Adaptive protection against the induction of oxidative DNA damage after hyperbaric oxygen treatment

Transcription

1 Carcinogenesis vol.19 no.11 pp , 1998 Adaptive protection against the induction of oxidative DNA damage after hyperbaric oxygen treatment Andreas Rothfuß, Claudia Dennog and Günter Speit 1 Universitätsklinikum Ulm, Abteilung Medizinische Genetik, D Ulm, Germany 1 To whom correspondence should be addressed guenter.speit@medizin.uni-ulm.de Hyperbaric oxygen (HBO) treatment (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total of three 20 min periods) of human subjects caused clear and reproducible DNA effects in the comet assay with leukocytes. Interestingly, DNA damage was detected only after the first treatment and not after further treatments under the same conditions, indicating an increase in antioxidant defences. We now demonstrate that blood taken 24 h after HBO treatment is well protected against the in vitro induction of DNA damage by hydrogen peroxide (H 2 O 2 ). H 2 O 2 treatment caused a significant induction of DNA effects in the comet assay and chromosome breakage in the micronucleus test in the blood of volunteers before HBO. The same treatment did not cause genotoxic effects 24 h after HBO. This protective effect lasted for at least 1 week. Experiments with isolated lymphocytes gave similar results, indicating that the adaptive response is a cellular effect. The cells were not comparably protected against the genotoxic effects of γ-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA. concentrations of oxygen may cause damaging effects to humans and it has been postulated that the known toxic effects of excessive exposure to oxygen are caused by an increased production of ROS (11). An increase in free radicals in the blood of persons undergoing HBO treatment was directly demonstrated by low temperature electron spin resonance (ESR) spectroscopy (12). Using the comet assay in conjunction with an enzyme (FPG protein) specific for oxidized DNA bases (8-oxo-guanines and formamidopyrimidines), we provided evidence for the induction of oxidative DNA base damage by HBO (9). Immediately after a single HBO treatment, clear and reproducible effects on DNA strand breakage and oxidative base damage were detected in peripheral blood of all subjects tested. Interestingly, DNA damage was detected only after the first HBO treatment and not after further treatments under the same conditions, indicating an increase in antioxidant defences. DNA damage also did not occur when HBO treatment was started with a reduced treatment time (1 20 min), which was then increased stepwise. To further characterize the antioxidant defence mechanisms after HBO we treated blood from subjects before and after HBO with ROS-producing mutagens. We used H 2 O 2 and γ-irradiation, which both cause DNA and other cellular damage through the formation of hydroxyl radicals (8,13). The genotoxic effects induced by H 2 O 2 and γ-irradiation were investigated with the comet assay and the micronucleus test. The comet assay has already been successfully used to demonstrate the induction of DNA strand breakage and oxidative base damage after treatment of blood cells with H 2 O 2 and γ-irradiation with high sensitivity (14 16). Since the biological significance of DNA effects seen in the comet assay is not yet clear (10,17) we also used the micronucleus assay for the evaluation of chromosome breakage. Both H 2 O 2 and γ-irradiation efficiently induce micronuclei in human blood in vitro (18,19). Experiments were performed to see whether there was a difference in the sensitivity of human blood against the genotoxic action of ROS-producing chemicals after HBO treatment of human subjects. Introduction DNA damage induced by reactive oxygen species (ROS) seems to play an important role in the induction of mutations and cancer (1,2). Endogenous damage to DNA caused by ROS liberated during normal respiration as well as oxidative damage induced exogenously by a variety of carcinogens may significantly contribute to the aetiology of cancer (3,4). To protect cells and organisms against ROS, various antioxidant defences have been evolved, which include enzymes and low molecular mass radical scavengers (5 7). When antioxidant defences are not completely efficient, increased free radical formation in the body is likely