Toxicology in Vitro 26 (2012) 1209–1215

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Toxicology in Vitro

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DNA integrity in patients undergoing hyperbaric oxygen (HBO) therapy

Aylin Üstündag a,⇑, Kemal S�ims�ek b, Hakan Ay c, Kadir Dündar d, Sinan Süzen a, Ahmet Aydın e,Yalçın Duydu a

a Ankara University, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, Tandogan, Ankara 06100, Turkeyb Gülhane Military Medical Academy, Underwater and Hyperbaric Medicine, Ankara, Turkeyc Gülhane Military Medical Academy, Haydarpas�a Hospital, Underwater and Hyperbaric Medicine Service, Kadıköy, _Istanbul 81010, Turkeyd Gölcük Military Hospital, _Izmit, Turkeye Yeditepe University, Faculty of Pharmacy, Department of Toxicology, 26 Agustos yerles�imi, Kayıs�dagı, Atas�ehir, _Istanbul 34755, Turkey

a r t i c l e i n f o

Article history:Received 3 January 2012Accepted 19 June 2012Available online 26 June 2012

Keywords:Hyperbaric oxygen therapyDNA damageComet assayAdaptive responseLymphocyte preservationFrozen lymphocytes

0887-2333/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.tiv.2012.06.007

⇑ Corresponding author. Tel.: +90 3122033122; faxE-mail address: dur@pharmacy.ankara.edu.tr (A. Ü

a b s t r a c t

Hyperbaric oxygen (HBO) therapy is successfully applied for a wide variety of diseases. However, recentstudies in humans undergoing (HBO) therapy have revealed that HBO is able to induce oxidative DNAdamage especially in lymphocytes while the biological significance of this outcome is still not clear.HBO mediated DNA damage in lymphocytes has been determined by using the alkaline version of thecomet assay in order to detect DNA strand breakages in patients undergoing HBO therapy. Blood sampleswere obtained from 100 voluntary patients and were drawn by venipuncture before and immediatelyafter the first session of HBO treatment. The DNA damaging effect of HBO has also been evaluated inthe fifth session of HBO therapy. DNA strand breakages were significantly increased after the first sessionof HBO treatment. However the elevated DNA strand breaks returned to their normal levels in lympho-cytes after two hours of in vitro incubation. The elevated DNA strand breaks consistently decreased andreached to the baseline levels after the fifth session of HBO therapy. The results of this study, conductedin patients undergoing HBO therapy, support the existence of the previously reported cellular adaptiveresponse against HBO mediated oxidative DNA damage in experimental studies.

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1. Introduction

Hyperbaric oxygen (HBO) therapy is increasingly used in anumber of areas of medical practice with symptoms caused by lackof oxygen in the target tissues. HBO implies the inhalation of 100%oxygen under a pressure greater than sea level (1 atmosphereabsolute, ATA) in a hyperbaric chamber for a total of 3 � 20 minperiods, interspersed with 5 min of air breathing (Gill and Bell,2004).

HBO therapy has been used for the treatment of a variety ofconditions. The approved indications for HBO therapy are air–gasembolism, decompression sickness, acute carbon monoxide intox-ication, soft tissue infections, radiation necrosis, chronic osteomy-elitis, skin graft and flaps, several acute ischemic conditions,gaseous gangrene, sudden hearing loss and impaired wound heal-ing (such as diabetic wounds) (Fujimura et al., 2007; UHMS, 2011).However, it is known that exposure to oxygen at high ambientpressure can cause damage to mammalian cells. Exposure to HBOleads to an increase in the amount of dissolved oxygen and there-fore reactive oxygen species (ROS) in the blood (Dennog et al.,1999; Speit et al., 2002). An increase in free radicals in the blood

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from persons undergoing HBO exposure was directly demon-strated by electron spin resonance spectroscopy (Yamaguchiet al., 1992; Narkowicz et al., 1993). When antioxidant defensesare not completely efficient, increased free radical formation inthe body is likely to increase damage. The term ‘‘oxidative stress’’is generally used to refer to this effect. Oter et al. (2005) revealedthat, after 2 h of HBO exposure at 3 ATA, the levels of the oxidativestress markers, thiobarbituric acid reactive substances (TBARS) andtotal superoxide dismutases (SOD) were elevated in the lung, brainand erythrocytes and glutathione peroxidase (GPx) and nitrate/ni-trite (NOx) activities were found to be elevated in the brain, in rats.

