Oxidative DNA damage and oxidative stress in subjects occupationally exposed to nitrous oxide (N2O)

Mutation Research 731 (2012) 58–63

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Oxidative DNA damage and oxidative stress in subjects occupationally exposed to nitrous oxide (N2 O) a,∗ ´ ˙ Teresa Wronska-Nofer , Jerzy-Roch Nofer b,c , Jolanta Jajte d , Elzbieta Dziubałtowska a , e f a a ˛ ´ Wiesław Szymczak , Wojciech Krajewski , Wojciech Wasowicz , Konrad Rydzynski a

Department of Toxicology and Carcinogeneis, Nofer Institute of Occupational Medicine, Lodz, Poland Center for Laboratory Medicine, University Hospital Münster, Münster, Germany c Department of Medicine, Endocrinology, Metabolism and Geriatrics, University of Modena and Reggio Emilia, Modena, Italy d Department of Toxicology, Medical University of Lodz, Lodz, Poland e Institute of Psychology, University of Lodz, Lodz, Poland f Department of Anaesthesiology, Polish Mother Memorial Hospital, Lodz, Poland b

a r t i c l e

i n f o

Article history: Received 18 June 2011 Received in revised form 27 September 2011 Accepted 25 October 2011 Available online 4 November 2011 Keywords: Nitrous oxide Genotoxicity DNA damage Oxidative stress Comet assay

a b s t r a c t Objectives: Occupational exposure to nitrous oxide (N2 O) and/or halogenated hydrocarbons has been suggested to induce damage of genetic material, but the underlying mechanisms remain obscure. This study investigated the role of oxidative processes in the genotoxicity associated with exposure to waste anaesthetic gases. Methods: The study was performed in 36 female nurses and in 36 unexposed female health care workers matched for age and employment duration. Genotoxic effects were examined by Comet test modification employing formamidopyrimidine glycosylase (FPG) that allows assessment of oxidative DNA damage. Reactive oxygen species (ROS) in leukocytes were investigated by fluorescence spectroscopy with 2 ,7 dichlorofluorescin diacetate. Oxidative stress markers including 8-iso-prostaglandin F2␣ (8-iso-PGF2␣ ), thiobarbituric acid-reacive substances (TBARS), ␣-tocopherol, and glutathione peroxidise (GPX) activity were measured immuno- or colorimetrically. N2 O, sevoflurane and isoflurane were monitored by gas chromatography and mass spectrometry. Results: The study documents for the first time the positive correlation between the oxidative DNA damage and the N2 O levels in the ambient air. By contrast, no association was observed between genotoxic effects and sevoflurane or isoflurane. In addition, ROS generation and plasma and urine concentrations of TBARS and 8-iso-PGF2␣ , respectively, were elevated, while GPX activity was reduced in nurses exposed to waste anaesthetic gases. Path analysis pointed to a causal relationship between N2 O exposure, oxidative stress and DNA damage. Conclusion: Occupational exposure to N2 O is associated with increased oxidative DNA damage and the level of exposure plays a critical role in this regard. Increased oxidative stress may represent a mechanistic link between chronic N2 O exposure and genotoxicity. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Occupational exposure to nitrous oxide (N2 O) exerts adverse effects on reproductive system of operating room personnel, which

Abbreviations: DAPI, 4,6-diamidino-2-phenyl-indol; DCF-DA, 2 ,7 dichlorofluorescin diacetate; FPG, formamidopyrimidine glycosylase; GPX, glutathion peroxidase; PBS, phosphate-buffered saline; RMSEA, root mean square error of approximation; ROS, reactive oxygen species; SCGE, single cell gel electrophoresis; TBARS, thiobarbarbituric acid-reactive substances. ∗ Corresponding author at: Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, 8 Sw. Teresy Str., 91-348 Lodz, Poland. Tel.: +48 42 6314 611; fax: +48 42 6314 610. ´ E-mail address: [email protected] (T. Wronska-Nofer). 0027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2011.10.010

