Mutation Research 666 (2009) 3943

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

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DNA damage induced by nitrous oxide: Study in medical personnel of operating rooms
a, Teresa Wronska-Nofer , Jadwiga Palus a , Wojciech Krajewski b , Jolanta Jajte c , a a a Magorzata Kucharska , Jan Stetkiewicz a , Wojciech Wasowicz , Konrad Rydzynski
a b c

Department of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, 8 Teresy St., 91-348 Lodz, Poland Department of Anaesthesiology, Polish Mathers Memorial Hospital, 281 Rzgowska St., 93-338 Lodz, Poland Department of Toxicology, Medical University, 1 Muszynskiego St., 90-151 Lodz, Poland

a r t i c l e

i n f o

a b s t r a c t
Occupational exposure to anaesthetics such as nitrous oxide (N2 O) and halogenated hydrocarbons has been suggested to increase risk of genetic damage. However, the dose-dependency of genotoxic effects has not been unequivocally established and their relation to occupational exposure limit (OEL) remain obscure. In this study, the genotoxicity associated with occupational exposure to anaesthetics has been investigated in a group of 55 female nurses and 29 male anaesthesiologists active for at least 5 years in a working environment containing variable concentrations of N2 O and halogenated hydrocarbons. 83 unexposed health care workers (52 female nurses and 31 male doctors) matched for age, gender, smoking habit and employment duration were included in the control group. Genotoxicity has been assessed using comet test. Concentrations of nitrous oxide, sevourane and isourane monitored by gas chromatography and mass spectrometry made possible to relate the extent of DNA damage to the level of exposure. Our results for the rst time document a positive correlation between the DNA damage and the N2 O levels in the ambient air. By contrast, no correlation has been observed between genotoxic effects and concentrations of sevourane and isourane. The extent of genetic injury was especially aggravated among nurses and anaesthesiologists exposed to N2 O in concentrations exceeding OEL (180 mg/m3 ). We conclude that occupational exposure to N2 O is associated with increased DNA damage and that the level of exposure plays a critical role in this regard. 2009 Elsevier B.V. All rights reserved.

Article history: Received 30 September 2008 Received in revised form 14 March 2009 Accepted 19 March 2009 Available online 5 April 2009 Keywords: Nitrous oxide Halogenated anaesthetics Genotoxicity DNA damage Comet assay

1. Introduction Occupational exposure to anaesthetics such as N2 O and halogenated hydrocarbons is thought to exert adverse effects on reproductive system of operating room personnel, which manifest as reduced fertility [1,2], spontaneous abortions [3,4] and birth defects [5,6]. The reproductive abnormalities observed in epidemiological investigations are usually attributed to changes produced by anaesthetics in genetic material of germ and somatic cells. However, the extent and mechanisms underlying deleterious effects of anaesthetics on human genetic material are controversially discussed and equivocal results were obtained in studies, in which genotoxic effects of exposure to N2 O together with halogenated hydrocarbons were examined. For instance, increased frequency of both chromosomal aberrations (CA) and sister chromatid exchange (SCE) was observed in some studies [713], whereas others did not conrm this effect [9,1417]. In case of micronuclei test, increased

Corresponding author. Tel.: +48 42 6314 611; fax: +48 42 6314 610. E-mail address: tewono@imp.lodz.pl (T. Wronska-Nofer). 0027-5107/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.03.012

frequency of micronuclei but not SCE was noted in operating room staff exposed either to N2 O only or to N2 O together with enurane, sevourane, or isourane [9,1720]. One reason accounting for these discrepant results could be the variable exposure level to anaesthetics. Unfortunately, the lack of anaesthetic concentration measurements in the ambient air of operating rooms in most of previous studies precluded the assessment of the extent of genetic material damage in relation to the actual magnitude of exposure. Additional reason of discrepancies was the presence of various confounding factors such as organic solvents or X-ray radiation, which might directly affect the genetic material and which were not adequately monitored in previous studies. The unclear association between the use of anaesthetics and genotoxicity prompted us to examine the extent of DNA damage in relation to the level of exposure to N2 O and halogenated anaesthetics. To this end DNA damage in peripheral blood leukocytes of operating room personnel as well as actual concentrations of N2 O and halogenated hydrocarbons in the ambient air were determined in parallel. In addition, the study was controlled for the presence of confounding factors affecting genetic material such as organic solvents and X-ray radiation. Our results for the rst time document

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T. Wro nska-Nofer et al. / Mutation Research 666 (2009) 3943 ipating in this study. The calculated coefcients of variation were 23.8% (n = 6) and 20.7% (n = 6).

