DNA damage after long-term repetitive hyperbaric oxygen exposure

Michael Groumlger1 Suumlkruuml Oumlter14 Vladislava Simkova15 Markus Bolten2 Andreas Koch2

Volker Warninghoff2 Michael Georgieff1 Claus-Martin Muth1 Guumlnter Speit3 Peter

Radermacher1

1Sektion Anaumlsthesiologische Pathopyhsiologie und Verfahrensentwicklung and 3Abteilung

Humangenetik Universitaumltsklinikum Ulm Germany 2Schifffahrtmedizinisches Institut der

Marine Kronshagen Germany 4Fizyoloji Anabilim Dali Guumllhane Askeri Tip Akademisi

Ankara Turkey 5Anesteziologicko-resuscitacni klinika Fakultni nemocnice u sv Anny Brnoacute

Czech Republic

Running title DNA damage and long-term repetitive HBO

Address for correspondence

Peter Radermacher MD

Sektion Anaumlsthesiologische Pathopyhsiologie und Verfahrensentwicklung

Universitaumltsklinikum

Parkstrasse 11

D-89073 Ulm

Telephone 49 731 500 60160

Fax 49 731 500 60162

e-mail peterradermacheruni-ulmde

Word count 2281 Word count abstract 240

Articles in PresS J Appl Physiol (November 20 2008) doi101152japplphysiol907372008

Copyright copy 2008 by the American Physiological Society

2

Abstract

A single exposure to hyperbaric oxygen (HBO) ie pure oxygen breathing at supra-

atmospheric pressures causes oxidative DNA damage both in humans in vivo as well in

isolated lymphocytes of human volunteers These DNA lesions however are rapidly repaired

and an adaptive protection is triggered against further oxidative stress caused by HBO

exposure Therefore we tested the hypothesis whether long-term repetitive exposure to HBO

would modify the degree of DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years Non-diving volunteers with and without endurance training served as controls In

addition to the measurement of DNA damage in peripheral blood (comet assay) blood

antioxidant enzyme activities and the ratio of the oxidized and reduced glutathione content

we assessed the DNA damage and superoxide anion radical (O2˙-) production induced by a

single ex vivo HBO-exposure of isolated lymphocytes All parameters of oxidative stress and

antioxidative capacity in vivo were comparable in the four different groups Exposure to HBO

increased both the level of DNA damage and O2˙- production in lymphocytes and this

response was significantly more pronounced in the cells obtained from the combat swimmers

than in all the other groups However in all groups DNA damage was completely removed

within one hour We conclude that at least in healthy volunteers with endurance training

long-term repetitive exposure to HBO does not modify the basal blood antioxidant capacity or

the basal level of DNA strand breaks The increased ex vivo HBO-related DNA damage in

isolated lymphocytes from these subjects however may reflect enhanced susceptibility to

oxidative DNA damage

3

Key words hyperbaric oxygen ndash combat swimmers ndash UDT divers ndash endurance training -

comet assay ndash superoxide radical ndash superoxide dismutase ndash catalase ndash glutathione peroxidase

4

Introduction

Exposure to hyperbaric oxygen (HBO) ie pure oxygen breathing at supra-atmospheric

pressures increases the formation of oxygen radical species [2127] which in turn results in

consumption of antioxidants [223] and reduces antioxidant enzyme activity [4] ultimately

causing lipid peroxidation [342631] organ injury [5] and DNA damage [101117202632-

3739-42] On the other hand HBO was reported to promote protective pre-conditioning

against ischemiareperfusion-induced oxidative organ injury in the brain spinal cord heart

and liver [16284546] Furthermore in healthy volunteers the HBO-induced DNA damage

not only rapidly disappeared after the end of the HBO exposure but a subsequent exposure

did not cause oxidative DNA damage any more [10] indicating the induction of antioxidant

defense [364041] that lasted for at least one week [32] In fact both a single HBO exposure

as well as a compressed-air dive to a depth of 40 meters ie at an inspiratory O2 partial

pressure of 1 bar increased the lymphocyte glutathione peroxidase activity [15] Finally in

healthy volunteers with a long-term diving experience and consequently repetitive diving-

associated exposures to hyperoxia HBO-induced DNA damage was less pronounced than in

non-diving controls [1126]

Therefore we tested the hypothesis whether long-term repetitive exposure to HBO would

modify the degree of DNA damage induced by a single ex vivo HBO-exposure of isolated

human peripheral lymphocytes In addition we investigated whether long-term repetitive

exposure to high inspiratory O2 partial pressures affects the lymphocyte total cellular

superoxide radical (O2˙-) production in response to a single well-defined HBO-induced

oxidative stress We studied combat swimmers and Underwater Demolition Team (UDT)

divers because over several years these subjects perform dives breathing pure O2 and O2-

enriched inspiratory gas mixtures using closed and semi-closed circuits respectively Thus

they represent a population with a particularly long-term repetitive exposure to HBO Combat

5

swimmers and UDT divers also undergo pronounced endurance training and most of their

diving activity also comprises strenuous exercise Exhaustive physical effort per se induces

oxidative stress-related DNA damage [1418192529] in particular beyond the anaerobic

threshold [1418] Since this response is markedly affected by the degree of endurance

training [29] a group of ldquoNaval Pentathlonrdquo athletes was also studied in order to compare the

divers with non-diving subjects with a comparable degree of endurance training Finally

isolated peripheral lymphocytes were investigated because i) isolated lymphocytes show more

pronounced HBO-induced DNA damage than whole blood samples and represent a well-

established model for the investigation of DNA damage in human biomonitoring [43] and ii)

allow for using HBO at O2 partial pressures and duration beyond those of the so-called

ldquoOxygen Tolerance Testrdquo [23] ie HBO at 28 bar for 30 minutes The in vivo central nervous

O2 toxicitiy [6] otherwise precludes exposing human volunteers to these HBO exposures The

level of HBO-induced DNA damage was assessed using the comet assay (single cell gel

electrophoresis) which allows measuring both the induction of DNA damage as well as its

repair [43] Previous investigations using the comet assay clearly demonstrated induction of

oxidative DNA damage (including oxidative DNA base damage as detected by the FPG

protein) in lymphocytes after exposure to a single HBO in vivo and in vitro as well as rapid

repair of the induced lesions [39]

6

Methods

The present study was part of a larger investigation entitled ldquoOxidative Stress during

Hyperoxia and Divingrdquo the protocol of which had been approved by the ethical committee of

the Christian-Albrecht-Universitaumlt Kiel All subjects gave their written informed consent to

participate in the investigation A total of 37 subjects were studied comprising combat

swimmers (n = 6) Underwater Demolition Team (UDT) divers (n = 13) and non-diving

volunteers with (ldquoNaval Pentathlon athletesrdquo n = 6) and without (controls n = 12) endurance

training The demographic data of the subjects are summarised in table 1 The UDT divers

had a diving experience of several years with a diving activity of approximately 10 hours

using O2 enriched gas mixtures and closed-circuit pure O2 breathing apparatus during the 15

months preceding the study (table 1) The combat swimmers had a median diving experience

using a closed-circuit pure O2 breathing apparatus of nearly one year with a median diving

time using this device of about 100 hours (table 1) The UDT divers and combat swimmers

had performed their last dives using O2 enriched gas mixtures or closed-circuit pure O2

breathing apparatus 4 ndash 6 and two weeks respectively prior to the investigation The Naval

Pentathlon athletes were investigated during their world championship preparation which had

started two months prior to the investigation All investigations took place during a two weeks

period Blood sampling took place in the morning after overnight fasting and was part of the

individual subjectsrsquo regular routine medical examination

Whole blood measurements

In all subjects whole blood samples were analysed for superoxide dismutase (SOD) catalase

and plasma gluthathion peroxidase (GPx) activities as well as the concentrations of reduced

(GSH) and oxidised (GSSG) glutathione respectively using commercially available test kits

as described previously [3] (SOD SOD Assay Kit-WST Dojindo Molecular Technologies

Inc Kumamoto Japan catalase Bioxytechreg Catalase-520trade OxisResearchtrade Portland OR