manifest as reduced fertility [1], spontaneous abortions [2], and birth defects [3]. These abnormalities seen in observational studies are usually attributed to changes produced by N2 O in genetic material of germ and somatic cells. Increased frequencies of both chromosomal aberrations and sister chromatid exchange were registered in several studies in operating room staff exposed either to N2 O alone or in combination with halogenated hydrocarbons [4–7]. In addition, increased frequency of micronuclei was repeatedly noted in a micronuclei test among the personnel of anaesthesiology wards [8–10]. We have recently scrutinized genotoxic effects in a large group of operating room personnel exposed to a broad range of N2 O concentrations and found a dose-dependent relationship between the extent of DNA damage as assessed by the single cell gel electrophoresis (SCGE, comet test) and the N2 O concentration in the

T. Wro´ nska-Nofer et al. / Mutation Research 731 (2012) 58–63

ambient air of operating theatre [11]. While these results provide strong support for the N2 O-induced genotoxicity, the mechanisms underlying the DNA damage in subjects occupationally exposed to N2 O have not been investigated to date. There is a large body of experimental evidence suggesting that oxidative processes may substantially contribute to genotoxicity associated with various pathological conditions such as cardiovascular disease, diabetes mellitus, and cancer as well as with the exposure to environmental and occupational pollutants [12–17]. Oxygen- and nitrogen-derived free radicals are major oxidants that react with DNA leading to the formation of various lesions including oxidized bases, abasic sites, and/or DNA strand breaks [18,19]. Oxidative damage to DNA can be specifically monitored in a modification of the comet assay, in which oxidized purines or pyrimidines are enzymatically converted to single strand breaks [19,20]. Whereas the standard comet assay was previously applied to assess genotoxicity associated with occupational exposure to anaesthetic waste gases [11,21–23], the oxidative DNA damage has not as yet been investigated in operating room personnel. This prompted us to assess the extent of oxidative DNA damage in relation to the level of exposure to N2 O and halogenated hydrocarbons and the extent of oxidative stress. To this end the oxidative DNA damage and generation of reactive oxygen species in peripheral blood lympho- or leucocytes, respectively, as well as biochemical markers of oxidative stress in erythrocytes, plasma and urine were determined in a group of anaesthesiology nurses exposed to various concentrations of N2 O and in a control group. In parallel, concentrations of N2 O and halogenated hydrocarbons in the ambient air of operating theatres were monitored. Our results for the first time document that occupational exposure to N2 O produces both oxidative stress and oxidative DNA damage that are interrelated and critically dependent on the magnitude of exposure.

59

times with 0.4 mol/L Tris buffer (pH 7.5), drained, exposed to 100% ethanol to dry and stained with fluorescent dye 4,6-diamidino-2-phenyl-indol (DAPI, 5.0 ␮g/mL). 2.4. Oxidatively generated DNA damage Oxidized DNA bases were detected in leukocytes with modified comet assay according to Collins et al. [20,26] after converting altered bases to strand breaks with formamidopyrimidine glycosylase (FPG), which rapidly removes its substrate lesions and plays a prominent role in the global repair of both purine and pyrimidine damage [27]. To determine oxidized bases, leucocytes were embedded in agarose and lysed as described above. Post-lysis slides were washed 3 times with the enzyme buffer containing 0.1 mol/L KCl, 0.5 mmol/L Na2 EDTA, 40 mmol/L HEPESKOH, 0.2 mg/mL bovine serum albumin, pH 8, treated with 50 ␮L of FPG or with buffer alone as a control and incubated at 37 ◦ C for 30 min. Subsequent steps including electrophoresis, neutralization and staining were carried out as described above. FPG used in this study was kindly provided by Dr. B. Tudek, Institute Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, aliquoted and stored at −70 ◦ C. To verify, whether FPG was sufficiently active to reveal oxidized base damage, cells were treated with 20 ␮M H2 O2 for 5 min on ice, lysed, treated with FPG, and processed by SCGE as described above. 2.5. Image analysis

