Carbachol

INHALATION OF COLD AIR through the mouth or nose induces an immediate increase in airway resistance in normal human subjects (1-5) and in animals. (6-8) The bronchomotor response in asthmatic human subjects (2,9,10) and in sensitized animals (7) is markedly accentuated. However, in order for the cold challenge to induce a bronchospasm, the subject must either hyperventilate subfreezing air (-5[degrees]C to -40[degrees]C) through his or her mouth, (1,2,9) to push moderately fresh air (+8[degrees]C to +15[degrees]C) directly into the upper airways, (6,7) or he or she must breathe subfreezing air (-5[degrees]C to -10[degrees]C) quietly through the nose. (5,10)

To date, there have been no studies in which the respiratory consequences of protracted occupational exposure to a cold environment in humans or animals have been analyzed. In the literature, we found data only on changes in respiratory function in long-time residents of the Canadian Arctic (11) and in healthy Indian males from the tropics living in the arctic region. (12) The results of both studies suggest that inhalation of cold air may be a primary factor in initiating chronic obstructive lung disease. Our experience with human deep-sea diving (13)--an activity that markedly increases respiratory heat loss and promotes bronchospasm and respiratory tract secretions--reveals that professional divers often present chronic airflow limitation associated with bronchial hyperresponsiveness to cholinergic agonists. These observations prompted us to examine the possibility of an occupational disease caused by long-time exposure to cold air in the workplace.

A group of subjects engaged by a hospital administration to prepare meals in cold stores was studied during their 1st year of exposure to a cold environment (+10[degrees]C to +3[degrees]C). We focused our attention on changes in baseline lung function, as well as on bronchomotor responses to a cholinergic agonist and to nasal inhalation of cold air. These data were compared with results for a control group of individuals who were engaged at the same time, but who worked at normal room air temperature (+22[degrees]C).

Subjects and Method

Study population. Eighteen subjects were studied. Six individuals (2 females and 4 males) constituted the control group and 12 (6 females and 6 males) worked in the cold stores. The subjects had never worked in a cold environment. The mean ages and weights of the 2 groups were not statistically different (age: 44 [+ or -] 2 yr vs. 47 [+ or -] 2 yr; weight: 68 [+ or -] 6 kg vs. 67 [+ or -] 5 kg for controls vs. subjects, respectively). Given that their occupational activities were linked to meal preparation, the subjects were nonsmokers or mild smokers (< 5 cigarettes/day). Their health histories are documented in the Results section. Subjects were told only that they would have the benefit of a medical check-up, including lung function tests and a questionnaire evaluated by a practitioner, before beginning their new job, and again 6 mo and 12 mo thereafter. They were not informed of the potential risks of a cold environment. Indeed, many of the subjects had previously worked under such conditions in our facility, and we felt that giving too much information might distort the reporting and interpretation of clinical symptoms.

Occupational activities of subjects exposed to cold. The room air temperature and relative humidity in the workplace were measured continuously. Frequent veterinary inspections ensured air quality; verification was provided that the environment was free of pollutants in the form of gases, molds, and allergens. The days and hours of exposure for each subject were similar, and all workers were exposed concurrently for 1 yr, commencing in October. The subjects worked 6 days per week, for 6 consecutive hours, preparing tray meals in large rooms flushed by an air stream at +10[degrees]C (relative humidity [RH] = 50-60%). Moreover, their daily tasks required them to remain for several 30- to 60-min periods in refrigerators where the ambient temperature was +3[degrees]C (RH = 48%). The mean daily duration of expo sure at +3[degrees]C was 110 min for each individual. Thus, these workers spent approximately 25% of their work day in an environment of +3[degrees]C. Although we had no information on the breathing rates of subjects in the refrigerators, observation revealed that their work level was relatively low and did not oblige them to carry heavy loads. In Marseilles, the mean outdoor temperature throughout the year is +20[degrees]C (winter: +12[degrees]C; summer: +26[degrees]C). Thus, we considered that the occupational minimal exposure temperature for our workers was rather cold.

The study questionnaire included antecedents of pulmonary and cardiovascular disease, ear-nose-throat (ENT) diseases (e.g., rhinitis, sore throat), and allergic manifestations (e.g., asthma, spasmodic tracheitis, hay fever, rash). We also asked the subjects about their activities outside the workplace--particularly any activity that might involve contact with irritants or allergenic agents. One subject who worked in the cold environment was eliminated from the study because his 6-mo questionnaire revealed that he frequently manipulated autopolymerized varnishes. Therefore, only 11 workers were studied.