to increase damage. The term oxidative stress is commonly used to refer to this effect (8). We have recently suggested that hyperbaric oxygen (HBO) Materials and methods Test persons and HBO treatment Fourteen healthy volunteers (non-smokers, aged years) gave informed treatment as used therapeutically might be a well-suited model consent to participate in this study. They were exposed to 100% oxygen at a pressure of 2.5 ATA in a hyperbaric chamber for a total of three 20 min for the investigation of oxidative stress in humans (9,10). HBO periods, interspersed with 5 min periods of air breathing, this being a standard implies the inhalation of 100% oxygen under a pressure of therapeutically used HBO treatment protocol. Venous blood samples were 2.5 ATA (atmospheres absolute) in a hyperbaric chamber for taken before HBO and 1, 3 and 7 days later. The blood samples were kept a total of three 20 min periods, interspersed with 5 min of air breathing. Under these conditions the amount of dissolved oxygen in the blood and also the formation of ROS are significantly increased. It is known that exposure to high on ice and used for the tests within 1 h. Mutagen treatment H 2 O 2, obtained from Sigma (München, Germany), was diluted in distilled water. For the comet assay, freshly collected blood was treated with H 2 O 2 by adding 100 µl H 2 O 2 to 200 µl of whole blood or isolated lymphocytes for 5 min on ice or for 1 h at 37 C. Peripheral blood lymphocytes were separated Abbreviations: ESR, electron spin resonance; HBO, hyperbaric oxygen; H 2 O 2, hydrogen peroxide; LMA, low melting agarose; MN, micronucleus; from whole blood on Histopaque gradients (Sigma), washed twice in 1 phosphate-buffered saline (PBS) and resuspended in PBS. For the micronucleus NDI, Nuclear Division Index; PBS, phosphate-buffered saline; ROS, reactive test, H 2 O 2 was added to the blood cultures at the times indicated and oxygen species. maintained until cell harvest. Oxford University Press 1913

2 A.Rothfuß, C.Dennog and G.Speit Table I. Induction of DNA migration (tail moment) in the comet assay with leukocytes of eight subjects by hydrogen peroxide (H 2 O 2 ; M) before and 1 and 7 days after HBO treatment Day 0 Day 1 Day 7 Control H 2 O 2 Control H 2 O 2 Control H 2 O 2 DK GA IJ JB KK PH RK RK Fig. 1. DNA migration (tail moment) in the comet assay with blood cells of human subjects after treatment with H 2 O 2 ( M) before and on the 1st and 7th day after hyperbaric oxygen (HBO) treatment. Mean SEM of eight subjects. u, control cells; j, treatment with H 2 O 2.*P 0.05; **P Either freshly collected whole blood or blood diluted in chromosome medium was exposed to 1 Gy and 2 Gy with Co-60 γ-rays (Gammacell 2000; Nuclear Data, Frankfurt, Germany) at 4 Gy/min. After irradiation, blood was kept on ice and both tests were performed immediately. Comet assay 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. After solidifying the LMA, slides were processed as described earlier (9). The time of alkali denaturation and electrophoresis (0.86 V/cm) was 25 min each. The presence of oxidative base damage was determined with a modified protocol using FPG protein (9). 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 of the 50 cells. Micronucleus test For the analysis of micronuclei, the procedure described by Surrallés et al. (20) was followed with modifications. Heparinized whole blood (0.3 ml) was added to 3 ml of chromosome medium A (Gibco BRL), supplemented with 2% PHA-L (Gibco BRL) and incubated at 37 C. Cytochalasin B (cyt B) (Sigma) was added to the cultures at a final concentration of 6 µg/ml 24 or 44 h after PHA stimulation. Cultures were harvested 24 h after cytochalasin B addition, giving a total culture time of 48 or 68 h, respectively. Cells were harvested by centrifugation at 120 g for 10 min. After hypotonic treatment with 0.56% KCl and centrifugation, cells were fixed by gently adding a methanol/glacial acetic acid (5:1) solution mixed with an equal volume of 0.9% NaCl. The cells were centrifuged and further fixed with icecold methanol/glacial acetic acid. This step was repeated twice. The resulting cells were transferred on a slide, dried at room temperature and stained with acridine orange (0.125 mg/ml in phosphate buffer). The frequency of micronuclei (MNs) was determined by scoring 1000 binucleated cells with