HBO-mediated DNA damage was shown with the alkaline ver-sion of the comet assay. The comet assay is a well established geno-toxicity test that detects DNA damage on the single cell level withhigh sensitivity. In alkaline version (pH > 13) DNA strand breaks,alkaline labile sites and incisions during DNA repair become appar-ent, and the amount of DNA migration indicates the amount of DNAdamage in the cell (Speit et al., 2002). The comet assay has beenmodified to enable the detection of specific classes of DNA damage.The formamido pyrimidine DNA-glycosylase (Fpg) protein is rec-ommended for the detection of oxidatively generated damage, inparticular, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG) (Speitet al., 2000). Fpg, is a DNA repair enzyme, which removes oxidizedpurines, and this results in abasic sites being converted into DNA

1210 A. Üstündag et al. / Toxicology in Vitro 26 (2012) 1209–1215

single strand breaks. This, additionally induced DNA damage leadsto additional DNA migration in the comet assay, and therefore pro-vides an indirect measure for the presence of oxidatively generatedbase damage (Speit et al., 2002). By the use of Fpg protein, the co-met assay becomes more specific as well as more sensitive.

By using the comet assay, the DNA damage after HBO was foundonly after the first HBO exposure but a subsequent exposure did notcause oxidatively generated damage any more in the treatments ofthe same individuals, indicating an increase in antioxidant defences(Speit et al., 2002; Gröger et al., 2009). It has been shown that treat-ment of individuals with a single HBO induces adaptive responsesin lymphocytes against the genotoxic effects of an oxidative agentsuch as hydrogen peroxide (H2O2). Adaptation can be caused by in-creased levels of DNA repair functions or enhancement of generaldefense against ROS. Various studies showed that there was no in-crease in the expression of the DNA repair enzymes APE and Pol b(Speit et al., 2000). Also there was no difference in the individualactivities of superoxide dismutase (SOD), catalase (CAT) and gluta-thione peroxidases (GPx) after HBO exposure (Dennog et al., 1999;Speit et al., 2000). However, the amount of heme oxygenase-1 (HO-1) in lymphocytes was clearly enhanced in HBO exposed individu-als (Speit et al., 2000; Rothfuss et al., 2001; Speit and Bonzheim,2003). HO-1 catalyzes the rate-limiting step in the degradation ofheme, producing bilirubin, ferrous iron and carbon monoxide(Rothfuss and Bonzheim, uy; Rothfuss et al., 2000; Rothfuss andSpeit, 2002). Enhanced cellular protection is commonly explainedby an increased production of bilirubin, a well-known antioxidant,and a subsequent increased sequestration of redox-active iron dueto increased ferritin levels (Rothfuss et al., 2001).

The aim of this study is to evaluate the DNA repair and cellularadaptive response in lymphocytes of patients (n = 100) undergoingHBO therapy by using the alkaline version of the comet assay. Therepair capacity of DNA was evaluated by in vitro post incubation oflymphocytes with or without H2O2 treatment. The cellular adap-tive response was investigated by comparing the levels of DNAdamage in patients undergoing the first and the fifth sessions ofHBO therapy. Additionally, this study will provide useful informa-tion on the DNA damaging effect of the freezing procedure that weused in lymphocyte preservation. From this point of few this is thefirst and the largest study that shows the increase of the oxidativeDNA damage and the adaptive response in lymphocytes from pa-tients undergoing HBO therapy concerning its.