Lung function tests. Spirometry was performed with a pneumotachograph (Masterscreen [Jaeger, Germany]) calibrated daily with a 5-l syringe. Forced expiratory volume in 1 sec (FE[V.sub.1.0]), vital capacity (VC), FE[V.sub.1.0]/VC ratio, and mean forced expiratory flow measured between 25% and 75% of VC (FE[F.sub.25-75%]) were calculated from maximal expiratory maneuvers. Test results were accepted if the difference between the best and the second-best tests was less than 5%--whichever was larger. Flow rates were expressed as a percentage of predicted values. (14) Central airway resistance ([R.sub.aw]) and specific conductance (S[G.sub.aw]) were measured in a pressure body plethysmograph (Masterlab [Jaeger, Germany]).

Airway response to carbachol. On separate days, a dose-response curve was obtained by plotting the value of S[G.sub.aw] against cumulative doses of carbachol in the range of 200 to 2,000 [micro]g. The sensitivity to carbachol was defined as the dose that induced a 50% decrease in S[G.sub.aw] (D50), and the reactivity was the slope of the S[G.sub.aw] vs. carbachol dose relationship. Carbachol challenge was repeated at 6 and 12 mo in 5 control subjects and in 7 individuals who worked in cold stores. A low D50 value and a high reactivity are common in asthmatic patients.

Airway response to nasal cold air breathing. The experimental setup was the same as has been used previously. (5,10) Each subject wore a mask firmly fitted to his or her nose and quietly breathed either room air or cold, dry air (-5[degrees]C, RH = 0.3%) via a 2-way valve, which prevented contamination of inspired air by expired gas. Before the trial period (i.e., during breathing of room air) we measured control values of interrupting resistance ([R.sub.int]) (Masterscreen [Jaeger, Germany]). [R.sub.int] values were measured every 2 breaths during single 100-ms occlusions at mid-tidal expiratory flow. [R.sub.int] measurements were repeated at 2, 5, and 10 min during the 10 min period of inspiring cold air, and then at 1 and 5 min after cold air inhalation had stopped. To measure [R.sub.int] during nasal inhalation of cold air, we asked each subject to breathe 1 time via the nose and 2 times via the mouth to maintain nasal stimulations between 2 successive [R.sub.int] measurements. Cold tests were repeated at 12 mo in the same individuals who underwent carbachol challenge (i.e., 5 controls and 7 individuals who worked in cold stores).

Statistical analysis. After the normality of data distribution was verified with the Kolmogorov-Smirnov test, we used a 1-way repeated-measures analysis of variance (ANOVA) to test for differences in the effects of exposure time (measurements at 6 mo in some individuals and at 12 mo in all subjects) on baseline lung function, dose response to carbachol, and airway response to nasal cold air breathing, by examining the changes in each individual. The chi-square test allowed us to compare symptom scores at the time of inclusion into the study, at 6 mo into the study, and at 12 mo into the study.

Results

Control group. At the beginning of the study, medical questionnaires revealed that 3 subjects had been asthmatics during their childhood. However, no subjects had presented pulmonary symptoms or rhinitis during the most recent 5 yr. Twelve months following the study, no symptoms of asthma or equivalent pathology were reported; however, a few more months after the study 1 individual suffered from obstructive rhinitis. At time zero, an airway obstruction was measured in 1 subject ([R.sub.aw] = 4.1 cm water ([H.sub.2]O)/I * sec; FE[F.sub.75-25%] = 69% of theoretical value) and was reversible with inhalation of a [beta]2 agonist ([DELTA][R.sub.aw] = -54%). The D50 to carbachol was low (500 [micro] g) in only 1 individual, who had antecedent symptoms of asthma. The data in Tables 1 and 2 show that there were no significant changes in baseline lung function or in airway responses to carbachol and cold air breathing throughout the 12-mo study.

Subjects exposed to cold environment. At time zero--before occupational exposure to cold--the questionnaires revealed that 1 subject was asthmatic and 2 suffered from hay fever. Thus, ENT and respiratory symptoms were found in 3 of 11 subjects. With the exception of the asthmatic subject, examinations at 6 mo indicated that ENT diseases were present in 3 of 11 individuals, and spasmodic tracheitis was present in 1 other subject (i.e., symptoms in 5 of 11 subjects). At 12 mo, 4 subjects suffered from rhinitis and sore throat and 2 others suffered from spasmodic tracheitis (symptoms in 7 of 11 subjects). The chi-square test indicated that the symptom scores at 6 mo and 12 mo were not significantly higher than at time zero. It must be noted that the symptoms were mild and that no subject lost work time during the study.