2. Material and methods

The DNA damage grade was assessed visually with fluorescence microscopy (Olympus BX) according to method described by Collins [26,28]. Briefly, at least 100 cells randomly selected on each gel slide were analyzed for DNA damage visually and assigned to one of five classes (0, 1, 2, 3, 4) according to the extent of damage: 0 - cells without visible damage, 1 - cells with minimal damage (slightly visible migration), 2 and 3 - cells with intermediate damage (migration clearly visible under microscope), 4 - cells with considerable DNA degradation (long and broad tail, poorly visible head). The total score has been computed for each slide according to the calculation formula 1 × n1 + 2 × n2 + 3 × n3 + 4 × n4 , where n represents cell number attributable to each damage class. This DNA damage score represents a weight averaged extent of DNA breakage, varies from 0 (all undamaged cells) to 400 (all damaged cells), and is expressed in arbitrary units. To roughly estimate the reproducibility of the comet assay, we assessed the DNA damage in regular time intervals in the same two healthy subjects not participating in this study. The calculated coefficients of variation were 23.8% (n = 6) and 20.7% (n = 6), respectively. The extent of DNA damage specifically attributable to oxidation (net enzyme-sensitive break sites) was estimated as a difference in DNA damage score calculated in the presence and absence of FPG.

2.1. Study subjects

2.6. Lymphocyte isolation and determination of reactive oxygen species (ROS)

The examined group included 36 female nurses working for 5–27 years in operating room at 10 hospitals, who had contact with anaesthetics such as N2 O and halogenated hydrocarbons during surgical procedures. 36 female nurses allocated to other wards of the same hospitals and without history of working in operating rooms were taken as a control group. All study subjects filled a questionnaire regarding demographic data and working activities in the past. Exposed and control groups were matched for age and employment duration. Smokers, past smokers and subjects with history of occupational exposure to X-rays were excluded from the study. All subjects were informed on the purpose of the study and gave a written consent to participate. All investigations were approved by the local Ethics Committee, and conformed to the current legislation in Poland.

Lymphocytes were separated from polymorphonuclear leukocytes and erythrocytes by layering 5 mL of whole blood onto 4 mL of Histopaque® (Sigma, St. Louis, MO) and centrifuging at 2000 rpm for 30 min at room temperature. Lymphocytes were aspirated from the gradient-plasma interfaces, washed twice and resuspended in phosphate-buffered saline (PBS). Viability of cells was checked by staining with 0.1% (v/v) trypan blue and only samples containing at least 95% viable lymphocytes were used for further experiments. ROS production in cells was measured using the fluorescent probe 2 ,7 -dichlorofluorescin diacetate (DCF-DA, 5.0 ␮g/mL). The changes of fluorescence (F) in time (30 min) were then calculated using the formula: F = (Ft30 –Ft0 )/Ft0 × 100%. The DCF fluorescence was monitored with a fluorescence spectrometer (LS-50B, Perkin-Elmer, Waltham, MA) with 488 nm excitation and 521 nm emission wavelengths. To minimize dye photooxidation samples were continuously kept in the dark.

2.2. Blood and urine sample collection Blood samples were collected from medical personnel of operating rooms and other wards during the working day. For DNA damage determination, a total of 100 ␮l of blood was taken from a finger prick into heparinized glass capillary, mixed with 1.0 mL ice-cold RPMI-1640 medium, transported on ice to the laboratory and processed within 3 h. For lymphocyte isolation and biochemical analysis blood was withdrawn by venipuncture into tubes containing heparin as anticoagulant. Samples were centrifuged immediately after blood drawing. Plasmas and erythrocytes were stored at −70 ◦ C until analysis. Overnight urine samples were collected into vials containing 5.0 mmol/L EDTA to prevent oxidative processes, divided into aliquots, and kept frozen at −70 ◦ C until analysis.