that occupational exposure to N2 O produces DNA damage that is critically dependent on the magnitude of exposure.
2. Material and methods 2.1. Study subjects and design The examined group included 84 medical staff members: 55 female nurses and 29 male anaesthesiologists working for 527 years in operating room at 10 hospitals in city area of Lodz. They had contact with anaesthetics such as N2 O and halogenated hydrocarbons during surgical and anaesthesia procedures. 83 medical staff members: 52 female nurses and 31 male physicians allocated to other, not surgical wards of the same hospitals and without history of working in operating room were taken as a control group. All study subjects were asked to ll a questionnaire regarding demographic data, place of residence, smoking habit, and working activities in the past. Smokers were classied according to number of cigarettes used per day (10, 15, 20, 25 and 30 or more cigarettes per day). Subjects smoking less than 10 cigarettes per day (3 persons) as well as subjects with history of occupational exposure to Xrays (2 persons) were excluded from the study. Exposed and control groups were matched for gender, age, smoking habit and employment duration. No relationship has been noted between the subject place of residence and the location of hospitals in the city area of Lodz. All subjects were informed on the scope and the purpose of the study and gave a written consent to participate. All investigations were carried out as a part of extended periodic medical examinations, were approved by the local Ethics Committee, and conformed to the current legislation in Poland. The study was performed in a cross-sectional format. Investigated parameters included evaluation of DNA damage (strand breaks) and monitoring of N2 O, izourane, and sevourane concentrations in the ambient air of operating rooms during anaesthetic/surgical procedures. 2.2. Blood sample collection

2.5. Analysis of N2 O, volatile anaesthetics and organic solvents in the ambient air of operating rooms N2 O and volatile anaesthetics present in the ambient air were evaluated in 24 operating rooms of 10 hospitals equipped with 1 of 3 ventilation systems differing with respect to number of air changes/h and efciency in removing exhaust gases from working environment: gravitational with supplementary pressure ventilation system (610 air changes/h) or pressure and exhaust ventilation system (1215 air changes/h) or complete laminar ow air-condition system (>15 air changes/h). Anaesthetic concentrations were measured during consecutive surgeries. Each monitoring session was carried out continuously for the duration of anaesthetic was being used but not shorter than for 75% of the working shift (6 h of non-stop work under conditions of anaesthetic exposure), which is in accordance with the rules of environmental monitoring and allows expressing the N2 O concentration in the air of operating rooms in terms of occupational exposure limit (OEL) value [25]. Subjects periodically active in operating room or active in generic theatres were excluded from the study. Static monitoring was carried out for N2 O measurement. Air from medical personnel breathing zone was directly sampled through a plastic tube to reservoir bag for N2 O measurement in accordance with National Institute Occupational Safety and Health (NIOSH) approved procedure. Individual dosimeters were applied for volatile anaesthetics as previously described [26,27]. Gas chromatographic separation coupled with mass spectrometry selective detection (GC/MS) was used for N2 O determination and partition gas chromatography with mass spectrometry (PGCMS) for the determination of halogenated anaesthetics and toxic solvents present in the air. The detection limits for the determination of N2 O, isourane, and sevourane were 6.5 mg/m3 , 0.186 mg/m3 and 0.073 mg/m3 , respectively.

2.6. Statistical analysis Blood samples were collected simultaneously from medical personnel of operating rooms and other wards. A total of 0.1 mL of blood was taken from a nger prick into heparinized glass capillary, mixed with 1 mL ice-cold RPMI 1640 medium, transported on ice to the laboratory and immediately used for comet assay. 2.3. Comet assay DNA damage in leukocytes was detected by comet assay (SCGEsingle cell gel electrophoresis) based on the method of Singh et al. [21] in modication of Mc Kelvey-Martin et al. [22] as described in our previous paper [23]. Briey, cells (0.1 mL whole blood in RPMI 1640 medium) were 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 incubated at 4 C for at least 1 h. Post-lysis slides were immersed 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 three times with 0.4 mol/L Tris buffer (pH 7.5), drained, exposed to ethanol (100%, v/v) to dry, and stained with uorescent dye 4,6-diamidino-2-phenyl-indol (DAPI, 5 g/mL). 2.4. Image analysis The DNA damage level was assessed visually with uorescence microscopy (Olympus BX) according to method described by Collins [23,24]. Briey, at least 100 cells on each gel slide randomly selected were analyzed for DNA damage visually and assigned to one of ve classes (04) according to the extent of damage: 0cells without visible damage; 1cells with minimal damage (slightly visible migration); 2 and 3cells with intermediate damage (migration clearly visible under microscope); 4cells 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 1n1 + 2n2 + 3n3 + 4n4 , 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 estimate the reproducibility of the comet assay, we assessed the DNA damage in regular time intervals in the same two healthy subjects not particA statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS-X). The results were expressed as mean SEM or as frequencies. Unpaired Students t-test was used for comparison of means. Univariate associations were assessed by Spearman rank correlation coefcients. Multivariate analysis was performed in order to test the relationship between N2 O levels and DNA damage score independently from potential confounders. A backward stepwise procedure was used with = 0.05 as a cut-off for entry or removal of variables. The multiple regression model was subsequently built considering DNA damage score to be dependent variable, whereas N2 O concentrations, age, sex, number of cigarettes smoked and hospital location were taken as independent variables. Hospital location was included into the model as dummy variable for each hospital (1 for individuals working in a given hospital, 0 for individuals working in other hospitals). p-Values less than 0.05 were considered signicant.

