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ANESTHETIC PHARMACOLOGY

High-Level, but Not Low-Level, Occupational Exposure to Inhaled Anesthetics Is Associated with Genotoxicity in the Micronucleus Assay

Gunther Wiesner, MD^*, Klaus Hoerauf, MD, Klaus Schroegendorfer, MD, Pawel Sobczynski, MD, Marion Harth, MD^*, and Hugo W. Ruediger, MD^§

*Department of Anesthesiology, University of Regensburg, Germany; Department of Anesthesiology, University of Vienna, Austria; Department of Anesthesiology, University of Poznan, Poland; and §Department of Occupational Medicine, University of Vienna, Austria

Address correspondence and reprint requests to Gunther Wiesner, MD, Department of Anesthesiology, University of Regensburg, Franz-Josef-Strauß-Allee 11, D-93053 Regensburg, Germany. Address e-mail to gunther.wiesner{at}klinik.uni-regensburg.de

	Abstract

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To minimize the possible health risks posed by waste anesthetic gases, the National Institute of Occupational Safety and Health (NIOSH) recommends exposure limits. We investigated the genotoxicity of a previously established occupational exposure exceeding these limits (high-level exposure) and of one within these limits (low-level exposure). Genotoxicity was assessed by the formation of micronucleated lymphocytes in 25 anesthetists and anesthetic nurses of an Eastern European (High-Level Exposure Group) and a German (Low-Level Exposure Group) university hospital. Each exposed group was compared with a group of nonexposed personnel of the same hospital. Compared with its Control Group, there was an increased fraction of micronucleated lymphocytes per 1000 binucleated cells in the High-Level Exposure Group (median 14.0, range 9.0–26.7 vs median 11.3, range 3.2–19.4; P < 0.05) but not in the Low-Level Exposure Group (median 9.8, range 4.2–20.0 vs median 10.5, range 5.0–20.5). We conclude that a high-level exposure to inhaled anesthetics is associated with an increase in chromosome damage, and measures are recommended to decrease exposure levels. As evidenced by the formation of micronucleated lymphocytes, the threshold values recommended by NIOSH appear to be safe.

Implications: A high level of occupational exposure to inhaled anesthetics is associated with genotoxicity (as defined by formation of micronucleated lymphocytes), whereas a low-level exposure (within National Institute of Occupational Safety and Health limits) is not.

	Introduction

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The health risks associated with occupational exposure to inhaled anesthetics are still debated. Epidemiological investigations in dental and operating room (OR) personnel exposed to nitrous oxide and volatile anesthetics revealed that there may be an increased risk for reproductive toxicity (1–5). To minimize the possible health risks, public health authorities recommend threshold values. The exposure limits recommended by the National Institute of Occupational Safety and Health (NIOSH) are 25 ppm nitrous oxide and 2 ppm for volatile anesthetics. The value for volatile anesthetics is reduced with the concomitant use of nitrous oxide (6).

With regard to reproductive toxicity, the genotoxicity of an occupational exposure to inhaled anesthetics is of major concern. In this study, genotoxicity was assessed by the formation of micronucleated lymphocytes in two groups of anesthetic personnel: one group with a high-level exposure to inhaled anesthetics exceeding these threshold values, and one group with a low-level exposure within these limits. The formation of micronucleated lymphocytes is an established cytogenetic procedure to assess chromosome damage in persons exposed to potentially genotoxic agents (7).

	Methods

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The study was performed at an Eastern European (High-Level Exposure Group) and a German (Low-Level Exposure Group) university hospital. Approval was obtained from both IRBs, and each participant gave written informed consent.

Twenty-five anesthetists and anesthetic nurses at the Eastern European and the German university hospital were studied. Each subject completed a questionnaire that asked about age, sex, smoking habits, acute and chronic disease, former or continued radiotherapy and chemotherapy, and the duration of exposure to waste anesthetic gases in the last month and year. At each hospital, 25 doctors and nurses not exposed to waste anesthetic gases and matched for age, sex, and smoking habits served as control groups.

The levels to which the anesthetic personnel were exposed are presented in detail elsewhere (8–10). In brief, exposure levels at the Eastern European university hospital, where most ORs were not equipped with scavenging and air-conditioning systems, were about 170 ppm nitrous oxide and 4 ppm of the volatile anesthetics halothane and isoflurane (8). At the German university hospital, which has a high standard regarding the removal of waste anesthetic gases, exposure levels were about 12 ppm nitrous oxide and <0.5 ppm of the volatile anesthetics isoflurane, sevoflurane, and desflurane (9,10).