well-preserved cytoplasm. The identification of MNs was performed according to Fenech (21). A total of 500 cells were scored to evaluate the percentage of cells with 1, 2, 3, 4 or 4 nuclei. A Nuclear Division Index (NDI) was calculated as follows: NDI (N1 2 N2 3 N3 4 N4)/500. Statistical analysis Differences between mean values were tested for significance (P 0.05 and P 0.01) using Student s t-test. Results Figure 1 shows the induction of DNA damage after treatment of whole blood with H 2 O 2 before and after HBO. The same treatment that caused a significant increase in DNA migration (tail moment) before HBO did not significantly induce DNA 1914 Fig. 2. The effects of H 2 O 2 (10 3 M) on micronucleus frequencies in human blood cells before and after the 1st and 7th day after HBO treatment. Mean and SEM of eight subjects. u, control cells; j, treatment with H 2 O 2. **P BNC, binucleated cells. damage 1 and 7 days after HBO. The statistical difference between the induction before and after HBO is indicated. The results for each individual donor are summarized in Table I. In these experiments, cells were treated with H 2 O 2 for 5 min on ice. In further tests, the same effect was found after H 2 O 2 treatment ( M) for 1 h at 37 C. Four subjects were additionally tested 3 days after HBO. Again, there was no significant induction of DNA migration in any of the subjects (data not shown). Figure 2 summarizes the results of the micronucleus tests for the same eight subjects. It can be seen that H 2 O 2 doubled the MN frequency before HBO, whereas 1 and 7 days after a single HBO MN frequencies were in the range of controls after an identical H 2 O 2 treatment. In each of the subjects tested, the H 2 O 2 -induced frequency of micronuclei was lower after HBO than it was before. Table II gives the individual MN data for the eight subjects. There was no indication of an influence of cytotoxicity in MN frequencies since the NDI was in controls and after HBO. The results with the MN were confirmed in a second experiment, where cytochalasin had been given after 24 h and cells were harvested after a total culture time of 48 h (data not shown). Using FPG protein we can demonstrate that H 2 O 2 induces significant oxidative DNA base damage. The comet assay with additional FPG treatment detects the DNA damaging effect of

3 Adaptive protection after HBO treatment Table II. Induction of micronuclei (per 1000 binucleated cells) in lymphocytes of eight subjects by hydrogen peroxide (H 2 O 2 ;10 3 M) treatment before and 1 and 7 days after HBO treatment Day 0 Day 1 Day 7 Control H 2 O 2 Control H 2 O 2 Control H 2 O 2 DK GA IJ JB KK PH RK RK Fig. 4. The effects of H 2 O 2 ( M) on DNA migration (tail moment) in isolated lymphocytes of human subjects before and 24 h after HBO treatment. Mean and SEM of eight subjects. u, control cells; j, treatment with H 2 O 2.*P Fig. 3. The effects of FPG protein on DNA migration (induced tail moment) in blood cells before and 24 h after HBO treatment. Mean and SEM of four subjects. Light shading, standard protocol of the comet assay after treatment with H 2 O 2 ( M); dark shading, modified protocol with additional formamidopyrimidine DNA glycosylase (FPG) treatment. *P 0.05; **P H 2 O 2 before HBO in whole blood with high sensitivity (Figure 3). One day after HBO, besides the effects in the standard comet assay, also in the presence of FPG protein, significantly less DNA damage was induced by H 2 O 2 (Figure 3). To directly compare the effects with and without FPG treatment, the respective control values were subtracted and the induced tail moment calculated. To further characterize the cause of the protection against the genotoxic effects of H 2 O 2, we treated isolated lymphocytes before and 24 h after HBO (Figure 4). Compared with whole blood, the increase in tail moment after H 2 O 2 treatment was much higher in lymphocytes; however, the same significantly reduced induction of DNA damage was found 1 day after HBO. As in whole blood, the protective effect seemed to last for at least 1 week in lymphocytes: blood from four subjects clearly indicated significantly reduced induction of DNA damage on days 3 and 7 after HBO (data not shown). In contrast to our results with H 2 O 2, human blood does not seem to be comparably protected against the genotoxic action of γ-irradiation (Figures 5 and 6). Before HBO, γ-irradiation induced a clear and dose-dependent induction of effects in the comet assay and the MN test. However, in contrast to the results with H 2 O 2, there was no protective effect 24 h after Fig. 5. DNA migration in the comet assay with whole blood after γ-irradiation (1 Gy; 2 Gy) before and 24 h after HBO treatment. Mean and SEM of eight subjects. u, control cells; shaded, treatment with 1 Gy; j, treatment with 2 Gy. HBO. Although the values seem to be somewhat lower after HBO, no significant difference was found in any of the tests. Discussion Our results indicate that a single HBO treatment leads to the induction of an adaptive response that protects human blood cells against the genotoxic effects of H 2 O 2.H 2 O 2 treatment of whole blood before HBO caused a significant increase in DNA- and chromosome damage as revealed by the comet assay and the MN test. The same treatment of blood samples from the same subjects 24 h after HBO caused significantly less induction of genotoxic effects, suggesting increased antioxidant defence. This induced protection lasted for at least 1 week. It was seen in all subjects studied, although with some degree of interindividual variation. The additional use of the FPG protein resulted in a much stronger induction of DNA effects by H 2 O 2 in the comet assay and gave indirect evidence for the induction of oxidative DNA base damage (9,22). At 24 h 1915

4 A.Rothfuß, C.Dennog and G.Speit Fig. 6. Frequencies of binucleated lymphocytes with micronuclei (MN) after γ-irradiation (1 Gy and 2 Gy) before and 24 h after HBO treatment. Mean and SEM of eight subjects. u, control cells; shaded, treatment with 1 Gy; j, treatment with 2 Gy. BNC, binucleated cells. after a single HBO treatment, significantly less oxidative base damage was induced. These results confirm the protective effect and suggest that the protection was not limited to specific types of DNA lesions but seemed to be a general prevention against the genotoxic action of H 2 O 2. Using isolated lymphocytes for the same type of experiments, we could show that the genotoxic effect of H 2 O 2 was much stronger compared with whole blood. This difference has been reported earlier and was explained by the high antioxidant capacity of the plasma and the erythrocytes (23,24). Importantly, the same protective effect that we found in experiments with whole blood is seen in isolated lymphocytes as well. These results clearly indicate that the adaptive protection against H 2 O 2 - induced genotoxicity is a cellular effect. In contrast, we could not demonstrate a comparable protection against the genotoxic effects of γ-irradiation. After HBO, the induction of genotoxic effects by γ-irradiation seems to be somewhat smaller, in particular in the comet assay, but there was no statistically significant difference. For both, H 2 O 2 and γ-irradiation, the ultimate DNA-damaging ROS is believed to be the hydroxyl radical. However, the mode of their DNA-damaging activity might explain the different results for H 2 O 2 and γ-irradiation. Besides a direct DNA-breaking effect, γ-irradiation produces hydroxyl radicals by radiolysis of water molecules. These radicals are only very reactive at the site of their formation, i.e. DNA damage is induced by hydroxy radicals produced in the vicinity of the chromosomal DNA. In contrast, H 2 O 2 is rather stable and can diffuse rapidly across cell membranes. In the presence of transition metal ions it forms highly reactive free radicals that induce DNA damage when they occur close to the chromosomal DNA. We therefore suggest that the induced antioxidant defence destroys H 2 O 2 in the cell or scavenges ROS that might induce a radical chain reaction in the cell before the nuclear DNA is reached. The biochemical mechanism of the induced antioxidant defence is not yet clear. It is well known that unwanted H 2 O 2 is removed from cells by the action of catalase, glutathione peroxidase (selenium containing) and certain other peroxidases (25). We measured the antioxidative status (26), including the concentrations of the main antioxidant enzymes catalase, 1916 glutathione peroxidase (Se) and superoxide dismutase, in blood samples of subjects before and 24 h after HBO but did not find any significant difference (C. Dennog et al., in preparation). One possible