7

GPx Gluthathione Peroxidase Assay Kit Cayman Chemical Ann Arbor MI GSH GSSG

GSHGSSG Ratio Assay Kit Calbiochemreg EMD Chemicals Inc San Diego CA) DNA

strand breaks were quantified with the alkaline version of the comet assay [44] Briefly cell

lysis for at least 1 hour and slide processing were performed as previously described in detail

[172026] using alkali denaturation and electrophoresis (086 Vmiddotcm-1 at a pH gt 13) to

transform alkali-sensitive parts of the DNA into DNA strand breaks Measurements were

made by image analysis determining the mean tail moment and the mean tail intensity of 100

cells per slide (two slides each per measurement in each individual) In none of the

experiments there was a relevant difference between these two parameters Therefore in

continuation of our previous studies we chose the tail moment for the presentation of our

results

Isolated lymphocytes

Lymphocytes were separated on Ficoll gradients washed in PBS buffer resuspended in

RPMI1640 buffer and thereafter exposed to HBO in a hyperbaric chamber containing 2

CO2 (to maintain pH 70-75) and 98 O2 as described previously (37 degC compression and

decompression rate 02 barsdotmin-1 2 hours isopression at 4 bar) [20] The comet assay was

performed in aliquots before as well as immediately one and two hours and after the HBO

exposure In addition the lymphocyte total cellular O2˙- concentration was measured at room

temperature after incubation with the spin probe 1-hydroxy-3-methoxycarbonyl-2255-

tetramethylpyrrolidine (CMH) before and immediately after the HBO exposure by electron

spin resonance using a Bruker EMX spectrometer [12] Results are expressed as nanomoles

O2˙- released by 106 cells In order to exclude any effect of the lymphocyte incubation

procedure on the comet assay results and the electron spin resonance measurements per se we

also determined the tail moment and the O2˙- in time control lymphocyte aliquots without

HBO-exposure (n = 6)

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

2

Abstract

A single exposure to hyperbaric oxygen (HBO) ie pure oxygen breathing at supra-

atmospheric pressures causes oxidative DNA damage both in humans in vivo as well in

isolated lymphocytes of human volunteers These DNA lesions however are rapidly repaired

and an adaptive protection is triggered against further oxidative stress caused by HBO

exposure Therefore we tested the hypothesis whether long-term repetitive exposure to HBO

would modify the degree of DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years Non-diving volunteers with and without endurance training served as controls In

addition to the measurement of DNA damage in peripheral blood (comet assay) blood

antioxidant enzyme activities and the ratio of the oxidized and reduced glutathione content

we assessed the DNA damage and superoxide anion radical (O2˙-) production induced by a

single ex vivo HBO-exposure of isolated lymphocytes All parameters of oxidative stress and

antioxidative capacity in vivo were comparable in the four different groups Exposure to HBO

increased both the level of DNA damage and O2˙- production in lymphocytes and this

response was significantly more pronounced in the cells obtained from the combat swimmers

than in all the other groups However in all groups DNA damage was completely removed

within one hour We conclude that at least in healthy volunteers with endurance training

long-term repetitive exposure to HBO does not modify the basal blood antioxidant capacity or

the basal level of DNA strand breaks The increased ex vivo HBO-related DNA damage in

isolated lymphocytes from these subjects however may reflect enhanced susceptibility to

oxidative DNA damage

3

Key words hyperbaric oxygen ndash combat swimmers ndash UDT divers ndash endurance training -

comet assay ndash superoxide radical ndash superoxide dismutase ndash catalase ndash glutathione peroxidase

4

Introduction

Exposure to hyperbaric oxygen (HBO) ie pure oxygen breathing at supra-atmospheric

pressures increases the formation of oxygen radical species [2127] which in turn results in

consumption of antioxidants [223] and reduces antioxidant enzyme activity [4] ultimately

causing lipid peroxidation [342631] organ injury [5] and DNA damage [101117202632-

3739-42] On the other hand HBO was reported to promote protective pre-conditioning

against ischemiareperfusion-induced oxidative organ injury in the brain spinal cord heart

and liver [16284546] Furthermore in healthy volunteers the HBO-induced DNA damage

not only rapidly disappeared after the end of the HBO exposure but a subsequent exposure

did not cause oxidative DNA damage any more [10] indicating the induction of antioxidant

defense [364041] that lasted for at least one week [32] In fact both a single HBO exposure

as well as a compressed-air dive to a depth of 40 meters ie at an inspiratory O2 partial

pressure of 1 bar increased the lymphocyte glutathione peroxidase activity [15] Finally in

healthy volunteers with a long-term diving experience and consequently repetitive diving-

associated exposures to hyperoxia HBO-induced DNA damage was less pronounced than in

non-diving controls [1126]

Therefore we tested the hypothesis whether long-term repetitive exposure to HBO would

modify the degree of DNA damage induced by a single ex vivo HBO-exposure of isolated

human peripheral lymphocytes In addition we investigated whether long-term repetitive

exposure to high inspiratory O2 partial pressures affects the lymphocyte total cellular

superoxide radical (O2˙-) production in response to a single well-defined HBO-induced

oxidative stress We studied combat swimmers and Underwater Demolition Team (UDT)

divers because over several years these subjects perform dives breathing pure O2 and O2-

enriched inspiratory gas mixtures using closed and semi-closed circuits respectively Thus

they represent a population with a particularly long-term repetitive exposure to HBO Combat

5

swimmers and UDT divers also undergo pronounced endurance training and most of their

diving activity also comprises strenuous exercise Exhaustive physical effort per se induces

oxidative stress-related DNA damage [1418192529] in particular beyond the anaerobic

threshold [1418] Since this response is markedly affected by the degree of endurance

training [29] a group of ldquoNaval Pentathlonrdquo athletes was also studied in order to compare the

divers with non-diving subjects with a comparable degree of endurance training Finally

isolated peripheral lymphocytes were investigated because i) isolated lymphocytes show more

pronounced HBO-induced DNA damage than whole blood samples and represent a well-

established model for the investigation of DNA damage in human biomonitoring [43] and ii)

allow for using HBO at O2 partial pressures and duration beyond those of the so-called

ldquoOxygen Tolerance Testrdquo [23] ie HBO at 28 bar for 30 minutes The in vivo central nervous

O2 toxicitiy [6] otherwise precludes exposing human volunteers to these HBO exposures The

level of HBO-induced DNA damage was assessed using the comet assay (single cell gel

electrophoresis) which allows measuring both the induction of DNA damage as well as its

repair [43] Previous investigations using the comet assay clearly demonstrated induction of

oxidative DNA damage (including oxidative DNA base damage as detected by the FPG

protein) in lymphocytes after exposure to a single HBO in vivo and in vitro as well as rapid

repair of the induced lesions [39]

6

Methods

The present study was part of a larger investigation entitled ldquoOxidative Stress during

Hyperoxia and Divingrdquo the protocol of which had been approved by the ethical committee of

the Christian-Albrecht-Universitaumlt Kiel All subjects gave their written informed consent to

participate in the investigation A total of 37 subjects were studied comprising combat

swimmers (n = 6) Underwater Demolition Team (UDT) divers (n = 13) and non-diving

volunteers with (ldquoNaval Pentathlon athletesrdquo n = 6) and without (controls n = 12) endurance

training The demographic data of the subjects are summarised in table 1 The UDT divers

had a diving experience of several years with a diving activity of approximately 10 hours

using O2 enriched gas mixtures and closed-circuit pure O2 breathing apparatus during the 15

months preceding the study (table 1) The combat swimmers had a median diving experience

using a closed-circuit pure O2 breathing apparatus of nearly one year with a median diving

time using this device of about 100 hours (table 1) The UDT divers and combat swimmers

had performed their last dives using O2 enriched gas mixtures or closed-circuit pure O2

breathing apparatus 4 ndash 6 and two weeks respectively prior to the investigation The Naval

Pentathlon athletes were investigated during their world championship preparation which had

started two months prior to the investigation All investigations took place during a two weeks

period Blood sampling took place in the morning after overnight fasting and was part of the

individual subjectsrsquo regular routine medical examination

Whole blood measurements

In all subjects whole blood samples were analysed for superoxide dismutase (SOD) catalase

and plasma gluthathion peroxidase (GPx) activities as well as the concentrations of reduced

(GSH) and oxidised (GSSG) glutathione respectively using commercially available test kits

as described previously [3] (SOD SOD Assay Kit-WST Dojindo Molecular Technologies

