive protection. Plasma contains a variety of low-molecular mass molecules with antioxidant activities like vitamins

and molecules with thiol groups. Several of these are dietary antioxidants and optimal intake of antioxidant nutrient is believed to be important in maintaining health w21x. We could not find any effect of HBO on this group of antioxidants. There was neither a change in vitamin levels nor in GSH concentration and also the total antioxidant power of the plasma as measured with the FRAP-assay was not significantly influenced. According to our previous findings demonstrating that the HBO-induced adaptive protection is a cellular effect w3x, an effect on cellular antioxidants could be expected. Our present results suggest that the antioxidant enzymes SOD, CAT and GPx did not increase in activity within the red cell concentrate fraction of blood and are not mainly responsible for the increased antioxidant defences in blood of humans after HBO. It is known, however, that in lung cells these enzymes are differentially regulated according to the type of oxidant injury inflicted. For example, hyperoxia caused an increase in SOD in rat lung cells in various studies w9x. In contrast to our findings, animal experiments with rats and guinea pigs indicated significant effects of HBO on antioxidant enzymes in brain and lung w10,22x. Possibly, blood is not well suited for the detection of changes in the activity of these enzymes because a possible induction in lymphocytes might be masked by the high background enzyme concentrations in plasma and erythrocytes. However, the large numbers of lymphocytes required for measurement of the activity of these antioxidant enzymes were too great to be practical. Another system that may contribute to protection against oxidative stress are families of stress-modulated proteins known as heat shock proteins HSPs.. The HSP70 family of proteins includes a constitutively expressed member and a stress-induced form. The synthesis of HSPs seems to play a significant role in cellular protection against oxidative damage w23,24x. We can now show that HSP70 is induced in lymphocytes of humans one day after HBO. Although the exact role of this group of stress response genes in the complex reaction of cells to oxidative stress remains to be elucidated, our results suggest that they contribute to the protection against ROS-induced DNA damage. Our attempts to modulate the cellular response to HBO by supplementation with vitamin E or NAC i.e., thiol groups. did not lead to significant effects.

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Vitamin E is a fat-soluble vitamin and a chain-breaking antioxidant which is very effective when incorporated into membranes. It has also been suggested that dietary supplementation with vitamin E may afford protection against substantial loss of cellular GSH levels. Experiments testing the resistance of rats to HBO-induced central nervous system toxicity found significantly higher levels of GSH in the liver, brain, lungs and blood of animals fed excess of vitamin E. However, HBO-induced toxicity was not influenced w25x. These authors also reported an increase in GPx-activity in rats after HBO which was higher in vitamin E supplemented animals than in the unsupplemented control group w22x. The efficiency of antioxidant supplementation in humans has been demonstrated in a study using the comet assay w26x. A supplement of vitamin C, b-carotene and vitamin E taken together for 20 weeks significantly decreased base oxidation in lymphocytes as well as rendering the lymphocytes more resistant to hydrogen peroxide-induced DNA damage in vitro. We have shown that vitamin E prevents DNA damage induced by exhaustive exercise which is known to induce free radical reactions w27x. Obviously, HBOinduced genotoxicity is due to different types of ROS and it remains to be shown whether an effective protocol for antioxidant vitamin supplementation exists. Another promising strategy might be the supplementation with NAC. NAC has the capacity to react directly with electrophiles and thus possesses antioxidant properties. NAC may also exert its antioxidant effect indirectly by facilitating GSH biosynthesis and supplying GSH for GPx-catalysed reactions. However treatment of rats with NAC did not protect against hyperoxia, led to decreased intracellular GSH levels and increased DNA damage w28x. Our treatment protocol with NAC did not increase GSH levels and did not influence HBO-induced DNA damage. Although there was considerable inter-individual variation in GSH levels, the mean values seem to indicate a decrease in plasmatic GSH concentrations after NAC supplementation while the cellular concentrations are unchanged. A single oral dose of NAC was given 1 h before start of the HBO treatment because it has been shown that the maximum plasma concentration is reached 12 h after oral intake w29x. However, the oral availability of NAC is only 610% w29x, but preliminary experi-

ments with intravenous application did not show any effect as well. Obviously, the applied protocols for antioxidant supplementation are not sufficient for providing protection against HBO-induced DNA damage in healthy volunteers. Whether or not more sophisticated protocols are effective remains to be shown.

Acknowledgements We would like to thank the volunteers for taking part in this study and Dr. H. Treiber for assistance in the HBO treatment. This study was financially supported by the program Environment and Health PUG. at the Forschungszentrum Karlsruhe with funds of the Department for Environment BadenWurttemberg, Germany. The investigations were part of the EU Concerted Action on HPRT Mutation EUCAHM; BMH4 CT96 0120..

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