After exposure levels were measured, blood samples for the micronucleus assay were obtained. All participants had been exposed to nitrous oxide and volatile anesthetics for at least 3 mo without interruption (excluding weekends). At the Eastern European university hospital, the study was done before measures to reduce exposure levels had been taken (8).

Five milliliters of whole blood was drawn from each subject ( NH4-Heparin Monovetten ; Sarstedt, Nuembrecht, Germany), and lymphocytes were isolated by density centrifugation ( Ficoll-Paque ; Pharmacia, Freiburg, Germany). Lymphocytes were washed twice with phosphate-buffered saline (Serva, Heidelberg, Germany) and cultured at 37°C for 72 h in 5 mL of chromosome medium 1A (Gibco, Vienna, Austria) that contained 10 mL/L phytohemagglutinin. After 44 h of incubation, 3 µg/mL cytochalasin B (Sigma, Deisenhofen, Germany) was added. After 72 h, the lymphocyte cultures were harvested, placed in a 0.075M potassium chloride solution for 8 min, fixed with methanol/glacial acetic acid (1:1 vol/vol) and put onto slides. The slides were dried and stained for 15 min in a 10 mg/L 4',6-diamidino-2-phenylindole (Serva, Heidelberg, Germany)/zinc chloride solution. Two thousand binucleated cells (BNC) per sample were scored blindly by the same reader for micronuclei, which had to meet the criteria defined by Fenech (7). The fraction of micronucleated lymphocytes per 1000 BNC (MN/1000 BNC) was calculated and used for data analysis. Cellular proliferation was evaluated by the mitotic index (MI), scoring the number of BNCs per 100 lymphocytes.

Normally distributed data are presented as mean ± SD. The comparisons between the two exposed groups regarding the duration of exposure and the comparisons between the exposed groups and their corresponding control groups regarding age and MI were performed by two-tailed, two-sample t-tests. Because MN/1000 BNC was not normally distributed, data are presented as median (range), and the comparisons between the exposed groups and their corresponding control groups were performed by the Mann-Whitney U-test. Nominal data were compared by ²-analysis. P < 0.05 was considered statistically significant. The SPSS for Windows , version 8.0, software package was used (SPSS Inc, Chicago, IL). By using an estimated effect size of 0.8, which was corrected for the Mann-Whitney U-test, the ß error level (11) was 0.2.

	Results

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The demographic data are presented in Table 1. There were no differences between exposed and control groups regarding age, sex, and smoking habits. No one suffered from significant acute or chronic disease, and no one had former or continuing radiotherapy or chemotherapy. In the exposed groups, the duration of exposure to waste anesthetic gases in the last month and in the last year did not differ from each other (Table 1).

View larger version (18K): [in this window] [in a new window]   Figure 1. Fraction of micronucleated lymphocytes in the High-Level Exposure Group of the Eastern European university hospital, the Low-Level Exposure Group of the German university hospital, and the corresponding Control Groups. The box represents the 25th–75th percentile, the line in the middle of the box is the median, the extended bars represent the 10th–90th percentile, and the circles represent values outside this range. *P = 0.012 vs the corresponding control group

	Discussion

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The micronucleus assay is an established cytogenetic end point that has been used for many years in the hazard identification of populations exposed to genotoxic agents. Micronuclei are generated by the loss of whole chromosomes or chromosome fragments at mitosis. Compared with other cytogenetic procedures, the micronucleus assay offers the advantage of rapid and easy analysis and the nonrequirement for metaphase cells (7,12). In 1996, Chang et al. (13) reported an increased micronucleus formation in pediatric anesthesia nurses with occupational exposure to nitrous oxide. Unfortunately, in their study, data on exposure levels are missing. The introduction to this study suggests that the nitrous oxide exposure was >25 parts per million—the threshold value recommended by NIOSH—and that there was also an exposure to volatile anesthetics (13). In veterinary surgeons exposed to 5.3 parts per million isoflurane (which is far above the limit recommended by NIOSH) and 12.3 parts per million nitrous oxide, the number of micronucleated lymphocytes was higher than in a nonexposed control group, although the difference failed to reach statistical significance (14). Nevertheless, both studies provide support for the result of our study, which shows that an occupational exposure to inhaled anesthetics above the limits recommended by NIOSH is associated with an increased formation of micronuclei.