Inc Kumamoto Japan catalase Bioxytechreg Catalase-520trade OxisResearchtrade Portland OR

7

GPx Gluthathione Peroxidase Assay Kit Cayman Chemical Ann Arbor MI GSH GSSG

GSHGSSG Ratio Assay Kit Calbiochemreg EMD Chemicals Inc San Diego CA) DNA

strand breaks were quantified with the alkaline version of the comet assay [44] Briefly cell

lysis for at least 1 hour and slide processing were performed as previously described in detail

[172026] using alkali denaturation and electrophoresis (086 Vmiddotcm-1 at a pH gt 13) to

transform alkali-sensitive parts of the DNA into DNA strand breaks Measurements were

made by image analysis determining the mean tail moment and the mean tail intensity of 100

cells per slide (two slides each per measurement in each individual) In none of the

experiments there was a relevant difference between these two parameters Therefore in

continuation of our previous studies we chose the tail moment for the presentation of our

results

Isolated lymphocytes

Lymphocytes were separated on Ficoll gradients washed in PBS buffer resuspended in

RPMI1640 buffer and thereafter exposed to HBO in a hyperbaric chamber containing 2

CO2 (to maintain pH 70-75) and 98 O2 as described previously (37 degC compression and

decompression rate 02 barsdotmin-1 2 hours isopression at 4 bar) [20] The comet assay was

performed in aliquots before as well as immediately one and two hours and after the HBO

exposure In addition the lymphocyte total cellular O2˙- concentration was measured at room

temperature after incubation with the spin probe 1-hydroxy-3-methoxycarbonyl-2255-

tetramethylpyrrolidine (CMH) before and immediately after the HBO exposure by electron

spin resonance using a Bruker EMX spectrometer [12] Results are expressed as nanomoles

O2˙- released by 106 cells In order to exclude any effect of the lymphocyte incubation

procedure on the comet assay results and the electron spin resonance measurements per se we

also determined the tail moment and the O2˙- in time control lymphocyte aliquots without

HBO-exposure (n = 6)

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

3

Key words hyperbaric oxygen ndash combat swimmers ndash UDT divers ndash endurance training -

comet assay ndash superoxide radical ndash superoxide dismutase ndash catalase ndash glutathione peroxidase

4

Introduction

Exposure to hyperbaric oxygen (HBO) ie pure oxygen breathing at supra-atmospheric

pressures increases the formation of oxygen radical species [2127] which in turn results in

consumption of antioxidants [223] and reduces antioxidant enzyme activity [4] ultimately

causing lipid peroxidation [342631] organ injury [5] and DNA damage [101117202632-

3739-42] On the other hand HBO was reported to promote protective pre-conditioning

against ischemiareperfusion-induced oxidative organ injury in the brain spinal cord heart

and liver [16284546] Furthermore in healthy volunteers the HBO-induced DNA damage

not only rapidly disappeared after the end of the HBO exposure but a subsequent exposure

did not cause oxidative DNA damage any more [10] indicating the induction of antioxidant

defense [364041] that lasted for at least one week [32] In fact both a single HBO exposure

as well as a compressed-air dive to a depth of 40 meters ie at an inspiratory O2 partial

pressure of 1 bar increased the lymphocyte glutathione peroxidase activity [15] Finally in

healthy volunteers with a long-term diving experience and consequently repetitive diving-

associated exposures to hyperoxia HBO-induced DNA damage was less pronounced than in

non-diving controls [1126]

Therefore we tested the hypothesis whether long-term repetitive exposure to HBO would

modify the degree of DNA damage induced by a single ex vivo HBO-exposure of isolated

human peripheral lymphocytes In addition we investigated whether long-term repetitive

exposure to high inspiratory O2 partial pressures affects the lymphocyte total cellular

superoxide radical (O2˙-) production in response to a single well-defined HBO-induced

oxidative stress We studied combat swimmers and Underwater Demolition Team (UDT)

divers because over several years these subjects perform dives breathing pure O2 and O2-

enriched inspiratory gas mixtures using closed and semi-closed circuits respectively Thus

they represent a population with a particularly long-term repetitive exposure to HBO Combat

5

swimmers and UDT divers also undergo pronounced endurance training and most of their

diving activity also comprises strenuous exercise Exhaustive physical effort per se induces

oxidative stress-related DNA damage [1418192529] in particular beyond the anaerobic

threshold [1418] Since this response is markedly affected by the degree of endurance

training [29] a group of ldquoNaval Pentathlonrdquo athletes was also studied in order to compare the

divers with non-diving subjects with a comparable degree of endurance training Finally

isolated peripheral lymphocytes were investigated because i) isolated lymphocytes show more

pronounced HBO-induced DNA damage than whole blood samples and represent a well-

established model for the investigation of DNA damage in human biomonitoring [43] and ii)

allow for using HBO at O2 partial pressures and duration beyond those of the so-called

ldquoOxygen Tolerance Testrdquo [23] ie HBO at 28 bar for 30 minutes The in vivo central nervous

O2 toxicitiy [6] otherwise precludes exposing human volunteers to these HBO exposures The

level of HBO-induced DNA damage was assessed using the comet assay (single cell gel

electrophoresis) which allows measuring both the induction of DNA damage as well as its

repair [43] Previous investigations using the comet assay clearly demonstrated induction of

oxidative DNA damage (including oxidative DNA base damage as detected by the FPG

protein) in lymphocytes after exposure to a single HBO in vivo and in vitro as well as rapid

repair of the induced lesions [39]

6

Methods

The present study was part of a larger investigation entitled ldquoOxidative Stress during

Hyperoxia and Divingrdquo the protocol of which had been approved by the ethical committee of

the Christian-Albrecht-Universitaumlt Kiel All subjects gave their written informed consent to

participate in the investigation A total of 37 subjects were studied comprising combat

swimmers (n = 6) Underwater Demolition Team (UDT) divers (n = 13) and non-diving

volunteers with (ldquoNaval Pentathlon athletesrdquo n = 6) and without (controls n = 12) endurance

training The demographic data of the subjects are summarised in table 1 The UDT divers

had a diving experience of several years with a diving activity of approximately 10 hours

using O2 enriched gas mixtures and closed-circuit pure O2 breathing apparatus during the 15

months preceding the study (table 1) The combat swimmers had a median diving experience

using a closed-circuit pure O2 breathing apparatus of nearly one year with a median diving

time using this device of about 100 hours (table 1) The UDT divers and combat swimmers

had performed their last dives using O2 enriched gas mixtures or closed-circuit pure O2

breathing apparatus 4 ndash 6 and two weeks respectively prior to the investigation The Naval

Pentathlon athletes were investigated during their world championship preparation which had

started two months prior to the investigation All investigations took place during a two weeks

period Blood sampling took place in the morning after overnight fasting and was part of the

individual subjectsrsquo regular routine medical examination

Whole blood measurements

In all subjects whole blood samples were analysed for superoxide dismutase (SOD) catalase

and plasma gluthathion peroxidase (GPx) activities as well as the concentrations of reduced

(GSH) and oxidised (GSSG) glutathione respectively using commercially available test kits

as described previously [3] (SOD SOD Assay Kit-WST Dojindo Molecular Technologies

Inc Kumamoto Japan catalase Bioxytechreg Catalase-520trade OxisResearchtrade Portland OR

7

GPx Gluthathione Peroxidase Assay Kit Cayman Chemical Ann Arbor MI GSH GSSG

GSHGSSG Ratio Assay Kit Calbiochemreg EMD Chemicals Inc San Diego CA) DNA

strand breaks were quantified with the alkaline version of the comet assay [44] Briefly cell

lysis for at least 1 hour and slide processing were performed as previously described in detail

[172026] using alkali denaturation and electrophoresis (086 Vmiddotcm-1 at a pH gt 13) to

transform alkali-sensitive parts of the DNA into DNA strand breaks Measurements were

made by image analysis determining the mean tail moment and the mean tail intensity of 100

cells per slide (two slides each per measurement in each individual) In none of the

experiments there was a relevant difference between these two parameters Therefore in

continuation of our previous studies we chose the tail moment for the presentation of our

results

Isolated lymphocytes

Lymphocytes were separated on Ficoll gradients washed in PBS buffer resuspended in

