Anesth Analg 2006;102:1573-1577
© 2006 International Anesthesia Research Society
doi: 10.1213/01.ane.0000204298.42159.0e
GENERAL ARTICLES
A Comparison of Sister Chromatid Exchanges in Lymphocytes of Anesthesiologists to Nonanesthesiologists in the Same Hospital
Ahmet Eroglu, MD,
Figen Celep, PhD, and
Nesrin Erciyes, MD
Departments of Anesthesiology, Medical Biology and Genetics, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkey
Address correspondence and reprint requests to Ahmet Eroglu, Karadeniz Technical University, Faculty of Medicine, Anesthesiology, 61080 Trabzon, Turkey. Address e-mail to aheroglu{at}hotmail.com.
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Abstract
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An increased incidence of sister chromatid exchanges (SCEs) in peripheral lymphocytes of operating room personnel exposed to waste anesthetic gases has been reported. We investigated whether the increase of SCEs in anesthesiologists was reversible. Twenty-five anesthesiologists exposed to waste anesthetic gases such as sevoflurane and nitrous oxide were compared with nonexposed internists working in the same hospital. The concentrations of sevoflurane and nitrous oxide in the operating rooms were measured. The incidence of SCE was measured in lymphocytes cultures of anesthesiologists before and after a 2-mo leave from the operating room. These values of SCE were compared with those of nonexposed physicians. Occupational exposure to sevoflurane and nitrous oxide in the operating rooms were above the threshold values. There was a significant difference in SCE values of the anesthesiologists compared with the nonexposed physicians (11.9 ± 4.4 versus 4.2 ± 1.1, P < 0.001). After a 2-mo leave from the operating room, the SCE values of the anesthesiologists were significantly lower compared with those taken before the leave (4.8 ± 1.8 and 11.9 ± 4.4, respectively, P < 0.001). We conclude that the increase of SCE in anesthesiologists exposed to increased environmental concentrations of waste anesthetics gases, such as sevoflurane and nitrous oxide, are reversible if they work free from exposure for 2 mo.
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Introduction
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The possibility of a health hazard resulting from exposure to anesthetics has been discussed extensively during recent decades (1–7). There is a great concern that patients, physicians, and the operating room (OR) personnel might be exposed to health risks with anesthetic gases such as nitrous oxide, halothane, and isoflurane (6,8). However, whether chronic exposure to waste anesthetic gases is hazardous to the health of anesthesiologists is still controversial. A meta-analysis suggested that chronic exposure to trace concentrations of anesthetic gases might cause mutations in DNA (7). Some studies (1,5–9) reported an association between occupational exposure of waste anesthetic gases and an increase in sister chromatid exchanges (SCEs) in lymphocytes for staff working in unscavenged ORs. Other studies (2–4,10,11) did not support the association between occupational exposure to waste anesthetic gases and an increase in SCEs in OR personnel. To minimize the possible health risks associated with occupational exposure to inhaled anesthetics, public health authorities recommended threshold values. The exposure limits recommended by the National Institute of Occupational Safety and Health (NIOSH) are 25 ppm for nitrous oxide and 2 ppm for volatile anesthetics. The limit for volatile anesthetics is reduced with the concomitant use of nitrous oxide (12). However, there are no data available to conclude whether the increases of SCE attributable to occupational exposure to waste anesthetic gases are reversible in anesthesiologists.
SCE analysis in peripheral blood lymphocytes is a sensitive cytogenetic technique widely used for the investigation of suspected human mutagens and carcinogens. An increased number of such SCE reflects the influence of mutagens. Its sensitivity and reliability have made this technique one of the most popular methods in toxicology and human biomonitoring (13–15).
The aim of this study was to investigate whether the increases of SCE in anesthesiologists exposed to waste anesthetic gases are reversible. Therefore, we compared the values of SCE in anesthesiologists when they were working inside and outside the OR and those of internists at the same hospital.
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Methods
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The study was conducted after approval of the Ethical Committee of Karadeniz Technical University Hospital, written informed consent was obtained from all volunteers. Twenty-five anesthesia physicians working in the OR (group anesthesiologists, group A) were compared with 25 nonexposed physicians (group control, group C) working in other nonsurgical departments of the hospital. The surface area of the ORs was between 36 and 56 m2, and the air volume of the ORs was between 90 and 140 m3. The ORs have air conditioning, but they do not have a central high-flow scavenging system, low leakage anesthesia machines, and preventative maintenance of equipment. OR anesthesia machines have sevoflurane vaporizers. The inhaled anesthetic was administered to the patients in a nitrous oxide and oxygen mixture during the study periods. Gases were at high flow. The induction of general anesthesia was performed inside the ORs. The anesthesiologists had to have worked full time (at least 8 h per day and at least 72 h per week), and they only worked in the ORs during the study period. The characteristics of the ORs where occupational exposure was measured are summarized in Table 1.