2. Materials and methods

2.1. Test subjects and HBO treatment

This study was approved by the Ethical Committee of the Gülh-ane Military Medical Academy (83, 26.12.2006). One hundred vol-unteer patients provided informed consent to participate in thisstudy. The subjects were 12–79 years of age and the general charac-teristics of the study group are shown in Table 1. The patients wereexposed to 10 consecutive HBO treatments (1 session/day), accord-ing to a routine therapy protocol. The treatment consisted of expo-sure to 100% oxygen at a pressure of 2,5 ATA in a hyperbaric chamber(for 12 patients) for 3 � 20 min periods, interspersed with 5 minperiods of breathing air. Heparinized venous blood samples were ta-ken before the first session of HBO treatment and immediately uponexit from the chamber after the first session. Therefore, the patientsacted as their own controls. The blood samples were kept on ice andprocessed within 1 h. Oxidative damage was introduced by 50 lMH2O2 in lymphocytes taken from the patient’s just before and imme-diately after the first and fifth sessions of the HBO therapy. In fifthsession, it was possible to take blood samples from only 5 patientsdue to the difficulties of following the patients during the therapy.

2.2. Blood samples

From each of the 100 patients, 5 mL of heparinized blood sam-ples was collected. Lymphocytes were isolated by using the leuco-sep tubes (12 mL, Greiner bio-one). The cells suspended in a totalvolume of 1 mL, were counted with a hemocytometer (Neubauerimproved, Marienfeld) by trypan blue staining, and cell concentra-tions adjusted to 104cells/slide for each 50 lL of suspension in co-met assay. The comet assay was performed in lymphocytes fromthe 100 patients before and after undergoing the first therapy ofHBO. To observe the DNA repair, the lymphocytes were incubatedat 37 �C for 2 h. After 15, 30, 60, and 120 min the comet assay wasperformed in lymphocytes taken from the patients after the firstHBO treatment. Furthermore, comet assay was performed on allfrozen lymphocytes at a later time for the total of 100 patients be-fore and after HBO therapy. Figs. 2 and 3 shows the whole proce-dure that was used in this study. Lymphocytes were frozenslowly to �80 �C in a storage solution (40% RPMI 1640, 10% DMSO,50% heat inactivated fetal calf serum). For use they were thawedquickly, diluted with PBS, and immediately centrifuged to removethem from the DMSO (Collins, 2004).

2.3. Comet assay

The basic alkaline technique of the comet assay was followed(Singh et al., 1988; Anderson et al., 1994; Collins et al., 1997) withsome modifications (Aydin et al., 2005). The microscope slides(with frosted ends) had been covered with normal melting pointagarose (1% NMA, in distilled water) before the experiment. Forthe second layer, 50 lL of lymphocyte suspension mixed with100 lL of low melting point agarose (0.5% LMA, in PBS, Sigma)were rapidly pipetted onto the slides, spread using a cover slip,and maintained on an ice-cold flat tray for 5 min for final aggluti-nation. After removal of the cover slip, the slides were immersed incold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, 1%sodium sarcosinate, pH 10) with 1% Triton-X 100 and 10% DMSOadded just before the reaction for at least 1 h at 4 �C. H2O2 wasused as positive control at concentrations of 50 and 100 lM (theresults were presented as the average of three replicate assays inFig. 4). The cells treated with H2O2 and embedded on slides wereimmersed in other cold lysing solution. The presence of oxidativebase damage was determined with a modified protocol using Fpgprotein (Speit et al., 2004). Slides were washed after lysis 3 times(for 5 min each) in enzyme buffer (40 mM Hepes, 0.1 M KCl,0.5 mM EDTA, 0.2 mg/mL BSA; pH 8.0) and covered with 100 lLof either buffer, sealed with a coverslip and incubated for 30 minat 37 �C. The slides were removed from the lysing solution,drained, and placed side by side avoiding space and with the aga-rose ends facing each other nearest the anode in a horizontal gelelectrophoresis tank. The tank was filled with fresh electrophoresissolution (1 mM Na2EDTA and 300 mM NaOH, pH 13) to a levelapproximately 0.5 cm above the slides. The time of alkali denatur-ation and electrophoresis (24 V, 300 mA) was 20 min each, in 4 �C.After electrophoresis, the slides were taken out of the tank. Trisbuffer (0.4 M Tris, pH 7.5) was added dropwise and gently to neu-tralize the excess alkali, and the slides were allowed to stand for5 min (This step was repeated 3 times). For staining, 65 lL of EtBr(20 lg/mL) was added to each slide. Slides were covered with acover slip, placed in a humidified airtight container to prevent dry-ing of the gel, and analyzed within 3–4 h. Slides were examined ona fluorescence microscope (Leica DM 1000). Images of 100 ran-domly selected lymphocytes were analyzed from each sample,and the DNA damage was scored visually as shown in Fig. 1. A totaldamage scored for the slide was derived by multiplying the num-ber of cells assigned to each grade of damage by the numeric valueof the grade and summing over all grades. Arbitrary Units (AU) was

