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Canadian Journal of Anesthesia 47:350-353 (2000)
© Canadian Anesthesiologists' Society, 2000
Brief Report

Does chronic occupational exposure to volatile anesthetic agents influence the rate of neutrophil apoptosis?

Yukiko Goto, MD*, John Gallagher , FFMMI AFOM{dagger}, Noel Fanning , MB FRCSI{ddagger}, Jianghuai Wang , MD PhD{ddagger}, Sean McCusker , BSc DIP OCC HYG MIOH§, Paul Redmond , BSc MCH FRCSI FRCS{ddagger} and George Shorten , MD PhD*

* From the Department of Anaesthesia and Intensive Care Medicine,
{dagger} Occupational Medicine,
{ddagger} Department of Surgery, Cork University Hospital, University College Cork, Cork, Ireland, and
§ McCusker Environmental, Co. Clare, Ireland.

Address correspondence to: Prof. G. Shorten, Department of Anaesthesia and Intensive Care Medicine, Cork University Hospital, University College Cork, Cork, Ireland. Phone: 353-21-546400 (Ext: 2566); Fax: 353-21-546434; E-mail: shorteng{at}shb.ie


   Abstract
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 Abstract
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Purpose: The purpose of this preliminary investigation was to determine whether the rate of neutrophil apoptosis in health care workers is influenced by exposure to volatile anesthetic agents.

Methods: Percentage neutrophil apoptosis (Annexin-V FITC assay) was measured in health care workers (n = 20) and unexposed volunteers (n = 10). For the health care workers, time weighted personal exposure monitoring to N2O, sevoflurane and isoflurane was carried out.

Results: The sevoflurane and isoflurane concentrations to which health care workers were exposed were less than recommended levels in all 20 cases. Percent apoptosis was less at 24 (but not at one and 12) hr culture in health care workers {50.5 (9.7)%; P = 0.008} than in unexposed volunteers {57.3 (5.1)%}.

Conclusion: Inhibition of neutrophil apoptosis at 24 hr culture was demonstrated in health care workers chronically exposed to volatile anesthetic agents. Exposure was well below recommended levels in the both scavenged and unscavenged work areas in which the study was carried out. Further study is required to assess the effect of greater degrees of chronic exposure to volatile anesthetic agents on neutrophil apoptosis.


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HEALTH care workers (HCW) who work in unscavenged operating theatres or in recovery rooms may be chronically exposed to low concentrations of volatile anesthetic agents. It has been reported that such chronic exposure influences immune status.1 Chronic exposure to volatile anesthetic agents has been implicated in increased incidence of carcinogenesis and teratogenesis, both processes in which apoptosis has a fundamental role.2 Apoptosis is a process of genetically regulated cell deletion, which is integral to normal and pathological processes including embryological development and cell proliferation.3,4

It is hypothesized that chronic occupational exposure of health care workers to low concentrations of volatile anesthetic agents may inhibit the rate of neutrophil apoptosis. To test this hypothesis, we compared the rate of neutrophil apoptosis in unexposed volunteers and HCWs who work in scavenged and in unscavenged areas.


   Methods
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With institutional ethical approval, and informed consent from each, 30 subjects were studied: healthy unexposed volunteers (n = 10), HCWs who work in scavenged operating theatres (scavenged group) (n = 12) and in unscavenged operating theatres or recovery areas (unscavenged group) (n = 8). Volunteers and HCWs were excluded if they had undergone general anesthesia within the previous three months.

Personal gas exposure monitors were placed on the shoulders or cap of each HCW for one day from 8:30 am to 2:00 pm. This monitoring was carried out using charcoal (volatile anesthetic agents) and Drager diffusion (nitrous oxide) sampling tubes followed by thermal desorption and quantitative infra-red analysis. The latter procedures were carried out in a National Measurement Accreditation Scheme (NAMAS) accredited laboratory.5

Heparinised venous blood (9 ml) was withdrawn from each volunteer and from the HCWs at 2:00 pm from personnel who had been working in the operating theatre or recovery area since 8:30 am. Neutrophils were isolated by sequential sedimentation in 6% Dextran (mol.wt. 520,000, Sigma, UK) in sodium chloride 0.9% for 45 min, centrifugation in Ficoll-Paque (Pharmacia LKB Biotechnology, Piscataway, NJ) at 300 g for 30 min to pellet granulocytes and remaining erythrocytes, and centrifugation of the resuspended pellet over an 81% isotonic Percoll (Sigma, UK) gradient at 350 g for 15 min to pellet erythrocytes. The diffuse layer at the interface containing neutrophils was harvested, washed, resuspended in medium and counted. Preparations of isolated neutrophils were maintained in RPMI1640 supplemented with 10% autologous platelet poor plasma, and L-glutamine, penicillin, streptomycin and amphotericin B, at a concentration of 1 x 106•ml–1 in polypropylene tubes at 37°C in a humidified CO2 incubator. After 1, 12 and 24 hr in culture, neutrophils (0.5 x 106 •ml–1 cells) were dual stained with propidium iodide (sigma, UK) (final concentration 10 µg•ml–1) and Annexin V-FITC (Bender MedSystems, Austria) (final concentration 0.6 µg•ml–1).6 Neutrophils were analysed on a Becton Dickinson FACScan flow cytometer equipped with Cell Quest (FigureGo). Ten thousand events were collected while gating on physical parameters to exclude cell debris.