RPMI1640 buffer and thereafter exposed to HBO in a hyperbaric chamber containing 2

CO2 (to maintain pH 70-75) and 98 O2 as described previously (37 degC compression and

decompression rate 02 barsdotmin-1 2 hours isopression at 4 bar) [20] The comet assay was

performed in aliquots before as well as immediately one and two hours and after the HBO

exposure In addition the lymphocyte total cellular O2˙- concentration was measured at room

temperature after incubation with the spin probe 1-hydroxy-3-methoxycarbonyl-2255-

tetramethylpyrrolidine (CMH) before and immediately after the HBO exposure by electron

spin resonance using a Bruker EMX spectrometer [12] Results are expressed as nanomoles

O2˙- released by 106 cells In order to exclude any effect of the lymphocyte incubation

procedure on the comet assay results and the electron spin resonance measurements per se we

also determined the tail moment and the O2˙- in time control lymphocyte aliquots without

HBO-exposure (n = 6)

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

4

Introduction

Exposure to hyperbaric oxygen (HBO) ie pure oxygen breathing at supra-atmospheric

pressures increases the formation of oxygen radical species [2127] which in turn results in

consumption of antioxidants [223] and reduces antioxidant enzyme activity [4] ultimately

causing lipid peroxidation [342631] organ injury [5] and DNA damage [101117202632-

3739-42] On the other hand HBO was reported to promote protective pre-conditioning

against ischemiareperfusion-induced oxidative organ injury in the brain spinal cord heart

and liver [16284546] Furthermore in healthy volunteers the HBO-induced DNA damage

not only rapidly disappeared after the end of the HBO exposure but a subsequent exposure

did not cause oxidative DNA damage any more [10] indicating the induction of antioxidant

defense [364041] that lasted for at least one week [32] In fact both a single HBO exposure

as well as a compressed-air dive to a depth of 40 meters ie at an inspiratory O2 partial

pressure of 1 bar increased the lymphocyte glutathione peroxidase activity [15] Finally in

healthy volunteers with a long-term diving experience and consequently repetitive diving-

associated exposures to hyperoxia HBO-induced DNA damage was less pronounced than in

non-diving controls [1126]

Therefore we tested the hypothesis whether long-term repetitive exposure to HBO would

modify the degree of DNA damage induced by a single ex vivo HBO-exposure of isolated

human peripheral lymphocytes In addition we investigated whether long-term repetitive

exposure to high inspiratory O2 partial pressures affects the lymphocyte total cellular

superoxide radical (O2˙-) production in response to a single well-defined HBO-induced

oxidative stress We studied combat swimmers and Underwater Demolition Team (UDT)

divers because over several years these subjects perform dives breathing pure O2 and O2-

enriched inspiratory gas mixtures using closed and semi-closed circuits respectively Thus

they represent a population with a particularly long-term repetitive exposure to HBO Combat

5

swimmers and UDT divers also undergo pronounced endurance training and most of their

diving activity also comprises strenuous exercise Exhaustive physical effort per se induces

oxidative stress-related DNA damage [1418192529] in particular beyond the anaerobic

threshold [1418] Since this response is markedly affected by the degree of endurance

training [29] a group of ldquoNaval Pentathlonrdquo athletes was also studied in order to compare the

divers with non-diving subjects with a comparable degree of endurance training Finally

isolated peripheral lymphocytes were investigated because i) isolated lymphocytes show more

pronounced HBO-induced DNA damage than whole blood samples and represent a well-

established model for the investigation of DNA damage in human biomonitoring [43] and ii)

allow for using HBO at O2 partial pressures and duration beyond those of the so-called

ldquoOxygen Tolerance Testrdquo [23] ie HBO at 28 bar for 30 minutes The in vivo central nervous

O2 toxicitiy [6] otherwise precludes exposing human volunteers to these HBO exposures The

level of HBO-induced DNA damage was assessed using the comet assay (single cell gel

electrophoresis) which allows measuring both the induction of DNA damage as well as its

repair [43] Previous investigations using the comet assay clearly demonstrated induction of

oxidative DNA damage (including oxidative DNA base damage as detected by the FPG

protein) in lymphocytes after exposure to a single HBO in vivo and in vitro as well as rapid

repair of the induced lesions [39]

6

Methods

The present study was part of a larger investigation entitled ldquoOxidative Stress during

Hyperoxia and Divingrdquo the protocol of which had been approved by the ethical committee of

the Christian-Albrecht-Universitaumlt Kiel All subjects gave their written informed consent to

participate in the investigation A total of 37 subjects were studied comprising combat

swimmers (n = 6) Underwater Demolition Team (UDT) divers (n = 13) and non-diving

volunteers with (ldquoNaval Pentathlon athletesrdquo n = 6) and without (controls n = 12) endurance

training The demographic data of the subjects are summarised in table 1 The UDT divers

had a diving experience of several years with a diving activity of approximately 10 hours

using O2 enriched gas mixtures and closed-circuit pure O2 breathing apparatus during the 15

months preceding the study (table 1) The combat swimmers had a median diving experience

using a closed-circuit pure O2 breathing apparatus of nearly one year with a median diving

time using this device of about 100 hours (table 1) The UDT divers and combat swimmers

had performed their last dives using O2 enriched gas mixtures or closed-circuit pure O2

breathing apparatus 4 ndash 6 and two weeks respectively prior to the investigation The Naval

Pentathlon athletes were investigated during their world championship preparation which had

started two months prior to the investigation All investigations took place during a two weeks

period Blood sampling took place in the morning after overnight fasting and was part of the

individual subjectsrsquo regular routine medical examination

Whole blood measurements

In all subjects whole blood samples were analysed for superoxide dismutase (SOD) catalase

and plasma gluthathion peroxidase (GPx) activities as well as the concentrations of reduced

(GSH) and oxidised (GSSG) glutathione respectively using commercially available test kits

as described previously [3] (SOD SOD Assay Kit-WST Dojindo Molecular Technologies

Inc Kumamoto Japan catalase Bioxytechreg Catalase-520trade OxisResearchtrade Portland OR

7

GPx Gluthathione Peroxidase Assay Kit Cayman Chemical Ann Arbor MI GSH GSSG

GSHGSSG Ratio Assay Kit Calbiochemreg EMD Chemicals Inc San Diego CA) DNA

strand breaks were quantified with the alkaline version of the comet assay [44] Briefly cell

lysis for at least 1 hour and slide processing were performed as previously described in detail

[172026] using alkali denaturation and electrophoresis (086 Vmiddotcm-1 at a pH gt 13) to

transform alkali-sensitive parts of the DNA into DNA strand breaks Measurements were

made by image analysis determining the mean tail moment and the mean tail intensity of 100

cells per slide (two slides each per measurement in each individual) In none of the

experiments there was a relevant difference between these two parameters Therefore in

continuation of our previous studies we chose the tail moment for the presentation of our

results

Isolated lymphocytes

Lymphocytes were separated on Ficoll gradients washed in PBS buffer resuspended in

RPMI1640 buffer and thereafter exposed to HBO in a hyperbaric chamber containing 2

CO2 (to maintain pH 70-75) and 98 O2 as described previously (37 degC compression and

decompression rate 02 barsdotmin-1 2 hours isopression at 4 bar) [20] The comet assay was

performed in aliquots before as well as immediately one and two hours and after the HBO

exposure In addition the lymphocyte total cellular O2˙- concentration was measured at room

temperature after incubation with the spin probe 1-hydroxy-3-methoxycarbonyl-2255-

tetramethylpyrrolidine (CMH) before and immediately after the HBO exposure by electron

spin resonance using a Bruker EMX spectrometer [12] Results are expressed as nanomoles

O2˙- released by 106 cells In order to exclude any effect of the lymphocyte incubation

procedure on the comet assay results and the electron spin resonance measurements per se we

also determined the tail moment and the O2˙- in time control lymphocyte aliquots without

HBO-exposure (n = 6)