Table 1The level of DNA migration (expressed as arbitrary units) in lymphocytes of patients undergoing HBO therapy (HBOT).

Classification of patientsinto categories

Comet assay results (Arbitrary units)

Before HBOT CV Immediately after the firstsession of HBOT (0 min)

CV After in vitro incubation (120th min) CV

Healthy donors**

Female (n = 5) 10.6 ± 1.35 (9–13) 12.74 – – – –Male (n = 5) 9.6 ± 1.2 (8–11)a 12.50 – – – –GenderFemale (n = 23) 10.11 ± 1.95 (8–14) 19.29 34.82 ± 2.83 (30–42)* 11.00 10.60 ± 1.83 (7–13) 17.26Male (n = 77) 10.08 ± 1.74 (7–15) 17.26 34.61 ± 2.07 (28–39)* 5.98 11.09 ± 2.23 (6–15)* 20.11Age 41 ± 17 (12–79)<30 (n = 28) 10.5 ± 1.59 (7–14) 15.14 35.1 ± 2.17 (30–40)* 6.18 11.2 ± 2.26 (6–15) 20.1830–45 (n = 34) 10.6 ± 1.84 (8–15) 17.36 34.4 ± 2.41 (28–42)* 7.01 11.5 ± 2.03 (7–15) 17.6546–60 (n = 25) 9.1 ± 1.77 (7–13) 19.45 34.8 ± 2.36 (30–39)* 6.78 9.9 ± 2.18 (6–15) 22.02>60 (n = 13) 9.6 ± 2.12 (7–15) 22.08 34 ± 1.61 (32–38)* 4.74 10.6 ± 1.63 (8–14) 15.38Smoking habitsNon smoker (n = 72) 10.1 ± 1.93 (7–15) 19.11 34.4 ± 2.34 (28–42)* 6.80 10.9 ± 2.23 (6–15) 20.46Smoker (n = 28) 9.8 ± 1.82 (7–13) 18.57 35.1 ± 1.96 (30–40)* 5.58 10.9 ± 2.03 (7–15) 18.62Alcohol consumptionNo (n = 88) 10.21 ± 1.95 (8–11) 19.10 34.68 ± 2.28 (28–42)* 6.57 11.12 ± 2.14 (7–15) 19.24Yes (n = 12) 9.33 ± 1.1 (7–15) 11.79 34.75 ± 2.16 (30–38)* 6.22 9.83 ± 1.77 (7–13) 18.01Coffee consumptionNo (n = 69) 10 ± 1.97 (7–15) 19.07 34.3 ± 1.95 (28–38)* 5.69 10.8 ± 2.34 (6–15) 21.67Yes (n = 31) 10.2 ± 1.74 (7–14) 17.06 35.3 ± 2.71 (30–42)* 7.68 11 ± 1.74 (8–15) 15.82Previous X-ray exposureYes (n = 34) 9.5 ± 1.83 (7–14) 19.26 34.7 ± 2.38 (30–40)* 6.86 10.5 ± 2.01 (6–15) 19.14No (n = 66) 10.3 ± 1.88 (7–15) 18.25 34.6 ± 2.19 (28–42)* 6.33 11.1 ± 2.22 (7–15) 20.00DiseasesDiabetic foot (n = 23) 10.1 ± 2.03 (7–15) 20.10 34.27 ± 2.74 (30–42)* 8.00 10.81 ± 1.89 (7–15) 17.48CO intoxication (n = 2) 9 ± 1 (8–10) 11.11 34.5 ± 0.70 (34–35)* 2.03 10 ± 3 (7–13) 30.00Sudden audition loss (n = 37) 9,7 ± 1.83 (7–15) 18.87 34.43 ± 1.74 (30–38)* 5.05 10.54 ± 2.03 (7–15) 19.26Other diseases (n = 38) 10.35 ± 1.87 (7–14) 18.07 35.10 ± 2.34 (28–40)* 6.67 11.4 ± 2.28 (6–15) 20