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  		FIGURE The percentage apoptosis was assessed by dual staining with Annexin V-FITC and propidium iodide at one, 12 and 24 hr culture. Data are reported as the percentage of Annexin V-FITC positive and propidium iodide negative cells.

Propidium iodide stains the cell when cell membrane has been damaged. Thus, cells which are stained with only Annexin V indicates apoptotic condition in the early phase where the cell membrane is still intact.

The percentage in the upper left quadrant indicates the percentage of Annexin V-FITC positive viable neutrophils.

 
Data relating to rate of apoptosis were analyzed with unpaired, one tail Student's t tests.


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The age and weight of unexposed volunteers and HCWs were 32.3 (9.7) yr, 38.8 (8.5) yr respectively and 74.6 (11.4) kg, 65.3 (8.4) kg respectively. The proportion of male patients was greater in the unexposed volunteers (8 / 10) than in HCWs (4 / 20).

The study periods were selected as representative of a normal working day, therefore no attempt was made to delineate the proportion of each study period during which subject was or was not exposed to anesthetic.

The results of personal gas monitoring are shown in Table IGo. Time weighted exposures to anesthetic gases were similar in the scavenged and unscavenged groups.


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  		TABLE I Time weighted average exposures (Mean ± SD) and recommended exposure limits
 
Rates of neutrophil apoptosis in HCWs and unexposed volunteers were similar after one and 12 hr culture (Table IIGo). At 24 hr culture, compared with unexposed volunteers {57.3 ± 5.1%}, percent apoptosis was less in HCWs {50.5 ± 9.7%; P = 0.008}.


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  		TABLE II Percentage neutrophil apoptosis. Data are mean ± SD. * P < 0.05 compared to volunteers.
 

   Discussion
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In this study, we demonstrated a reduction of the rate of neutrophil apoptosis only at 24 hr in culture, in comparing HCWs who are occupationally exposed to volatile anesthetic agents with unexposed volunteers. These findings must be interpreted in the light of the small number of subjects studied and the relatively low degrees of exposure to which the HCWs were subjected. Several other investigators have reported degrees of environmental contamination with anesthetic gases that are far in excess of those reported in this study, and of the occupational exposure standards.1 A power analysis was not performed in this study because these data are preliminary and represent an attempt to identify whether a full investigation of the effects of volatile agents on apoptosis is warranted. Furthermore, it is not clear why the moderate effect seen at 24 hr in culture was not present earlier.

Recently, the role of apoptosis in many physiological and pathophysiological processes has been demonstrated.3,4 Normally, apoptosis of neutrophils is responsible in part for termination of the inflammatory response to tissue injury (such as surgery). Although the functional state of neutrophils, in which apoptosis has been inhibited, has not been defined, it is possible that an augmented or prolonged inflammatory response may result. One possibility is if apoptosis is inhibited, neutrophils may follow the alternative death pathway, necrosis, which would be associated with a greater inflammatory response.

These preliminary results suggest that chronic occupational exposure to volatile anesthetic agent may influence neutrophil apoptosis. Further investigation is warranted to assess the effect of chronic exposure to greater concentrations of anesthetic gases and the implication of such an effects on regulation of the inflammatory response.


   Footnotes
 
These studies were carried out with the support of Abbott Laboratory Ireland, Inc.

Accepted for publication December 5, 1999.


   References
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 Abstract
 Introduction
 Methods
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 Discussion
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1 Peric M, Vranes Z, Marusic M. Immunological disturbances in anaesthetic personnel chronically exposed to high occupational concentrations of nitrous oxide and halothane. Anaesthesia 1991; 46: 531–7.[Medline]

2 Guirguis SS, Pelmear PL, Roy ML, Wong L. Health effects associated with exposure to anaesthetic gases in Ontario hospital personnel. Br J Ind Med 1990; 47: 490–7.[Medline]

3 Pratt RM, Martin GR. Epithelial cell death and cyclic AMP increase during palatal development. Pro Natl Acad Sci USA 1975; 72: 874–7.[Abstract/Free Full Text]

4 Raff MC. Social controls on cell survival and cell death. Nature 1992; 356: 397–9.[Medline]

5 Jerome SM. Environmental radioactivity measurement intercomparisons in the UK. Int J Rad Appl Instrum [A] 1992; 43: 191–9.

6 Homburg CHE, de Haas M, van dem Borne AEG, Verhoeven AJ, Reutelingsperger CP, Roos D. Human neutrophils lose their surface FcRIII and acquire annexin V binding sites during apoptosis in vitro. Blood 1995; 85: 532–40.[Abstract/Free Full Text]




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