2.7. Biochemical analysis Vitamin E (␣-tocopherol) levels in plasma were measured by fluorescence spectrometry according to Taylor and Tappel [29]. Glutathion peroxidase (GPX) activities in erythrocytes were measured photometrically as described previously [30], using t-butyl-hydroperoxide as a substrate. Plasma levels of lipid peroxidation products (thiobarbarbituric acid-reactive substances, TBARS) were measured colorimetrically ˛ et al. [31] using malondialdehyde as a standard. Concentraaccording to Wasowicz tions of 8-iso-prostaglandin F2␣ (8-iso-PGF2␣ ) in urine were determined using a commercially available enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) according to instructions of the supplier.

2.3. Endogenous DNA damage 2.8. Quantitative analysis of anaesthetics in the ambient air of operating rooms Endogenous DNA damage in leucocytes was detected by SCGE (comet test) based on the method of Singh et al. [24] as described previously [25]. Briefly, a small aliquot of the sample of blood in RPMI was embedded in agarose on a microscope slide, lysed with a cold solution containing 2.5 mol/L NaCl, 100 mmol/L Na2 EDTA, 10 mmol/L Tris base, pH 10, and Triton X 100 (1%, v/v) added just before use, and allowed to stay at 4 ◦ C for at least 1 h. Post-lysis slides were unwound in alkaline electrophoretic solution (1 mmol/L Na2 EDTA, 300 mmol/L NaOH, pH > 13, prepared at the time of use) to produce DNA strand breakage, and electrophoresis was carried out at 4 ◦ C for 30 min. at approximately 300 mA and 25 V. Thereafter, slides were neutralized by rinsing 3

Static monitoring of N2 O concentrations was carried out during consecutive surgeries as previously described [32]. Each monitoring session was carried out continuously for the duration of anaesthetic being used but not shorter than for 75% of the working shift, which is in accordance with the rules of environmental monitoring [33]. Individual dosimeters were applied for volatile anaesthetics as previously described [34,35]. Gas chromatographic separation coupled with mass spectrometry selective detection was used for N2 O determination and partition gas chromatography with mass spectrometry for the determination of halogenated anaesthetics

T. Wro´ nska-Nofer et al. / Mutation Research 731 (2012) 58–63

β1

40

β2

β3 Score SSB, FPG

and toxic solvents present in the air. The detection limits for determination of N2 O, isoflurane, and sevoflurane were 6.5 mg/m3 , 0.186 mg/m3 and 0.073 mg/m3 , respectively. 2.9. Statistical analysis An exploratory statistics was performed using the Statistical Package for the Social Sciences (SPSS-X). The results were expressed as mean ± SEM or as frequencies. Two-tailed Student’s t-test was used for comparison of means. Spearman’s rank correlation test was used to examine serial correlations. Differences or correlations with a p value <0.05 were considered significant. The interrelationships between the exposure to N2 O, oxidative stress, and the endogenous or oxidatively generated DNA damage were assessed using path analysis, which is an extension of the multiple linear regression allowing for discrimination between causal and coincidental correlations among a set of variables. A path diagram was constructed using SPSS® AMOS 6.0 software as shown in Fig. 1 assuming direct or indirect, oxidative stressmediated effects of N2 O on DNA damage. Path coefficients (ˇi ) were computed thereafter based on hypothesized models using the maximum likelihood method and approximated t-tests to estimate individual path significance. The validity of the model fitting was assessed by the root mean square error of approximation (RMSEA).