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

5

swimmers and UDT divers also undergo pronounced endurance training and most of their

diving activity also comprises strenuous exercise Exhaustive physical effort per se induces

oxidative stress-related DNA damage [1418192529] in particular beyond the anaerobic

threshold [1418] Since this response is markedly affected by the degree of endurance

training [29] a group of ldquoNaval Pentathlonrdquo athletes was also studied in order to compare the

divers with non-diving subjects with a comparable degree of endurance training Finally

isolated peripheral lymphocytes were investigated because i) isolated lymphocytes show more

pronounced HBO-induced DNA damage than whole blood samples and represent a well-

established model for the investigation of DNA damage in human biomonitoring [43] and ii)

allow for using HBO at O2 partial pressures and duration beyond those of the so-called

ldquoOxygen Tolerance Testrdquo [23] ie HBO at 28 bar for 30 minutes The in vivo central nervous

O2 toxicitiy [6] otherwise precludes exposing human volunteers to these HBO exposures The

level of HBO-induced DNA damage was assessed using the comet assay (single cell gel

electrophoresis) which allows measuring both the induction of DNA damage as well as its

repair [43] Previous investigations using the comet assay clearly demonstrated induction of

oxidative DNA damage (including oxidative DNA base damage as detected by the FPG

protein) in lymphocytes after exposure to a single HBO in vivo and in vitro as well as rapid

repair of the induced lesions [39]

6

Methods

The present study was part of a larger investigation entitled ldquoOxidative Stress during

Hyperoxia and Divingrdquo the protocol of which had been approved by the ethical committee of

the Christian-Albrecht-Universitaumlt Kiel All subjects gave their written informed consent to

participate in the investigation A total of 37 subjects were studied comprising combat

swimmers (n = 6) Underwater Demolition Team (UDT) divers (n = 13) and non-diving

volunteers with (ldquoNaval Pentathlon athletesrdquo n = 6) and without (controls n = 12) endurance

training The demographic data of the subjects are summarised in table 1 The UDT divers

had a diving experience of several years with a diving activity of approximately 10 hours

using O2 enriched gas mixtures and closed-circuit pure O2 breathing apparatus during the 15

months preceding the study (table 1) The combat swimmers had a median diving experience

using a closed-circuit pure O2 breathing apparatus of nearly one year with a median diving

time using this device of about 100 hours (table 1) The UDT divers and combat swimmers

had performed their last dives using O2 enriched gas mixtures or closed-circuit pure O2

breathing apparatus 4 ndash 6 and two weeks respectively prior to the investigation The Naval

Pentathlon athletes were investigated during their world championship preparation which had

started two months prior to the investigation All investigations took place during a two weeks

period Blood sampling took place in the morning after overnight fasting and was part of the

individual subjectsrsquo regular routine medical examination

Whole blood measurements

In all subjects whole blood samples were analysed for superoxide dismutase (SOD) catalase

and plasma gluthathion peroxidase (GPx) activities as well as the concentrations of reduced

(GSH) and oxidised (GSSG) glutathione respectively using commercially available test kits

as described previously [3] (SOD SOD Assay Kit-WST Dojindo Molecular Technologies

Inc Kumamoto Japan catalase Bioxytechreg Catalase-520trade OxisResearchtrade Portland OR

7

GPx Gluthathione Peroxidase Assay Kit Cayman Chemical Ann Arbor MI GSH GSSG

GSHGSSG Ratio Assay Kit Calbiochemreg EMD Chemicals Inc San Diego CA) DNA

strand breaks were quantified with the alkaline version of the comet assay [44] Briefly cell

lysis for at least 1 hour and slide processing were performed as previously described in detail

[172026] using alkali denaturation and electrophoresis (086 Vmiddotcm-1 at a pH gt 13) to

transform alkali-sensitive parts of the DNA into DNA strand breaks Measurements were

made by image analysis determining the mean tail moment and the mean tail intensity of 100

cells per slide (two slides each per measurement in each individual) In none of the

experiments there was a relevant difference between these two parameters Therefore in

continuation of our previous studies we chose the tail moment for the presentation of our

results

Isolated lymphocytes

Lymphocytes were separated on Ficoll gradients washed in PBS buffer resuspended in

RPMI1640 buffer and thereafter exposed to HBO in a hyperbaric chamber containing 2

CO2 (to maintain pH 70-75) and 98 O2 as described previously (37 degC compression and

decompression rate 02 barsdotmin-1 2 hours isopression at 4 bar) [20] The comet assay was

performed in aliquots before as well as immediately one and two hours and after the HBO

exposure In addition the lymphocyte total cellular O2˙- concentration was measured at room

temperature after incubation with the spin probe 1-hydroxy-3-methoxycarbonyl-2255-

tetramethylpyrrolidine (CMH) before and immediately after the HBO exposure by electron

spin resonance using a Bruker EMX spectrometer [12] Results are expressed as nanomoles

O2˙- released by 106 cells In order to exclude any effect of the lymphocyte incubation

procedure on the comet assay results and the electron spin resonance measurements per se we

also determined the tail moment and the O2˙- in time control lymphocyte aliquots without

HBO-exposure (n = 6)

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

6

Methods

The present study was part of a larger investigation entitled ldquoOxidative Stress during

Hyperoxia and Divingrdquo the protocol of which had been approved by the ethical committee of

the Christian-Albrecht-Universitaumlt Kiel All subjects gave their written informed consent to

participate in the investigation A total of 37 subjects were studied comprising combat

swimmers (n = 6) Underwater Demolition Team (UDT) divers (n = 13) and non-diving

volunteers with (ldquoNaval Pentathlon athletesrdquo n = 6) and without (controls n = 12) endurance

training The demographic data of the subjects are summarised in table 1 The UDT divers

had a diving experience of several years with a diving activity of approximately 10 hours

using O2 enriched gas mixtures and closed-circuit pure O2 breathing apparatus during the 15

months preceding the study (table 1) The combat swimmers had a median diving experience

using a closed-circuit pure O2 breathing apparatus of nearly one year with a median diving

time using this device of about 100 hours (table 1) The UDT divers and combat swimmers

had performed their last dives using O2 enriched gas mixtures or closed-circuit pure O2

breathing apparatus 4 ndash 6 and two weeks respectively prior to the investigation The Naval

Pentathlon athletes were investigated during their world championship preparation which had

started two months prior to the investigation All investigations took place during a two weeks

period Blood sampling took place in the morning after overnight fasting and was part of the

individual subjectsrsquo regular routine medical examination

Whole blood measurements

In all subjects whole blood samples were analysed for superoxide dismutase (SOD) catalase

and plasma gluthathion peroxidase (GPx) activities as well as the concentrations of reduced

(GSH) and oxidised (GSSG) glutathione respectively using commercially available test kits

as described previously [3] (SOD SOD Assay Kit-WST Dojindo Molecular Technologies

Inc Kumamoto Japan catalase Bioxytechreg Catalase-520trade OxisResearchtrade Portland OR

7

GPx Gluthathione Peroxidase Assay Kit Cayman Chemical Ann Arbor MI GSH GSSG

GSHGSSG Ratio Assay Kit Calbiochemreg EMD Chemicals Inc San Diego CA) DNA

strand breaks were quantified with the alkaline version of the comet assay [44] Briefly cell

lysis for at least 1 hour and slide processing were performed as previously described in detail

[172026] using alkali denaturation and electrophoresis (086 Vmiddotcm-1 at a pH gt 13) to

transform alkali-sensitive parts of the DNA into DNA strand breaks Measurements were

made by image analysis determining the mean tail moment and the mean tail intensity of 100

cells per slide (two slides each per measurement in each individual) In none of the

experiments there was a relevant difference between these two parameters Therefore in

continuation of our previous studies we chose the tail moment for the presentation of our

results

Isolated lymphocytes

Lymphocytes were separated on Ficoll gradients washed in PBS buffer resuspended in

RPMI1640 buffer and thereafter exposed to HBO in a hyperbaric chamber containing 2

CO2 (to maintain pH 70-75) and 98 O2 as described previously (37 degC compression and

decompression rate 02 barsdotmin-1 2 hours isopression at 4 bar) [20] The comet assay was

performed in aliquots before as well as immediately one and two hours and after the HBO

exposure In addition the lymphocyte total cellular O2˙- concentration was measured at room

temperature after incubation with the spin probe 1-hydroxy-3-methoxycarbonyl-2255-

tetramethylpyrrolidine (CMH) before and immediately after the HBO exposure by electron

spin resonance using a Bruker EMX spectrometer [12] Results are expressed as nanomoles