3. Results 3.1. Characteristics of examined populations The characteristics of the exposed and the control groups are shown in Table 1. Both groups did not differ signicantly with respect to age, smoking habit and employment duration. 3.2. Effect of occupational exposure to anaesthetics on DNA damage The levels of DNA damage in peripheral blood leukocytes of subjects occupationally exposed to anaesthetics and their unexposed counterparts are shown in Fig. 1. Cells obtained from both female and male operation room personnel were characterized by signicantly increased DNA damage score.

Table 1 Characteristic of subjects enrolled in the study with respect to age, duration of employment (control group) or exposure to anaesthetics (exposed group) and smoking habit. Women Control (n = 52) Age (years) Employment duration/time of exposure (years) Smokers (%) 41 (2453) 15.8 (526) 53.8 Exposed (n = 55) 39 (2555) 14.6 (526) 49.2 Men Control (n = 31) 42 (2953) 16.5 (530) 47.8 Exposed (n = 29) 44 (3055) 18 (531) 40.2

Shown are mean values or percentage. Values in brackets represent range. Smokers were consuming more than 10 cigarettes per day.

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Fig. 1. Endogenous DNA damage in female and male individuals occupationally exposed to N2 O. DNA damage was determined with comet test as described under Section 2 and is expressed as DNA damage score. Error bars denote SEM. *p < 0.001.

3.3. DNA damage in relation to anaesthetic concentrations in the ambient air Monitoring of anaesthetics in operating theatres revealed that N2 O prominently contributed to ambient air pollution. N2 O levels varied over three orders of magnitude and ranged from 35.8 mg/m3 to 1502 mg/m3 depending on the ventilation system (Table 2). In 18 out of 24 operating rooms examined in this study, concentrations of N2 O during operations exceeded the OEL value of 180 mg/m3 adopted in most of European countries including Germany, Great Britain, Italy, Switzerland, Netherlands and Sweden. By contrast, in none of operating theatres concentrations of volatile anaesthetics including isourane and sevourane exceeded the threshold limit values legally binding in Germany and Poland (Table 2). In all operating theatres examined in this study, concentrations of organic solvents remained below the threshold limit values recommended by US and European public health authorities (Table 2). Single regression analysis of the relationship between DNA damage in peripheral blood of examined individuals and anaesthetic concentrations in the ambient air revealed a signicant positive correlation between the N2 O exposure levels and the DNA damage score (Fig. 2A). Similar correlation between DNA damage score and N2 O exposure was found in a subgroup of non-smoking study participants (Fig. 2B). The association between N2 O concentrations

Fig. 2. Relationship between the extent of DNA damage and the magnitude of N2 O exposure. Shown is the correlation between the DNA damage expressed as DNA damage score and N2 O concentrations determined in the ambient air in operating rooms. Panel A: all N2 O-exposed subjects examined in the study and panel B: non-smoking subjects exposed to N2 O. Solid linescorrelation and dotted linescondence intervals.

and the DNA damage was further assessed in a multiple linear regression model, in which following parameters were additionally included as independent variables: age, gender, smoking status and hospital location. In this model, DNA damage score maintained a statistically signicant association with N2 O concentrations and the resulting standardized -coefcient of regression relating the two variables was 1.10 (p < 0.001). In contrast to N2 O exposure levels, no signicant correlations were observed between the DNA damage score and the exposure levels to halogenated hydrocarbons (sevourane and/or isourane; r = 0,17; p = 0.37).