3. Results 3.1. Effect of occupational exposure to anaesthetics on DNA damage Total and oxidatively generated DNA damage scores were 27.1 ± 1.33 and 20.2 ± 1.38, respectively, in the whole population examined. The DNA damage grade in blood leukocytes of subjects occupationally exposed to anaesthetics and their unexposed counterparts is shown in Fig. 2. Cells obtained from operation room personnel were characterized by significantly increased DNA damage score. Moreover, treatment of peripheral leucocytes with FPG led to more extensive induction of DNA breakage and thereby to increased DNA damage scores attributable to oxidative damage in a study group occupationally exposed to anaesthetics. 3.2. Effect of occupational exposure to anaesthetics on oxidative stress The results of the assessment of oxidative stress in operating room personnel and in the control group are shown in Table 1. Both, the generation of ROS directly determined in the peripheral blood leukocytes and plasma and urine levels of TBARS and F2 isoprostanes, respectively, which reflect free radicals-induced lipid peroxidation processes, were significantly increased in subjects occupationally exposed to anaesthetics. In addition, significantly lower activities of GPX, a major antioxidative enzyme, were observed in erythrocytes obtained from this study group. No

A

*

20

0

30

Formamidopyrimidine

Fig. 1. Path diagram reflecting the interrelationship between endogenous or oxidative DNA damage and N2 O concentration and oxidative stress. - Single strand break (SSB) and formamidopyrimidine glycosylase (FPG)-induced DNA damage scores were taken as dependent variables. N2 O concentrations in the ambient air and ROS generation in peripheral blood leukocytes were included as primary and secondary explanatory variables, respectively. Arrows reflect causal relationships in default models assuming indirect N2 O effects on DNA damage mediated by ROS (solid arrows) or direct N2 O effects on DNA damage (dotted arrows). ˇ’s represent path coefficients (regression weights) attributable to each path.

(DNA damage score)

Single Strand Breaks

ROS

Glycosylase

N2O

(DNA damage score)

60

Control

Exposed

B

*

15

0

Control

Exposed

Fig. 2. Endogenous DNA damage in female individuals occupationally exposed to N2 O. – DNA damage was determined with Comet test without (A) or with treatment with formamidopyrimidine glycosylase (B) as described under Section 2 and is expressed as DNA damage score. Error bars denote SEM. *p < 0.01. Table 1 Oxidative stress markers in subjects occupationally exposed to N2 O. Exposed (n = 36) ROS (arbU) 8-iso-PGF2␣ (pg/mL) TBARS (␮mol/L) GPX (␮mol NADPH/g Hb) ␣-Tocopherol (mg/L)

47.8 394.3 4.01 20.2 7.70

± ± ± ± ±

4.8 34.1 0.22 0.6 0.58

Control (n = 36) 33.0 278.0 3.50 24.5 8.31

± ± ± ± ±

2.9 24.3 0.15 1.3 0.38

p <0.005 <0.01 <0.05 <0.01 n.s.

ROS: reactive oxygen species; TBARS: thiobarbituric acid reactive substances; GPX: glutathione peroxidase activity; n.s.: not significant.

differences, however, were noted between the anaestheticexposed and non-exposed groups with regard to plasma ␣tocopherol. 3.3. Oxidative stress and DNA damage in relation to N2 O concentrations in the ambient air Monitoring of anaesthetic concentrations in operating theatres revealed N2 O levels ranging from 185 to 1502 mg/m3 and thereby exceeding the occupational exposure limit of 180 mg/m3 . By contrast, isoflurane and sevoflurane concentrations ranged between 0.4–15.0 mg/m3 and 0.5–14.0 mg/m3 , respectively, which is below occupational exposure limits of 55.0 mg/m3 and 32.0 mg/m3 , respectively. Analysis of the relationship between the oxidative stress and N2 O concentrations in the ambient air showed a significant positive correlation between the exposure levels to this anaesthetic and the ROS generation in peripheral blood leukocytes (r = 0.85; p < 0.001) (Fig. 3). Further analysis revealed that the ROS generation in leukocytes was significantly correlated with DNA damage score in peripheral blood cells of examined individuals either before the enzyme treatment (r = 0.51; p < 0.005) or after the strand break induction with FPG (r = 0.40; p < 0.05) (Fig. 3).