O2˙- released by 106 cells In order to exclude any effect of the lymphocyte incubation

procedure on the comet assay results and the electron spin resonance measurements per se we

also determined the tail moment and the O2˙- in time control lymphocyte aliquots without

HBO-exposure (n = 6)

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

7

GPx Gluthathione Peroxidase Assay Kit Cayman Chemical Ann Arbor MI GSH GSSG

GSHGSSG Ratio Assay Kit Calbiochemreg EMD Chemicals Inc San Diego CA) DNA

strand breaks were quantified with the alkaline version of the comet assay [44] Briefly cell

lysis for at least 1 hour and slide processing were performed as previously described in detail

[172026] using alkali denaturation and electrophoresis (086 Vmiddotcm-1 at a pH gt 13) to

transform alkali-sensitive parts of the DNA into DNA strand breaks Measurements were

made by image analysis determining the mean tail moment and the mean tail intensity of 100

cells per slide (two slides each per measurement in each individual) In none of the

experiments there was a relevant difference between these two parameters Therefore in

continuation of our previous studies we chose the tail moment for the presentation of our

results

Isolated lymphocytes

Lymphocytes were separated on Ficoll gradients washed in PBS buffer resuspended in

RPMI1640 buffer and thereafter exposed to HBO in a hyperbaric chamber containing 2

CO2 (to maintain pH 70-75) and 98 O2 as described previously (37 degC compression and

decompression rate 02 barsdotmin-1 2 hours isopression at 4 bar) [20] The comet assay was

performed in aliquots before as well as immediately one and two hours and after the HBO

exposure In addition the lymphocyte total cellular O2˙- concentration was measured at room

temperature after incubation with the spin probe 1-hydroxy-3-methoxycarbonyl-2255-

tetramethylpyrrolidine (CMH) before and immediately after the HBO exposure by electron

spin resonance using a Bruker EMX spectrometer [12] Results are expressed as nanomoles

O2˙- released by 106 cells In order to exclude any effect of the lymphocyte incubation

procedure on the comet assay results and the electron spin resonance measurements per se we

also determined the tail moment and the O2˙- in time control lymphocyte aliquots without

HBO-exposure (n = 6)

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

8

Statistics

All data are presented as median (quartiles) unless otherwise stated After exclusion of normal

distribution of the main criteria (tail moment) using the Kolmogorov-Smirnov test

differences between the four groups were tested using a Kruskal-Wallis one way analysis of

variance on ranks and a subsequent Dunn test The time course of the tail moment in the

isolated lymphocytes before and after HBO exposure was tested using a Friedman repeated

measures analysis of variance on ranks and a subsequent Dunn test the difference between

the lymphocyte O2˙- release before and after the HBO exposure was tested using a Wilcoxon

signed rank test

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

9

Results

Table 2 summarizes the results of the whole blood DNA damage (tail moment) antioxidant

enzyme activity and gluthathione measurements There were no statistically significant inter-

group differences in any of these parameters

Figures 1 and 2 summarize the results of the ex vivo HBO exposure of the isolated

lymphocytes The tail moment of time control lymphocyte preparations (n = 6) without HBO

exposure (010 (009011) 012 (008012) 011 (009012) 010 (008012) at 0 2 3 and

5 hours respectively which correspond to the time points before and immediately as well as

1 and 2 hours after HBO respectively p=0319) did not show any time-dependent difference

The same holds true for the lymphocyte O2˙- production (041 before vs 048 nMol per 106

cells p=0067) HBO induced a significant increase in DNA damage in all groups (p=0006

plt0001 p=001 and plt0001 in the combat swimmers UDT divers Naval Pentathlon

athletes and untrained controls respectively) but was nearly twice as high in the combat

swimmers (p=0039 versus the other three groups investigated) Nevertheless in all groups

the increased tail moment returned to normal values at one hour after the HBO exposure

again without any inter-group difference Lymphocyte O2˙- production was significantly

higher in the two diving populations (p=0003 versus both Naval Pentathlon athletes and

untrained controls) HBO comparably increased the lymphocyte O2˙- production by

approximately 4 nmol per 106 cells (30 nmol per 106 cells p=0094 48 nmol per 106 cells

plt0001 49 nmol per 106 cells p=0027 and 34 nmol per 106 cells p=0002 versus before

HBO in the combat swimmers UDT divers Naval Pentathlon athletes and untrained controls

respectively) Lymphocyte O2˙- production was significantly higher in the lymphocytes of the

two diving populations after the cell HBO exposure (p=0019 versus both Navy Pentathlon

athletes untrained controls)

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

10

Discussion

The present study tested the hypothesis that long-term repetitive exposure to HBO would

modify the degree of HBO-induced DNA damage Combat swimmers and UDT divers were

investigated because their diving practice comprises repetitive long-term exposure to HBO

over years none-diving volunteers with (ldquoNavy Pentathlon athletesrdquo) and without endurance

training served as controls We studied isolated lymphocytes ex vivo in order to allow for O2

partial pressures and exposure times beyond the threshold of central nervous O2 toxicity [6]

The key findings were that i) there was no inter-group difference in the incidence of blood

cell DNA damage nor in antioxidant enzyme activity or capacity that ii) ex vivo HBO

exposure was associated with more pronounced O2˙- formation in isolated lymphocytes from

the UDT divers and combat swimmers than in those from control subjects and Naval

Pentathlon athletes and iii) lymphocytes from combat swimmers showed the most

pronounced increase in HBO-induced DNA damage which in turn was rapidly removed

within one hour

Air breathing under hyperbaric conditions results in exposure to hyperoxia since according to

Boylersquos law the inspiratory PO2 increases in direct proportion to the rise in ambient pressure

Nevertheless whereas ample literature is available demonstrating that both hyperbaric or

prolonged normobaric pure O2 breathing cause oxidative DNA damage [10111732-3739-

42] lipid peroxidation and reduction of both enzymatic and non-enzymatic anti-oxidant

capacity [124232731] contradictory results have been reported on the effect of hyperoxia

associated with diving per se after a dry chamber dive to simulated depth of 250 meters with

maximum inspiratory O2 partial pressures of 70 kPa Djurhuus et al found a decrease in the

blood content of reduced GSH while no DNA single strand breaks could be detected [13] In

contrast both a water immersion to 40 meters breathing air (ie at an inspiratory PO2 of 100

kPa) and a dry chamber HBO exposure to 22 bar (ie at an inspiratory PO2 of 220 kPa) not

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

11

only caused increased lymphocyte H2O2 production but also resulted in enhanced intracellular

GPx activity and in particular expression of heme oxygenase-1 (HO-1) mRNA [15] The

latter finding is of particular interest both in vivo and in vitro enhanced HO-1 expression

assumes crucial importance as an adaptive protection against HBO-induced oxidative DNA

damage [1735-37]

Our finding that prior to any HBO exposure blood samples from the four different groups

studied presented with a comparable degree of DNA damage and red blood cell or plasma

antioxidant capacity confirms data described by other authors as well as our own group Niess

et al [29] demonstrated that the tail moment in whole blood samples obtained from well-

trained long distance runners before an exhaustive treadmill exercise was similar to that in

sedentary control subjects We previously reported that prior to an exposure to HBO

experienced divers [26] showed a nearly identical level of DNA damage in whole blood

samples as non-diving volunteers [1011] Complementary to these observations other

authors reported that both SOD catalase and GPx activities as well as the blood GSH content

and the GSHGSSG ratio did not differ between trained subjects and untrained controls

[3038] Furthermore divers with long-term diving activity also presented with similar values

for these parameters as non-divers [101126] Nevertheless our present observations contrast

with data reported by Knez et al [22] who found significantly higher resting catalase and GPx

activities and lower malondialdehyde concentrations in ironman triathletes than in matched

control subjects We can only speculate on this discrepancy in particular since the training

level of the Naval Pentathlon athletes in our study (maximum VO2 74 (range 73 ndash 77)

mLmiddotmin-1middotkg-1) was even slightly higher than that reported to induced inducing an adaptive

increase in antioxidant enzyme activity in other endurance athletes ie ironman triathletes by

other authors (maximum VO2 65 plusmn 6 [22] and 66 plusmn 4 mLmiddotmin-1middotkg-1 [30] respectively)