a Mean ± SD (Range).* Significantly different (p < 0.05) from the background level (before HBOT).

** Healthy individuals that were not under therapy with HBO.

A. Üstündag et al. / Toxicology in Vitro 26 (2012) 1209–1215 1211

calculated with the given formula leading into the following clas-sifications: [0 � Type 0 (no damage)] + [1 � Type 1 (low dam-age)] + [2 � Type 2 (medium damage)] + [3 � Type 3 (highdamage)] + [4 � Type 4 (very high damage)] (Fig. 1). The slideswere scored by one reader in order to minimize the variability.

2.4. Individual variability

Healthy, non smoker, male [n = 5, age: 33 ± 5.49 (28–41)] andfemale [n = 5, age: 41.2 ± 6.61 (33–48)] donors were used to deter-mine the individual variability of background DNA damage of lym-phocytes in alkaline Comet assay. Blood samples from these fivevolunteers were drawn in order to isolate peripheral blood lym-phocytes. DNA damage in lymphocytes was determined by usingalkaline Comet assay as was described under Section 2.3. The var-iation of background DNA damage in lymphocytes of healthy male/female volunteers (healthy donors) was expressed by means ofcoefficient of variation (CV) in Table 1. Individual variability ofbackground DNA damage of peripheral blood lymphocytes wasalso evaluated in voluntarily participated patients (n = 100) havingdifferent characteristics and diseases as shown in Table 1.

Fig. 1. Appearance of undamaged and damaged cells under fluorescent microscope (TypType 4 very high damage).

2.5. Statistical procedures

SPSS for Windows Release 11 was used for all data analysis. Allresults were expressed as mean ± standard deviations. Statisticalcomparisons of the results from the comet assay, AU of controland exposed groups, were carried out by one-way analysis of var-iance (ANOVA) test. Post hoc analysis of group differences was per-formed by the Fisher’s least significant difference (LSD) test. Thelimit for statistical significance was fixed as p < 0.05. The interindi-vidual variation was expressed by the means of coefficient of var-iation (CV) and calculated by the following formula; CV = [Standarddeviation (SD)/Mean] � 100.

3. Results

The entire procedures followed in this study are show in Figs. 1and 2. Visual scoring method was performed to measure the extentof DNA damage of lymphocytes in comet assay and the resultswere expressed as arbitrary units. The appearance of undamagedand/or damaged lymphocytes under fluorescence microscope hasbeen shown in Fig. 3. H2O2 was used as the positive control at 50

e 0 undamaged, Type 1 low damage, Type 2 medium damage, Type 3 high damage,

Fig. 2. The entire procedure performed on the blood samples taken before and after the first session of the HBO therapy.

Fig. 3. The entire procedure performed on blood samples taken within the first and fifth sessions of the HBO therapy.