explanation for this result might be that an induction in leukocytes is masked by high background enzyme concentrations in plasma and erythrocytes. These conflicting findings might also indicate that measurements of the antioxidant status in whole blood might not be sensitive enough to detect cellular adaptive protection. Various animal experiments have shown an increase in antioxidant enzymes after hyperoxia (27) but only a few studies have investigated the effects of HBO. In experiments with rats and guinea pigs, exposure to HBO had marked effects on antioxidant enzymes in brain and lung (28). In another study, where rats were exposed to HBO, an increase in the content of GSH (reduced glutathione) in lung tissue was observed (29). However, the available data are far from explaining the adaptive response to HBO in humans. We are, therefore, about to study various antioxidants at the cellular level. Our present data suggest that HBO treatment of humans is an excellent model system to elucidate at least one pathway of induced antioxidant defence in humans. Although our results point to increased protection by intracellular antioxidants of leukocytes they do not rule out an increase in extracellular antioxidants. An alternate explanation might also be an inducible change in the levels of free transition metals. It has been shown that superoxide enhances DNA damage by elevating free iron levels (30). If iron is sequestered more efficiently as a result of adaptation to the initial HBO treatment, the effect might be resistance to H 2 O 2 rather than ionizing radiation. Furthermore, an adaptive increase in DNA repair mechanism(s) might be induced. However, our findings cannot solely be explained by increased repair since the effects in the comet assay were determined immediately after a short H 2 O 2 exposure so that repair effects could not occur (31). Our findings also cannot be explained by induced shifts in the blood cell population. It has been reported earlier that after HBO no change in the total count and percentage of T cells, B cells and NK cells was seen but shifts in T cell subpopulations occurred immediately on exit from the chamber (32). The shift in T cell subpopulations was partially cancelled 24 h after HBO. We made blood cell counts before and after HBO (without determining T-cell subpopulations) and could also not find any significant deviation from normal values (data not shown). Using the comet assay to determine oxidative DNA damage in subjects, Duthie et al. (33) found that a supplement of vitamin C, β-carotene and vitamin E taken together for 20 weeks significantly decreased base oxidation in lymphocytes as well as rendering the lymphocytes more resistant to H 2 O 2 -induced DNA damage in vitro. However, the physiological mechanism of this effect is obviously different from what was happening after HBO. Vitamin supplementation seems to be less specific, because a single uptake of vitamin C also led to reduced induction of DNA strand breakage in the comet assay after in vitro irradiation of blood samples with ionizing radiation (34). Taken together, our results indicate that HBO induces an adaptive response that protects human blood cells against oxidative stress. This increased protection lasts for several days and is a cellular effect. The biochemical basis of this effect still has to be elucidated but antioxidants that scavenge ROS distant from nuclear DNA seem to be involved.

5 Adaptive protection after HBO treatment Acknowledgements We would like to thank the volunteers for taking part in this study and Dr Serge Boiteux, Institut Gustave Roussy, Villejuif, France, for the generous gift of the FPG protein. The authors are grateful to 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. References 1. Halliwell,B. (1994) Free radicals, antioxidants, and human disease: curiosity, cause or consequence? Lancet, 344, Loft,S. and Poulsen,H.E. (1996) Cancer risk and oxidative DNA damage in man. J. Mol. Med., 74, Ames,B.N. (1983) Dietary carcinogens and anticarcinogens, oxygen radicals and degenerative diseases. Science, 221, Ames,B.N. (1989) Endogenous DNA damage as related to cancer and aging. Mutat. Res., 214, Stocker,R. and Frei,B. 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Mutat. Res., 316, Received on June 8, 1998; revised on July 23, 1998; accepted on July 27,