T. Wro´ nska-Nofer et al. / Mutation Research 731 (2012) 58–63

150

A

ROS (%)

100

50

r=0.85 P<0.001

0

1000

0

Single Strand Breaks (DNA damage score)

4. Discussion

(mg/m3)

B

30

r=0.51 P<0.005 0

0

50

100

150

(DNA damage score)

Glycosylase

Formamidopyrimidine

ROS (%) 60

C

30

r=0.40 P<0.05

0

0

path coefficients were both significant. Likewise, these factors combined significantly affected the variability of oxidized DNA damage as determined by FPG-induced DNA breakage. RMSEA values indicated an excellent goodness of fit to the observed data for models assuming indirect effects of N2 O on DNA damage via ROS. By contrast, the path coefficient associated with direct effects of N2 O exposure on oxidative DNA damage was not significant. Moreover, models assuming direct effects of N2 O on DNA damage failed to show satisfactory fit to the observed data.

2000

Nitrous oxide 60

61

50 100 ROS (%)

150

Fig. 3. Relationship between the magnitude of N2 O exposure, the oxidative stress and the extent of DNA damage. – Shown are the correlations between N2 O concentrations determined in the ambient air in operating rooms and the ROS generation in peripheral blood leukocytes (A) and between the ROS generation and the DNA damage expressed as DNA damage score without (B) or with prior treatment formamidopyrimidine glycosylase (C) as described under Section 2. Solid lines – correlations; dotted lines – confidence intervals.

Significant correlation was also found between N2 O concentrations and total DNA damage (DNA strand breaks before treatment (r = 0.54; p < 0.01), whereas the correlation between N2 O concentrations and the FPG-induced DNA damage was of borderline significance (r = 0.31; p = 0.079). By contrast, no significant correlations were observed between sevoflurane or isoflurane levels in operating theatres and parameters reflecting either oxidative stress or DNA damage (not shown). 3.4. Causal links between N2 O concentration and DNA damage In order to identify causal relationships linking N2 O exposure to oxidative stress and DNA damage and to exclude coincidental correlations, the path analysis was performed. As shown in Table 2, N2 O exposure and oxidative stress as reflected by the ROS generation levels in peripheral blood leukocytes were most influential factors for the endogenous DNA damage, and their

Substantial body of evidence accumulated over recent years suggests that N2 O, a waste anaesthetic gas present in the ambient air of operating rooms, increases the risk of genetic damage. However, mechanisms underlying the genotoxic effects of N2 O remain obscure. The present study provides several pieces of evidence pointing to oxidative stress as a link between the occupational exposure to N2 O and the DNA damage. First, application of the comet assay modification that employs enzyme recognizing oxidized DNA and thereby allows the assessment of DNA damage specifically attributable to oxidative stress revealed increased presence of oxidized purines in study subjects exposed to volatile anaesthetics. Furthermore, a dose-dependence relationship could be observed between the extent of oxidative DNA damage and N2 O concentrations but not halogenated hydrocarbon concentrations in the ambient air of operating theatres. Second, evidence for the enhancement of pro-oxidative processes as documented directly by the increased ROS production in peripheral blood leukocytes and indirectly by elevated plasma and urine concentrations of lipid peroxidation products has been found in study subjects exposed to N2 O. Similarly to oxidative DNA damage, a close positive correlation between ROS generation and N2 O concentrations in operating rooms has been noted. Third, the path analysis of interrelationships between N2 O concentration, ROS generation, and DNA breakage suggested that oxidative stress is a critical intermediate linking N2 O exposure to genotoxicity, while oxidative stress-independent effects of N2 O on DNA integrity are less likely. Pro-oxidative effects of inhalable anaesthetics were previously demonstrated in several studies. Halogenated hydrocarbons were shown to enhance generation of oxygen free radicals in neutrophils and isolated hearts under in vitro conditions [36,37]. Moreover, indices of enhanced oxidative damage to lipids, proteins and DNA were reported in animals exposed to isoflurane or sevoflurane or in patients undergoing desflurane anaesthesia [38–41]. Less attention, however, was devoted towards the assessment of oxidative stress in subjects occupationally exposed to anaesthetic waste gases. Partial exhaustion of the total antioxidative reserve of plasma as well as the reduced activity of enzymes of anti-oxidative defence such as GPX and superoxide dismutase were noted in two studies in operating room personnel [42,43]. The present study not only extends these previous results by demonstrating that parameters reflecting enhanced oxidative stress including ROS generation, TBARS concentration in plasma, F2 isoprostanes concentration in urine as well as DNA base oxidation are significantly increased in subjects occupationally exposed to waste anaesthetic gases, but is the first to document dose-dependent relationships between the extent of genetic damage, oxidative stress and the N2 O concentration in the ambient air of operating theatre. By contrast, no such relationships were noted in case of halogenated hydrocarbons. We interpret these observations to mean that N2 O rather than halogenated hydrocarbons is an air pollutant specifically responsible