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and 100 lM concentrations in order to induce DNA damage inperipheral blood lymphocytes. The increase in DNA damage wasdetermined by means of both alkaline comet assay and Fpg-mod-ified alkaline comet assay as shown in Fig. 4. Statistically signifi-cant increase (p < 0.05) in DNA damage was determined in both50 and 100 lM H2O2 in in vitro treatment of lymphocytes. TheDNA damage in lymphocytes was significantly lower in comet as-say than the Fpg-modified comet assay (Fig. 4). This result reflectsthe higher sensitivity of Fpg-modified comet assay.

Fig. 5 summarizes the comet assay results in lymphocytes takenfrom 100 patients, before (BT) and immediately after (AT) the firstsession of HBO therapy. The isolated lymphocytes were incubatedin vitro for 2 h immediately after the first session of HBO therapy.The mean DNA damage levels of 100 patients were determined atthe 15th, 30th, 60th and 120th min of in vitro post-incubation.The DNA damage in lymphocytes was sharply increased to its high-est level immediately after the first HBO therapy in each patient.The elevated mean DNA damage, however, decreased toward the

Chart A Chart B

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Fig. 4. DNA damage in lymphocytes treated with H2O2 (Chart A) and H2O2 + Fpg (Chart B) as positive control. The data are representing the average of three replicate assays(p < 0.05, one-way ANOVA).

Fig. 5. DNA damage in lymphocytes before and after the HBO therapy. The decreasein DNA damage with increasing time reflects the DNA repair in in vitro conditions.BT1/AT1: Before and after the first session of HBO therapy. � Outliers, ⁄significantlyhigher from the background (BT1) level (p < 0.05, one-way ANOVA).

A. Üstündag et al. / Toxicology in Vitro 26 (2012) 1209–1215 1213

background level within the in vitro incubation period as shown inFig. 5. In spite of the consistent decrease in DNA damage within theincubation period, the mean DNA damage in lymphocytes was stillsignificantly higher (p < 0.05) than the mean background DNA dam-age level at the end (120th min) of the in vitro incubation (Fig. 5).

The DNA damage levels were also compared between freshlyisolated and frozen (�80 �C) lymphocytes. The DNA damage levelswere determined before and after the first session of HBO therapyin fresh and frozen lymphocytes of patients (n = 100). The meanDNA damage determined by the comet assay was statistically notdifferent (p > 0.05) between freshly isolated and frozen lympho-cytes as shown in Fig. 6.

The mean DNA damage levels of patients (n = 100) are compiledin Table 1. The patients were categorized according to their gender,age, smoking habits, coffee consumption, alcohol consumption anddiseases in Table 1. The mean levels of DNA damage in lymphocytesdetermined before and immediately after the first session of HBOtherapy were compared within these categories. Statistically signif-icant (p < 0.05) increases in the mean levels of DNA damage wereobserved immediately after the first session of HBO therapy in eachcategories. After 2 h in vitro incubation the HBO induced DNA dam-age was completely removed in all except one category. The onlyexception was the male (n = 77) patients. When patients were clas-sified according to their gender, the mean level of DNA damage inlymphocytes of male patients was still significantly higher(p > 0.05) than the background level after 2 h in vitro incubation(Table 1).

The mean DNA damage levels of lymphocytes were also com-pared between the first and the fifth session of the HBO therapy.However, it was possible to take blood samples from only 5 patients(1 diabetic foot, 4 sudden audition losses) at the fifth session of HBOtherapy. Therefore the results shown in Fig. 7 are not representingthe whole patients. According to the comet assay results the meanDNA damage increased significantly (p < 0.05) within the first HBOtherapy in these 5 patients as was expected. The same significant in-crease in the mean DNA damage, however, was not observed imme-diately after the fifth session of HBO therapy. Additionally, H2O2

induced mean DNA damage in lymphocytes obtained from the pa-

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Fig. 7. H2O2 induced mean DNA damage levels (n = 5 patients). H2O2 induced DNAdamage in fifth session was significantly (p < 0.05) lower than the first session ofthe HBO therapy. H2O2 induced DNA damage level decreased to its backgroundlevel after 2 h in vitro incubation in lymphocytes obtained from the patients afterthe fifth session of the HBO therapy. White bars: first session of HBO therapy. Blackbars: fifth session of HBO therapy. ⁄Significantly higher from the background (BT1or BT5) level (p < 0.05, one-way ANOVA).