Table 2 Concentrations of N2 O, halogenated anaesthetics and organic solvents in the ambient air of operating rooms. Operating rooms Anaesthetics (mg/m ) Nitrous oxide Sevourane Isourane Organic solvents (mg/m3 ) Ethanol Isopropanol Propanol Ethyl ether Light petrold
a b 3

Concentration 440 (35.81502) 4.7 (0.415.0) 5.2 (0.514.0) 22.3 (2.576.1) 9.6 (0.438.9) 7.1 (0.052.8) Trace amount Trace amount

OEL Germanya 180.0 n.a. 80.0 1900 500 n.a. 1200 180

OEL USAb 90.0 n.a. n.a. 1800 492 246 1210 170

OEL Polandc 90.0 55.0 32.0 1900 900 200 300 100

24 24 12 24 24 24 24 10

List of hazardous materials (Source: Technische Regeln fur Gefahrstoffe (TRGS 900), BarbBl 2006/1, p. 41; BarbBl 2000/10, p. 34). American Conference of Governmental Industrial Hygienists (ACGIH)Occupational Safety and Health Administration www.osha.gov/dts/anestheticgases/index.html and guide to occupational exposure values, Cinncinati, 2006. c Ordinance of the Minister of Labour and Social Policy on the Maximum Admissible Concentrations and Intensities of Harmful Agents in the Work Environment, Ofcial Journal of Law of the Republic of Poland Nr. 212/2002, position 1769 with amendments 212/2005, Nr. 217/2002, position 1833, and Nr. 261/2007 position 1142. n.a.not announced. d Light petrol (pharmaceutical grade) measured as the sum of hexane isomers; OEL values are given for n-hexane. Anaesthetics and organic solvents concentrations expressed as time-weighted average (TWA) over an 8 h working shift measured in the ambient air of operating rooms equipped with different ventilation systems. Details are given in Section 2. Shown are mean values and range in brackets.

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Table 3 The extent of DNA damage in the peripheral blood leukocytes of subjects exposed to low and high N2 O concentrations (low exposure and high exposure groups) and in a control group1 . N2 O concentration (mg/m3 ) Control group (n = 52) Non-smokers (n = 28) Smokers (n = 24) Low exposure group (n = 22) Non-smokers (n = 9) Smokers (n = 13) High exposure group (n = 33) Non-smokers (n = 20) Smokers (n = 13) DNA damage score (arb U) 24.0 1.54 25.7 1.49 22.0 1.66 29.5 1.94a 27.1 1.97 31.2 1.84 34.3 2.73b,c 31.2 2.18 37.7 3.16

175.4 (35.8320.4)

704 (447.41502)

No statistical differences were observed between smokers and non-smokers in each group. 1 Low exposure and high exposure groups included individuals working in operating rooms, exposed to N2 O at concentration below or above 2 OEL values (2 180 = 360 mg/m3 ), respectively. Cells with damage were measured with comet assay and the extent of damage is expressed as DNA damage score as described under Section 2. Shown are mean values SEM. Values in brackets represent range. a p = 0.12 (low exposure vs. control). b p < 0.001 (high exposure vs. control). c p = 0.2 (low exposure vs. high exposure).

In the next, we aimed to assess the impact of the N2 O exposure on the DNA damage in relation to mandatory exposure limits. The analysis of the distribution of N2 O levels in polluted areas revealed that in operating theatres equipped with complete air condition or pressure/exhaust ventilation systems N2 O concentrations were below, equal or exceeded the OEL value less than twice (range 35.8320.4 mg/m3 ). By contrast, N2 O concentrations substantially higher than OEL (range 447.41502 mg/m3 ) were registered in operating rooms equipped only with gravitational ventilation. Therefore, to assess the impact of the N2 O exposure on genotoxicity in relation to OEL value, the DNA damage score was evaluated in two arbitrary subsets of study subjects: low exposure group included individuals working in operating rooms with ventilation system maintaining >12 air changes/h and N2 O exposure below 2 OEL (2 180 = 360 mg/m3 ) whereas high exposure group included subjects working in operating rooms with ventilation system maintaining >610 changes/h and with N2 O exposure above 2 OEL (2 180 = 360 mg/m3 ). As shown in Table 3, DNA damage score was signicantly increased in the high exposure but not in the low exposure group. 4. Discussion Current knowledge regarding the genotoxic effects of occupational exposure by inhalation to anaesthetics such as N2 O or halogenated hydrocarbons is incomplete. Both increased and unchanged occurrence of genotoxic effects has been reported in exposed groups using various cytogenetic endpoints such as sister chromatid exchange or micronuclei formation [720]. Insufcient matching for age, gender and smoking habits as well as lack of environmental monitoring of anaesthetic concentrations might likely account for these discrepant results. Wiesner et al. [19] assessed the genotoxic effect of anaesthetics in carefully matched groups of operating room personnel of German and Eastern European hospitals and concluded that a high-level but not a low-level occupational exposure was associated with increased chromosome damage. However, even these authors were unable to distinguish between the potential deleterious effects of N2 O and halogenated hydrocarbons as well as to demonstrate the dose-depending effect of exposure, because the study groups were too small and the examined subjects were exposed, exclusively, to very low or to very high