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Table 2 Path analysis of links between N2 O exposure, oxidative stress and DNA damage.

Path

β) Path coefficients (β

Significance (p)

N2O ROS

β1: 0.81

<0.001

ROS SSB-score

β2: 0.49

<0.01

N2O SSB-score

β3: 0.44

<0.05

N2O ROS

β2: 0.81

<0.001

ROS FPG-score

β2: 0.40

<0.05

N2O FPG-score

β2: 0.34

n.s.

RMSEA

0.03 0.71

0.00 0.72

RMSEA: root mean square error of approximation; SSB-score: endogenous DNA damage score; FPG-score: formamidopyrimidin glycosylase-induced DNA damage score.

for oxidative stress and DNA damage in operating room personnel occupationally exposed to inhalational anaesthetics. It has to be noted, however, that in contrast to N2 O the concentrations of isoflurane and sevoflurane in all operating theatres examined here were relatively low. Hence, our results do not preclude pro-oxidative and/or genotoxic effects of these anaesthetics at concentrations exceeding respective exposure limits. The mechanisms underlying deleterious effects of N2 O on DNA integrity have not been examined to date. Based on results of the present study, the direct influence of N2 O on DNA cannot be entirely dismissed. The outcome of the path analysis, however, does not seem to favour the contention that N2 O, which is chemically rather an inert compound, directly interacts with genetic material to produce DNA breakage. In our view, it is more likely that some intermediates are required for N2 O to effectively induce DNA damage and that oxidative stress plays an important role in this respect. Actually, a single electron transfer-mediated reduction of N2 O to nitrogen with ensuing production of reactive oxygen species was postulated in previous studies [44–46]. Moreover, N2 O was demonstrated to elevate cytosolic concentration of low molecular weight iron species, which catalyze oxygen free radicals formation in Haber-Weiss and Fenton reactions [47]. In addition, N2 O was shown to promote a one-electron reduction of the vitamin B12 molecule of methionine synthase followed by the formation of superoxide and hydroxyl radicals and the inactivation of the enzyme that catalyzes the remethylation of homocysteine to methionine [46,48,49]. In this context it is worth notice that decreased vitamin B12 levels and increased concentrations of homocysteine in plasma were previously reported by us in subjects exposed to N2 O at concentrations substantially exceeding occupational exposure limit [50]. As homocysteine decreases the expression of antioxidant enzymes including GPX, the elevation of its concentration may represent another mechanism linking the occupational exposure to N2 O to oxidative stress. It may also explain the decreased GPX activity, which has been observed in N2 O-exposed subjects in the present study. In conclusion, the present study for the first time documents the oxidative DNA damage in subjects occupationally exposed to high concentrations of N2 O and identifies oxidative stress as a link between the N2 O exposure and the genotoxicity. Further studies are necessary to precisely define molecular mechanisms underlying deleterious effects of N2 O on DNA integrity.

Conflict of interest The authors declare to have no competing interests.

Acknowledgements This study was supported by Polish Ministry of Science and Higher Education within a Project 3PO5D.025.24 (to T. W.-N.) and by the intramural grant from the Nofer Institute of Occupational Medicine (to T. W.-N.).

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