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

12

Prolonged normobaric hyperoxia was shown to cause oxidative stress which is associated

with DNA damage [78] that can be detected using the comet assay [44] Short-term exposure

to HBO leads to comparable oxidative stress and thus it is used as an appropriate model to

investigate oxidatively generated DNA damage and its repair [41] In good agreement with

this rational exposure of isolated lymphocytes to HBO induced DNA damage in our

experiment and this response was twice as high in the blood samples obtained from the

combat swimmers than in all the other groups Hence our data suggest that HBO exposure

induced more DNA damage in these subjects than in the UDT divers non-diving endurance

athletes and non-diving sedentary controls On the other hand background levels of DNA

damage were measured in these samples one hour after HBO exposure Near-complete

removal of the HBO-induced DNA damage within one hour was also observed in the samples

from the combat swimmers despite the higher levels of DNA damage immediately after the

HBO exposure It is unclear whether this finding indicates higher DNA repair capacity of

lymphocytes from combat swimmers because no measurements were performed at earlier

time points It should be noted that due to the duration of the HBO exposure itself (two hours

of isopression plus 20 minutes each of compression and decompression respectively) it is

impossible to determine any influence of DNA repair on the comet assay effects during this

period of time and to establish exact repair kinetics Our results are in agreement with

previous findings that also reported rapid repair of DNA damage in human lymphocytes after

exposure to HBO in vivo and in vitro [39]

Interestingly there was no difference in the removal of DNA damage after the end of the

HBO exposure between the Naval Pentathlon athletes and the sedentary control subjects

Since the HBO-related O2˙- radical production did not differ between these two groups these

findings suggest that at least under our experimental conditions endurance-trained athletes

and sedentary controls had a nearly identical response to HBO-induced oxidative stress This

result is in contrast to literature reports on the effect of exhaustive effort in trained and

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

13

untrained subjects depending on the type of activity both increased and decreased antioxidant

enzyme activities and increased levels of DNA damage were observed after strenuous effort

but this response was always less pronounced in well-trained athletes than in non-fit subjects

[92938]

Limitations of the study

All investigations took place during a two weeks period in order to eliminate any seasonal

influences In turn this short time frame however limited the volunteer recruitment in

particular among the combat swimmers and the Naval Pentathlon athletes Thus the sample

size of the individual groups was fairly variable In addition albeit we included only male

non-smokers in order to avoid the impact of gender andor smoking habits we cannot rule out

selection bias as a confounding factor in our study due to the fact that vulnerable andor non-

fit subjects would of course stay away from activities such as diving and or extreme

endurance

Conclusion

Combat swimmers and UDT divers ie subjects with repetitive and prolonged exposures to

HBO over years and non-diving volunteers both with (ldquoNaval Pentathlon athletesrdquo) and

without endurance training did not show any difference in the incidence of blood cell DNA

damage nor in antioxidant enzyme activity or capacity While combat swimmers showed the

most pronounced O2˙- formation and increase of DNA damage after ex vivo HBO exposure of

isolated lymphocytes complete removal of DNA damage was at least as rapid as in the other

groups

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

14

Acknowledgements

Suumlkruuml Oumlter was supported by a research grant of the Turkish National Air Force and the

Scientific and Technological Research Council of Turkey We are indebted to Bruno Fink

MD Noxygen GmbH Elzach Germany for performing the electron spin resonance

measurements

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

15

References

1 Bader N Bosy-Westphal A Koch A Mueller MJ Influence of vitamin C and E

supplementation on oxidative stress induced by hyperbaric oxygen in healthy men

Ann Nutr Metab 50 173-176 2006

2 Bader N Bosy-Westphal A Koch A Rimbach G Weimann A Poulsen HE

Muumlller MJ Effect of hyperbaric oxygen and vitamin C and E supplementation on

biomarkers of oxidative stress in healthy men Br J Nutr 98 826-833 2007

3 Barth E Bassi G Maybauer DM Simon F Groumlger M Oumlter S Speit G Nguyen

CD Hasel C Moumlller P Wachter U Vogt JA Matejovic M Radermacher P

Calzia E Effects of ventilation with 100 oxygen during early hyperdynamic

porcine fecal peritonitis Crit Care Med 36 495-503 2008

4 Benedetti S Lamorgese A Piersantelli M Pagliarani S Benvenuti F Canestrari

F Oxidative stress and antioxidant status in patients undergoing prolonged exposure

to hyperbaric oxygen Clin Biochem 37312-7 2004

5 Bernareggi M Radice S Rossoni G Oriani E Berti F Hyperbaric oxygen

increases plasma exudation in rat trachea involvement of nitric oxide Br J Pharmacol

126 794-800 1999

6 Carraway MS Piantadosi CA Oxygen toxicity Respir Care Clin N Am 5 265-295

1999

7 Cacciuttolo MA Trinh L Lumpkin JA Rao G Hyperoxia induces DNA damage in

mammalian cells Free Radic Biol Med 14267-276 1993

8 Conger AD Fairchild LM Breaking of chromosomes by oxygen Proc Nat Acad

Scie USA 38289-299 1952

9 Deacutekaacuteny M Nemeskeacuteri V Gyoumlre I Harbula I Malomsoki J Pucsok J Antioxidant

status of interval-trained athletes in various sports Int J Sports Med 27112-6 2006

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

16

10 Dennog C Hartmann A Fey G Speit G Detection of DNA damage after

hyperbaric oxygen (HBO) therapy Mutagenesis 11 605-609 1996

11 Dennog C Radermacher P Barnett YA Speit G Antioxidant status in humans

after exposure to hyperbaric oxygen Mutat Res 428 83-89 1999

12 Dikalov SI Li W Mehranpour P Wang SS Zafari AM Production of

extracellular superoxide by human lymphoblast cell lines comparison of electron spin

resonance techniques and cytochrome C reduction assay Biochem Pharmacol 73972-

80 2007

13 Djurhuus R Segadal K Svardal AM Glutathione in blood cells decreases without

DNA breaks after a simulated saturation dive to 250 msw Aviat Space Environ Med

77 597-604 2006

14 Fehrenbach E Veith R Schmid M Dickhuth HH Northoff H Niess AM Inverse

response of leukocyte heat shock proteins and DNA damage to exercise and heat Free

Radic Res 37975-82 2003

15 Ferrer MD Sureda A Batle JM Tauler P Tur JA Pons A Scuba diving enhances

endogenous antioxidant defenses in lymphocytes and neutrophils Free Rad Res 41

274-281 2007

16 Freiberger JJ Suliman HB Sheng H McAdoo J Piantadosi CA Warner DS A

comparison of hyperbaric oxygen versus hypoxic cerebral preconditioning in neonatal

rats Brain Res 1075213-22 2006

17 Groumlger M Speit G Radermacher P Muth CM Interaction of hyperbaric oxygen

nitric oxide and heme oxygenase on DNA strand breaks in vivo Mutat Res 572 167-

172 2005

18 Hartmann A Plappert U Raddatz K Gruumlnert-Fuchs M Speit G Does physical

activity induce DNA damage Mutagenesis 9 269-272 1994

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

17

19 Hartmann A Pfuhler S Dennog C Germadnik D Pilger A Speit G Exercise-

induced DNA effects in human leukocytes are not accompanied by increased

formation of 8-hydroxy-2rsquo-deoxyguanosine or induction of micronuclei Free Radic

Biol Med 24 245-251 1998

20 Hauser B Groumlger M Ehrmann U Albicini M Bruumlckner UB Schelzig H

Venkatesh B Li H Szaboacute C Speit G Radermacher P Kick J The PARP-1

inhibitor INO-1001 facilitates hemodynamic stabilization without affecting DNA

repair in porcine aortic cross-clamping- induced ischemiareperfusion Shock 25 633-