N2 O concentration. In the present study we assessed the genotoxic effects of inhalable anaesthetics in a large group of operating room personnel exposed to entire range of N2 O concentrations from very low to very high. The examined cohort included female nurses and male anaesthesiologists employed for the period not shorter than 5 years and the presence of confounding factors such as X-ray radiation or organic solvents has been excluded. Our results for the rst time unequivocally document a dose-dependent relationship between the extent of genetic damage and the N2 O concentration in the ambient air of operating theatre. By contrast, no such relationship has been noted in case of halogenated hydrocarbons, though it has to be emphasized that in contrast to N2 O the exposure to isourane and sevourane in all operating theatres remained below respective exposure limits. We interpret these observations to mean that N2 O is an air pollutant primarily responsible for occupational genotoxicity in operating room personnel occupationally exposed to inhalational anaesthetics. Monitoring concentrations of N2 O and volatile anaesthetics in each operating room examined in this study allow relating present results to the currently valid occupational exposure limits. The threshold values recommended by regulatory authorities in various countries differ considerably. For instance, OEL value for N2 O in several European countries including Germany, Great Britain, Italy, Switzerland and Sweden is 180 mg/m3 , whereas 90 mg/m3 is recommended in Poland (since 2006) and United States American Conference of Governmental Industrial Hygienists (ACGIH). In the present study, only operating room personnel working in the environment, in which the OEL values of 180 mg/m3 were exceeded several folds presented with signicantly increased levels of DNA damage as compared to the control group. By contrast, no evidence of genotoxic effects could be noticed among operating room personnel exposed to N2 O in concentrations that were lower or exceeded the OEL binding in several European countries less than twice. Based on our observations, it may be assumed that maintaining the N2 O level below 180 mg/m3 is fully sufcient to minimize the risk of genetic damage. On the other hand, exceeding the OEL value may be associated with increased occurrence of genotoxic effects that were repeatedly brought in conjunction with increased carcino- and teratogenicity as well as with reduced fertility [28,29]. Our results points to the essential role of gas waste removing systems for the effective reduction of N2 O burden [30,31] and favour regular measurements of N2 O in occupational environment. The latter may be especially important in developing countries, in which the use of advanced gas waste removal systems in operating rooms is still limited. Few limitations of the present study have to be acknowledged. First, while individual dosimeters were used for the determination of exposure levels to halogenated hydrocarbons, N2 O concentrations were measured in ambient air samples collected from the breathing zone of medical personnel. Unfortunately, the specic adsorbent for N2 O is currently unknown and individual dosimeters measuring N2 O concentration at individual level are not available to date. Nevertheless, the method applied in present study for N2 O exposure assessment conforms to recommendations of National Institute Occupational Safety and Health (NIOSH Analytical Method 6600 [32]) and has been extensively used in previous studies examining adverse effects of occupational exposure to N2 O [12,13,19,20,35]. Second, while no relationship between the genotoxicity and the magnitude of exposure to halogenated hydrocarbons was observed in the present study, the concentrations of isourane and sevourane in all operating theatres were relatively low. Hence, our results do not preclude genotoxic effects of these anaesthetics at concentrations exceeding respective exposure limits. Actually, evidence for DNA damage was reported in patients anaesthetized with isourane, sevourane and/or desurane even under the absence of N2 O anaesthesia and very recently also in

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anaesthetists exposed to a single halogenated anaesthetic (sevourane) [3335]. However, other investigations do not support the link between genotoxicity and occupational exposure to halogenated hydrocarbons mandating further studies in this area [16,35,36]. In conclusion, this investigation provides the evidence that occupational exposure to N2 O is directly associated with increased DNA damage and that the level of exposure plays a critical role in this regard. By contrast, exposure to halogenated hydrocarbons at low concentrations (equal or below OEL values) does not induce DNA damage. Conict of interests The authors declare that there are no conicts of interest. Acknowledgements This study was supported by Polish Ministry of Science and Higher Education within a Project 3PO5D.025.24 and by the intramural grant from the Nofer Institute of Occupational Medicine. The authors would like to thank Dr. Helmut Schulte, Assmann Stiftung fr Prvention, Mnster, Germany, for his support with statistical analysis and most helpful discussion. References
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