By using other cytogenetic end points, such as chromosomal aberrations and sister chromatid exchanges, several investigators have observed genotoxic effects in anesthetic and OR personnel exposed to nitrous oxide and volatile anesthetics (15–17). Data on exposure levels in these studies are missing or at least incomplete. From their country of origin (India, Czechoslovakia, and Turkey), it may be assumed that they compare with those of our High-Level Exposure Group in Eastern Europe. Recently, the induction of sister chromatid exchanges by waste anesthetic gases was also demonstrated in a group of OR personnel with an exposure to 11.8 parts per million nitrous oxide and 0.5 parts per million isoflurane, i.e., within the limits of NIOSH (18).

Sister chromatid exchanges are interchanges between DNA replication products at apparently homologous loci. Their value in the risk assessment of populations exposed to potentially genotoxic agents is limited (12). In contrast, micronuclei result from chromosome breakage and, theoretically, there should be an association with cancer (19). Actually, in cancer patients before anticancer treatment, MN/1000 BNC was twice as high as in healthy controls (20). The occupational exposure of workers from industry and hospitals to the cytostatic drug cyclophosphamide, a known mutagen and carcinogen, led to an increase of micronucleated lymphocytes of 40% (21), which is slightly higher than the 25% increase in our study. In vitro, 10^-4M cyclophosphamide, which is a commonly used positive control (22), produced an increase of about 150% (23). However, the implications of an increased number of micronucleated lymphocytes in occupationally-exposed persons are not entirely clear. There is a large interindividual variability (Fig. 1) (7,22) and a minor intraindividual variability (24). In women, the micronucleus frequency is 1.4 times more than in men, and in people 60 years of age, it is twice as high as in people 30 years of age (7). A prospective study on the predictive value of micronuclei for cancer risk assessment failed to establish an association between micronucleus frequency and cancer incidence. The authors assumed that this negative result was caused by the limited number of cancer cases observed and by variations in the micronucleus assays applied (19). Prospective studies on the risk assessment of reproductive toxicity by the micronucleus assay are missing. In conclusion, the micronucleus assay has its value in the proof of an exposure to genotoxic agents in a group of persons, whereas its predictive value for health risk assessment remains to be established (12,19).

A limitation of our study, as of all other studies on this topic, is that it is not clear whether the observed genotoxic effect (measured as an increased MN/1000 BNC in the High-Level Exposure Group) is attributable to the exposure to nitrous oxide, volatile anesthetics, or a mixture of both. Another limitation, with regard to the volatile anesthetics, is that the High-Level Exposure Group was exposed to halothane and isoflurane, whereas the Low-Level Exposure Group was exposed to isoflurane, sevoflurane, and desflurane. Currently, no data conclusively demonstrate that halothane is a more genotoxic drug than the other volatile anesthetics. This also applies to nitrous oxide, although nitrous oxide may interfere with DNA synthesis by irreversibly oxidizing the cobalt atom of vitamin B₁₂ and reducing methionine and thymidylate synthetase activity (25). Further studies should be performed in personnel solely exposed to nitrous oxide or (single) volatile anesthetics.

Another limitation of our study is that we did not thoroughly evaluate whether the anesthetic personnel in the Eastern European and the German university hospital had been exposed to other potentially genotoxic agents. Although it was not obvious, there might have been differences in the exposure to radiation or to chemicals, such as ethylene oxide. Furthermore, there is a general lack of reliability in the data obtained from questionnaires.

Some of these limitations would have been of minor importance in a follow-up study on the anesthetic personnel of the Eastern European university hospital before and after measures to reduce occupational exposure had been taken (8). Although these measures reduced occupational exposure, the levels, especially of nitrous oxide, were still far above the limits recommended by NIOSH (8). Therefore, the measurement of micronucleated lymphocytes was not repeated.

Regarding the demographic data in Table 1, it is striking that more women and smokers were investigated in the Eastern European university hospital than in the German university hospital. This should not pose a problem, because each group of exposed anesthetic personnel was compared with a nonexposed group of personnel of the same hospital, and these groups did not differ from each other. This procedure furthermore takes into account that the living conditions, e.g., the pollution, may be different in Eastern Europe and Germany. In addition, in contrast to age and sex, smoking is not an important confounding factor for the induction of micronuclei (22).

To summarize, this investigation provides evidence that a high-level, but not a low-level, occupational exposure to inhaled anesthetics is associated with an increase in chromosome damage. Therefore, measures to effectively decrease exposure levels are recommended. With regard to the induction of micronuclei, the threshold values recommended by NIOSH appear to be safe.

	References

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999