640 2006

21 Jamieson D Chance B Cadenas E Boveris A The relation of free radical

production to hyperoxia Annu Rev Physiol 48703-19 1986

22 Knez WL Jenkins DG Coombes JS Oxidative stress in half and full Ironman

triathletes Med Sci Sports Exerc 39283-8 2007

23 Kot J Siacutecko Z Woźniak M Oxidative stress during oxygen tolerance test Internat

Marit Health 54 1-4 2003

24 Lemaicirctre F Meunier N Bedu M Effect of air diving exposure generally

encountered by recreational divers Oxidative stress Undersea Hyperb Med 29 39-

49 2002

25 Moslashller P Loft S Lundby C Olsen NV Acute hypoxia and hypoxic exercise induce

DNA strand breaks and oxidative DNA damage in humans FASEB J 151181-6 2001

26 Muth CM Glenz Y Klaus M Radermacher P Speit G Leverve X Influence of

an orally effective SOD on hyperbaric oxygen-related cell damage Free Radic Res

38927-32 2004

27 Narkowicz CK Vial JH McCartney PW Hyperbaric oxygen therapy increases free

radical levels in the blood of humans Free Rad Res Comm 19 71-80 1993

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

18

28 Nie H Xiong L Lao N Chen S Xu N Zhu Z Hyperbaric oxygen preconditioning

induces tolerance against spinal cord ischemia by upregulation of antioxidant enzymes

in rabbits J Cereb Blood Flow Metab 26666-74 2006

29 Niess AM Hartmann A Gruumlnert-Fuchs M Poch B Speit G DNA damage after

exhaustive treadmill running in trained and untrained men Int J Sports Med 17397-

403 1996

30 Palazzetti S Richard MJ Favier A Margaritis I Overloaded training increases

exercise-induced oxidative stress damage Can J Appl Physiol 28 588-604 2003

31 Pelaia P Rocco M De Blasi RA Spadetta G Alampi D Araimo FSM Niccolucci

S Evaluation of lipidic peroxidation during hyperbaric oxygen therapy Protective

role of N-acetylcysteine Minerva Anest 61 133-139 1995

32 Rothfuss A Dennog C Speit G Adaptive protection against induction of oxidative

DNA damage after hyperbaric oxygen treatment Carcinogenesis 19 1913-1917 1998

33 Rothfuss A Stahl W Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro Environ Mol Mutagen 34 291-296 1999

34 Rothfuss A Merk O Radermacher P Speit G Evaluation of mutagenic effects of

hyperbaric oxygen (HBO) in vitro II Induction of oxidative DNA damage and

mutations in the mouse lymphoma assay Mutat Res471 87-94 2000

35 Rothfuss A Radermacher P Speit G Involvement of heme oxygenase-1 (HO-1) in

the adaptive protection of human lymphocytes after hyperbaric oxygen (HBO)

treatment Carcinogenesis 22 1979-1985 2001

36 Rothfuss A Speit G Investigations on the mechanism of hyperbaric oxygen (HBO)-

induced adaptive protection against oxidative stress Mutat Res 508 157-165 2002

37 Rothfuss A Speit G Overexpression of heme oxygenase-1 (HO-1) in V79 cells

results in increased resistance to hyperbaric oxygen (HBO)-induced DNA damage

Environ Mol Mutagen 40 258-265 2002

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

19

38 Schneider CD Barp J Ribeiro JL Belloacute-Klein A Oliveira AR Oxidative stress

after three different intensities of running Can J Appl Physiol 30 723-734 2005

39 Speit G Dennog C Lampl L Biological significance of DNA damage induced by

hyperbaric oxygen Mutagenesis 13 85-87 1998

40 Speit G Dennog C Eichhorn U Rothfuss A Kaina B Induction of heme

oxygenase-1 and adaptive protection against the induction of DNA damage after

hyperbaric oxygen treatment Carcinogenesis 21 1795-1799 2000

41 Speit G Dennog C Radermacher P Rothfuss A Genotoxicity of hyperbaric

oxygen Mutat Res 512 111-119 2002

42 Speit G Bonzheim I Genotoxic and protective effects of hyperbaric oxygen in A549

lung cells Mutagenesis 18 545-8 2003

43 Speit G Hartmann A The comet assay a sensitive genotoxicity test for the detection

of DNA damage and repair Methods Mol Biol 314 275-286 2006

44 Vangala RR Kritzler K Schoumlch G Topp H Induction of single-strand breaks in

lymphocyte DNA of rats exposed to hyperoxia Arch Toxicol 72 247-248 1998

45 Yogaratnam JZ Laden G Madden LA Seymour AM Guvendik L Cowen M

Greenman J Cale A Griffin S Hyperbaric oxygen a new drug in myocardial

revascularization and protection Cardiovasc Revasc Med 7146-54 2006

46 Yu SY Chiu JH Yang SD Yu HY Hsieh CC Chen PJ Lui WY Wu CW

Preconditioned hyperbaric oxygenation protects the liver against ischemia-reperfusion

injury in rats J Surg Res 12828-36 2005

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

20

Figure 1

Tail moment of the comet assay in ex vivo HBO-exposed isolated lymphocytes of the control

subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box plots)

the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots) the

combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

Figure 2

Superoxide radical (O2-) production in ex vivo HBO-exposed isolated lymphocytes of the

control subjects (n = 12 open box plots) the Naval Pentathlon athletes (n = 6 light grey box

plots) the Underwater Demolition Team (UDT) divers (n = 13 light grey hatched box plots)

and the combat swimmers (n = 6 dark grey box plots) All data are median (quartiles range)

depicts p lt 005 versus before HBO exposure sect depicts p lt 005 versus control subjects

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

21

Table 1 Demographic data of the subjects studied

Demographic data maximum O2 uptake (VO2max) in the non-diving subjects without

(controls) and with endurance training (Naval Pentathlon athletes) UDT divers and combat

swimmers All data are median (range) depicts p lt 005 versus the other groups Note that

there was no intergroup difference for age gender height and weight except for the markedly

higher anaerobic threshold (VO2max) in the Naval Pentathlon athletes

Number

of subjects

Age

[years]

Weight

[kg]

Height

[cm]

VO2max

[mLmiddotmin-1middotkg-1]

Diving experience with

pure O2 or O2-enriched

gas mixtures

Controls 12 25

(1847)

78

(58109)

181

(165191)

not determined oslash

Naval Pentathlon

athletes

6 29

(2237)

76 (7082) 178

(173192)

74

(7377)

oslash

UDT divers 13 29

(2248)

90

(66105)

185

(163194)

45

(2859)

125 hours O2-enriched

gas mixtures 75 hours

pure O2 over 8 years

Combat

swimmers

6 25

(2428)

80 (6492) 178

(175190)

48

(4260)

Pure O2 diving 101

hours over 11 months

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

22

Table 2 Blood DNA damage and antioxidant enzyme activities

Tail moment in the comet assay of whole blood cells blood antioxidant enzyme activities as

well as in the four groups studied UDT Underwater Demolition Team SOD superoxide

dismutase GPx glutathione peroxidase GSH reduced GSSG oxidised glutathione All data

are median (range)

Controls

(n =12)

UDT divers

(n = 13)

Combat swimmers

(n = 6)

Naval Pentathlon

athletes (n= 6)

Tail

moment

018

(014024)

017

(011023)

017

(014021)

018

(011019)

SOD

[kUmiddotgHb-1]

61

(4476)

98

(42170)

56

(48169)

55

(4786)

Catalase

[kUmiddotgHb-1]

745

(5601038)

682

(569827)

761

(554787)

727

(658802)

SOD

[kUmiddotgprotein-1]

112

(62179)

109

(58392)

88

(75114)

81

(64111)

Catalase

[kUmiddotgprotein-1]

238

(97368)

125

(69268)

164

(50365)

138

(61687)

GPx

[Umiddotgprotein-1]

24

(2028)

25

(2034)

23

(2128)

28

(2235)

GSH

[micromolmiddotL-1]

1495

(9301808)

1377

(11591655)

1345

(7731438)

1174

(7781469)

GSSG

[micromolmiddotL-1]

80

(31276)

93

(65312)

84

(83141)

66

(46389)

GSHGSSG

ratio

173

(52478)

155

(37231)

147

(54162)

151

(12286)

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10

23

pre HBO post HBO 1 hour 2 hours post HBO post HBO

tail

mom

ent

00

02

04

06ControlsNaval pentathlon athletesUDT-diversCombat swimmers

sect

Figure 1TailMoment

06

04

02

06

pre HBO post HBO

Supe

roxi

de [micro

Mol

10^

6 Ly

mph

ocyt

es]

0000

0001

0002

0003

0004ControlsNaval pentathlon athletesUDT-diversCombat swimmers

O2-

[nMol106 lymphocytes]Figure 2

sect sect

sect sect p=0094 vs

bevor HBO

40

30

20

10