Fig. 6. The mean DNA damage levels of lymphocytes in different applications. Themean background DNA damage levels were statistically not significant (p > 0.05) ineach pair wise (BT1/BT1–80 �C/BT1 + Fpg) comparisons. BT1/AT1: Before and afterthe first session of HBO therapy. � Outliers, ⁄significantly higher from thebackground level (p < 0.05, one-way ANOVA).

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tients after the fifth HBO therapy was significantly (p < 0.05) lowerthan the mean DNA damage in lymphocytes obtained from the pa-tients after the first session of HBO therapy. H2O2 induced DNAdamage in lymphocytes reduced significantly after 2 h in vitro incu-bation. However, the mean level of DNA damage was still signifi-cantly higher (p < 0.05) than the mean background DNA damagelevel of lymphocytes obtained from the patients after the first ses-sion of the HBO therapy. On the other hand, the mean level ofH2O2 induced DNA damage reduced to the mean background levelin lymphocytes obtained from the patients after the fifth sessionof the HBO therapy (Fig. 7).

4. Discussion

The present study provides additional information on the DNA-damaging effect of HBO treatment and freezing process (�80 �C) inlymphocytes. HBO mediated DNA damage was analyzed in 100 pa-tients by comparing the mean level of DNA damage of patientswithin the first and fifth sessions of therapy.

HBO treatment is applied as a therapy for a wide variety of dis-eases with symptoms caused by lack of oxygen in the target tis-sues. But exposure to HBO leads to an increase in the amount ofdissolved oxygen and also reactive oxygen species in the blood(Dennog et al., 1996; Speit et al., 1998). It is thought that the tox-icity of HBO can be mediated by the production of oxygen free rad-icals, which lead to lipid peroxidation and tissue damage (Pabloset al., 1997). Eken et al. (2005) investigated the effects of HBO onoxidative stress and genetic toxicity. This study revealed thatHBO treatment did not cause significant changes in erythrocyteantioxidant capacity and lipid peroxidation, however it could in-duce genotoxicity.

In the present study, the DNA damaging-effect of HBO in lym-phocytes was investigated by means of the alkaline version ofthe comet assay. Our results revealed that HBO treatment increasesignificantly the DNA damage in peripheral blood lymphocytesafter the first session of therapy. Alkaline version of the comet as-say detects majorly the strand breaks and alkali-labile sites in DNA.As well known, however, comet assay in combination with Fpgprotein provide additional information on some specific classesof DNA damage. The Fpg protein of Escherichia coli is a DNA repairenzyme with DNA glycosylase, abasic site nicking, and deoxyriboseexcising activities (Timothy et al., 1993). This repair enzyme iswidely used for the detection of oxidatively generated base dam-age, in particular 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxo-dG) and other oxidatively damaged guanines (Collins et al., 1993;Speit et al., 2004). Although it is known that Fpg protein also de-tects AP sites and various kinds of ring-opened N-7 guanine ad-ducts, 8-oxo-dG has been proposed to be the most relevantsubstrate (Speit et al., 2002; Tchou et al., 1994).

In this study, we detected significantly induced oxidative basedamage immediately after the first session of HBO therapy byusing the alkaline comet assay in combination with Fpg protein(Fig. 6). This high level of DNA damage probably stems from thesignificant increase of 8-oxo-dG adducts. As well known, 8-oxo-dG is one of the mutagenic base modifications produced in DNAby the reaction of reactive oxygen species (ROS) (Haghdoostet al., 2005). According to our results, however, the increased levelof DNA damage in lymphocytes reduced nearly to the backgroundlevel after 2 h in vitro incubation as shown in Fig. 5. These resultsindicate a rapid DNA repair in human lymphocytes after HBO treat-ment. However, the comet assay only measure the kinetics ofstrand break rejoining (speed) but not the accuracy of DNA repair(Speit et al., 2002). Therefore rapid repair does not mean that theDNA is correctly repaired.