To measure the occupational exposure, standard solutions of sevoflurane 1 to 100 ppm were prepared in chloroform. Sevoflurane concentrations were assayed using an FT-IR spectrometer (Perkin Elmer, England). The samples of gases in the ORs were taken using a gas cell (IR GAS CELL, Perkin Elmar, Beaconsfield, England) after the instrument was in the ORs for 3-4 h. The samples were taken close to the anesthesiologists, and there were multiple samples for the ORs. Infrared spectrums of sevoflurane and nitrous oxide were done using an IR spectrometer (spectrum one FT-IR spectrometer, Perkin Elmer), and then the concentrations of sevoflurane and nitrous oxide were measured.
Each subject was interviewed using a standardized questionnaire with questions on drug intake, contraception, diseases and vaccinations during the previous 3 mo, alcohol intake, exposure to indoor/outdoor pollutants, and diagnostic and therapeutic radiographs. Group C subjects were selected among the physicians who had not previously worked in ORs, radiology or radiation oncology units, nuclear medicine units, sections of angiography, or in units responsible for disinfection and sterilization. Subjects who smoked were excluded from the study.
Heparinized peripheral venous blood samples (lithium heparin 15 U/mL) were obtained from each subject at the end of the working day and at the end of the working week in group A (sample 1). Sample 2 was obtained from anesthesiologists after working 2 mo outside the OR. They worked in an anesthesia or pain polyclinic without exposure to anesthetic gases during these 2 mo. The venous blood samples from group C were taken at the same time as those from the anesthesiologists.
Laboratory analyses of blood samples taken from anesthesiologists and control subjects were performed randomly. Whole blood (0.5 mL) cultures were established in 5 mL of chromosome medium (PBMax Karyotyping medium, GIBCO Invitrogen Corporation, NY), containing 5-bromo-2'-deoxyuridine (Sigma Chemical, St. Louis, MO) 50 µmol/L for 72 h. Two hours before harvest, 0.1 mg colcemid was added. The cultures were harvested and chromosome preparations were made according to routine chromosomal analysis (16,17). Bromodeoxyuridine-incorporated metaphase chromosomes were stained by the Hoechst-Giemsa method. Metaphases with few or no overlaps were selected for analysis. The incidence of SCE was measured by examining 30 complete second metaphases, counting one SCE each time two adjacent segments of one of the chromatids in a chromosome were stained differently. The value of SCE for each specimen was taken as the mean rate of SCE per metaphase. Two different chromosome cultures for each person were performed to prevent negative technical conditions, such as using different culture medium, effect of concentration of bromodeoxyuridine, effect of colcemid, time and amount, or technical equipment. These cultures were scored at the same time and results are compared. When we observed different scores, SCE analyses were repeated. All scoring and counting procedures were performed by the same blinded investigator using a nonautomated light microscope (Olympus BX-50, Olympus Optical Co, Tokyo, Japan). To prevent excessive evaluation two different chromosome cultures were performed and measured for each sample. If there were different results from both cultures of the same sample, a new culture was done and the results were compared.
A power analysis was performed based on the data from a previous report (8). The intention was to detect a difference between groups of 1.5 SCE per subject (sd 1.3 SCE per cell) when scoring 30 metaphases per subject. We calculated 16 subjects per group required using a two-tailed 
error of 5% and ß error of 10%. Our study reached a power of 95% because we enrolled 25 subjects per group (18).
After data acquisition, the arithmetic mean ± sd was calculated for each group. Group characteristics for age, weight, height, sex, and duration of work in the hospital were compared with the Student's t-test and 
2 test (for sex and alcohol intake). Statistical significance of differences in SCE between groups was analyzed using the Student's t-test. Differences of SCE within the same group were compared by the paired Student's t-tests. A value of P 
0.05 was taken as statistically significant.
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Results
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A description of the ORs and multiple measurements of occupational exposure to sevoflurane and nitrous oxide for each OR are shown in Table 1. Mean values of sevoflurane and nitrous oxide in the ORs were 8.9 ± 5.6 ppm and 119 ± 39 ppm, respectively (Table 1).
There were no significant differences in subject characteristics (age, weight, height, sex, intake of alcohol, and duration of work in the hospital) between groups (Table 2). The duration of working in the hospital was 1–25 yr for group A (14 with 1–5 yr and 11 with 6–25 yr). There is no correlation between the duration of working in the hospital and frequency of SCE in group A when separated into 2 subgroups (the SCE values were 11.5 ± 4.3 and 12.3 ± 4.5, respectively, P = 0.920).
There was a significant difference between group A and group C for SCE values (11.9 ± 4.4 versus 4.2 ± 1.1) (P < 0.001). After a 2-mo leave from the OR, the SCE values of group A were significantly lower compared with the values taken before the leave (4.8 ± 1.8 and 11.9 ± 4.4, respectively; P < 0.001). The nonexposed SCE values of group A were similar to those of group C (4.8 ± 1.8 and 4.3 ± 1.1, P = 0.365) (Table 3).
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Discussion
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In this prospective study, we found that the incidence of SCE in anesthesiologists exposed to waste anesthetic gases was significantly more frequent than that of unexposed physicians in the same hospital. The SCEs in anesthesiologists decreased when they worked outside the OR for 2 months. This finding showed that the increase of SCE in anesthesiologists was reversible.
The health risks associated with occupational exposure to inhaled anesthetics are still debated. Chronic exposure to nitrous oxide and volatile anesthetics may result in adverse health effects in dental and OR personnel (19). Most of the published cytogenetic data in humans for anesthetic gases are on chromosome aberration and SCE. However, most of the human cytogenetic studies examined divinyl ether, fluroxene, nitrous oxide, halothane, isoflurane, enflurane, nd sevoflurane. Published data of these drugs in animals or in patients are clinical or laboratory evidence of organ toxicity after anesthesia (1,6–9,20–22). NIOSH recommends a 2 ppm threshold value for all volatile anesthetics and 25 ppm for nitrous oxide. The measurement levels in our study and eastern European university hospitals were above these limits (23–25).
SCE analysis in peripheral blood lymphocytes is a well established technique aimed at evaluating human exposure to toxic agents. SCEs are interchanges between DNA replication products at apparently homologous loci. Although the precise molecular mechanisms underlying SCE formation are not fully understood, it has been suggested that they reflect either DNA damage or DNA repair or both (15).
Hoerauf et al. (8) conclude that exposure to even small concentrations of waste anesthetic gases may result in an increased frequency of SCE in OR personnel. This genetic damage is also comparable with smoking 11-20 cigarettes per day. Sardas et al. (6) found an increased frequency of SCE in 18 anesthesiologists (smokers and nonsmokers) exposed to unknown amounts of halothane and nitrous oxide compared with 18 controls. However, they reported that a significant difference in the mean number of SCEs was not found between smoking and nonsmoking anesthesiologists. We excluded smoker subjects from our study and found significant increases of SCE in 25 anesthesiologists compared with 25 controls. This outcome is associated with our poorly equipped ORs (not having a central high-flow scavenging system and low leakage anesthesia machines, and not having facilities to use low-flow and closed-circuit anesthesia). Hobbhahn et al. (10) and Bozkurt et al. (11) reported that long-term occupational exposure to waste anesthetic gases may not result in an increased risk of genetic damage if an OR has sufficient air conditioning, a central high-flow scavenging system, low leakage anesthesia machines, and well maintained equipment. Hoerauf et al. (8) suggested that two major points should be considered when evaluating the current situation of occupational risks of inhaled anesthetics. First, following the recommendations based on the epidemiological data to reduce health risks by minimizing occupational exposure, the working environment can be improved technically, possibly resulting in small concentrations of waste anesthetic gases; and, second, halothane, which is classified as potentially embryotoxic, can be substituted with isoflurane. In our cases halothane was substituted with sevoflurane.
This is the first study of whether the increases of SCE values of anesthesiologists resulting from exposure to waste anesthetic gases is reversible. Erol et al. (26) reported that the frequency of chromosome aberrations, but not SCE, was increased after ionizing radiation exposure in cardiology laboratory workers. They also reported that these effects were reversible because after 2 months of a nonradiation period there were no chromosomal aberrations. Karab
yk et al. (22) reported that the comet assay of peripheral blood lymphocytes of patients during anesthesia with isoflurane or sevoflurane indicated the highest genotoxic effect at 120 minutes of anesthesia in patients undergoing lower abdominal minor surgery. This increased genotoxic effect began to decrease in the postoperative period and the cells were able to completely repair the induced DNA damage on the fifth postoperative day.
In this study, we found that the increase of SCE in lymphocytes of the anesthesiologists was reversible. We think that this outcome is associated with 2 months spent away from waste anesthetic gases. During this period, DNA may repair its own damage. However, the lifespan of lymphocytes is generally less than 2 months. Moreover, the mechanisms underlying SCE formation are not fully understood. It is thought that SCE formation reflects either DNA damage or repair or both. SCE also occur as a normal feature of cell division during DNA replication. However, we suggest that not only should OR equipment be technically improved but also that anesthesiologists should periodically minimize occupational exposure to waste anesthetic gases.
Unfortunately there are some shortcomings of the study. We could not use a direct reading instrument to measure the occupational exposure of anesthesiologists to inhaled anesthetics. On the other hand, SCE we measured were not specific to a particular anesthetic. All anesthesiologists were exposed to both sevoflurane and nitrous oxide at the same time. However, the levels of occupational exposure to sevoflurane and nitrous oxide we measured in our ORs were above the threshold values of NIOSH. Further studies should be performed in personnel solely exposed to sevoflurane or nitrous oxide.
In conclusion, this prospective study demonstrates that the increase of SCE in anesthesiologists exposed to increased environmental concentrations of waste anesthetic gases such as sevoflurane and nitrous oxide is reversible if they work free from exposure for 2 months.
We thank Muammer Erdol and Sevil Yilmaz for their technical assistance, and Eczacibasi- Baxter for technical support.
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Footnotes
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Accepted for publication December 21, 2005.
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References
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Abstract
Introduction