The influence of HBO exposure on the mean DNA damage levelsof patients (n = 100) was also evaluated within the generated cate-gories as shown in Table 1. The mean DNA damage levels were sig-nificantly increased in the first session of HBO therapy in aconsistent manner and decreased to their background levels after2 h of in vitro incubation in all except one category. In spite of asharp decrease in the mean DNA damage level of lymphocytes frommale patients after 2 h of in vitro incubation, it was still significantlyhigher (p < 0.05) than the pre-existing (background) mean DNAdamage level. This result indicates that the DNA repair in lympho-cytes from male patients is slower than the lymphocytes from fe-male patients.

The statistically significant increase (p < 0.05) in mean level ofDNA damage in 50 lM H2O2 treated lymphocytes obtained from

A. Üstündag et al. / Toxicology in Vitro 26 (2012) 1209–1215 1215

the patients (n = 5) after the first session of HBO therapy reducedsharply after 2 h in vitro incubation. However the mean level ofDNA damage was still significantly higher than the mean back-ground level of DNA damage in lymphocytes (Fig. 7). On the otherhand, HBO mediated increase in DNA damage was not observed afterthe fifth session of HBO therapy. Although the increase in the meanlevel of DNA damage in 50 lM H2O2 treated lymphocytes obtainedfrom the patients after the fifth session of HBO therapy was still sta-tistically significant (p < 0.05), the mean level of H2O2 induced DNAdamage was remarkably (p < 0.05) lower than the first session ofHBO therapy. Additionally, the H2O2 induced DNA damage in lym-phocytes was completely repaired within 2 h in vitro incubation.These results reflect an adaptive response at the cellular level.

Previously published studies showed that HBO treatmentcaused clear and reproducible DNA damage in lymphocytes as de-tected with the comet assay. Induction of DNA damage was foundonly after the first HBO exposure and not after further treatmentsof the same individuals (Dennog et al., 1996; Gröger et al., 2009).Similar results were also obtained from studies in V79 Chinesehamster cells (Rothfuss and Speit, 2002). Our study results are inagreement with these previously published studies (Rothfusset al., 1998, 1999, 2001; Speit et al., 2000) and support the occur-rence of the adaptive response after a single standard HBO therapy.

The present study provides some useful information also on thepossible DNA-damaging effect of lymphocyte preservation byfreezing (�80 �C). Long lasting preservation of blood samples col-lected in epidemiological studies is always of great importance.Freezing procedure is a widely used method for preserving a bloodsample for a further analysis. Our results suggest that freezing pro-cess (�80 �C) has no unfavorable effect on the pre-existing DNAdamage in peripheral blood lymphocytes obtained from the pa-tients undergoing HBO therapy. The mean levels of DNA damagebetween freshly isolated and frozen lymphocytes were statistically(p > 0.05) not different as shown in Fig. 6.

Consequently, the studies conducted in mammalian cells haverevealed that the HBO induced DNA damage can lead gross geneticalterations and chromosome aberrations (Guskov et al., 1990; Roth-fuss et al., 2000; Speit et al., 2002). On the other hand, mutationshave not been reported in humans undergoing HBO therapy sofar. However, it is important to note that the available human stud-ies were conducted in healthy humans. Therefore the mutagenicpotential of HBO exposure should be studied further in patientsundergoing HBO therapy concerning its approved indications.

Conflict of Interest

In this study, there is no conflict of interest.

Acknowledgements

This study was supported by Ankara University Research Fund(Project no: 20050803052). We gratefully thank Gülhane MilitaryMedical Academy, Underwater and Hyperbaric Medicine for theirsupport during the collection of blood samples.

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