In Table 1 are shown the mean values of pulmonary function data obtained at 0, 6, and 12 mo in the 11 individuals who were exposed to the cold environment. At time zero, a modest airflow limitation was present in 4 of 11 subjects ([R.sub.aw] = 3.7 [+ or -] 0.6 cm [H.sub.2]O/l * sec; FE[F.sub.25-75%] = 74 [+ or -] 11%), and 2 of them had ENT and asthmatic symptoms. We tested these 4 "obstructive" individuals for airway response to a [beta]2 agonist (a 50% decrease in [R.sub.aw] was measured in 2 of 3 individuals), but not for reactivity to carbachol, which was explored only in the 7 remaining subjects. At 6 and 12 mo, we noted a modest but significant decrease in FE[V.sub.1.0] and FE[F.sub.25-75%] and an increase in the mean baseline Raw value. Plotting FE[V.sub.1.0] and [R.sub.aw] values--measured at 0, 6, and 12 mo--against time revealed significant correlations (Fig. 1); however, no correlation existed between FE[F.sub.25-75%] and time.

[FIGURE 1 OMITTED]

In 7 of 11 individuals who had normal lung function at time zero, the carbachol challenge was repeated at 0, 6, and 12 mo, and the airway response to cold air breathing was measured at 0 and 12 mo. Table 2 shows that the D50, the reactivity to carbachol, and the airway response to nasal inhalation of cold air were not significantly different in the 2 groups at time zero and that no change in these variables occurred in the control group after 12 mo. On the other hand, in subjects working in the cold environment we measured (after 6 mo, then after 12 mo of cold exposure) a significant increase in reactivity to carbachol; the changes were correlated with time (Fig. 2). No significant variation in sensitivity to carbachol was noted (Table 2). As is shown in Figure 3, the enhanced airway reactivity to carbachol after 12 mo of cold exposure was correlated significantly (r = .940; p = .001) with the corresponding increase in baseline Raw value. The significance of the relationship persisted (r = .801; p < .01)--even if we deleted results for the asthmatic subject, who had the highest reactivity at 12 mo. No significant changes in airway response to nasal inhalation of cold air were noted after 12 mo of cold exposure (Table 2).

[FIGURE 2 OMITTED]

Therefore, after 12 mo of cold exposure, airway obstruction was measured in 6 subjects, 5 of whom had an increased reactivity to carbachol. It must be emphasized that 2 of these workers had no ENT or pulmonary symptoms, and that no further changes in pulmonary function were found in the 3 subjects who suffered from ENT diseases or spasmodic tracheitis.

[FIGURE 3 OMITTED]

Discussion

The present study showed that 12 mo of daily occupation in a cold environment, with a requirement to spend 25% of that time at +3[degrees]C, increased the frequency of ENT and respiratory symptoms (rhinitis, sore throat, spasmodic tracheitis), and mainly elicited a modest but significant airway obstruction associated with bronchial hyperresponsiveness to a cholinergic agonist (carbachol). Decreased expiratory flow rates, elevated airway resistance, and increased bronchomotor response to carbachol occurred after 6 mo of cold exposure; further changes in lung function were noted at 12 mo. Thus, it seems that an altered respiratory function develops in parallel with duration of cold exposure. However, we cannot affirm the occurrence of cold-induced bronchial hyperresponsiveness in subjects who worked in the cold environment, inasmuch as the D50 to carbachol did not vary significantly with time. It is possible that the progressive increase in bronchial reactivity may result only from the enhanced baseline [R.sub.aw] value and not from any changes in the intrinsic properties of airway smooth muscle, as confirmed by the absence of any D50 change during the 1-yr study.

The changes in pulmonary function, as well as the increased frequency of ENT and respiratory symptoms, throughout our 1-yr study cannot be attributed to changes in smoking habits (the subjects were nonsmokers or very mild smokers) or to an occupational exposure to toxic or allergic agents (the environmental air quality was controlled by veterinary inspection). Moreover, given that the RH inside the refrigerators (50-60%) was slightly higher than that measured throughout the year in Marseilles (45-50%), we do not suspect any effect of dry air on the respiratory tract (ignoring the dependence of humidity on temperature).

Other mechanisms could be responsible for the changes in pulmonary function observed in response to breathing cold air. The progressive airflow limitation may result from the increased frequency of episodes of ENT disease (rhinitis and sore throat) and acute bronchitis and/or the repetition of cold-induced reflex bronchoconstriction. A study by Trigg et al. (15) showed that lower airway inflammation was present in atopic and nonatopic normal subjects with colds; however, it is difficult to attribute airway obstruction and increased airway reactivity to frequent viral infections. Changes in physiological variables throughout the 1-yr study appeared to be independent of the occurrence of ENT or pulmonary diseases. Cold-induced alteration of lung function in a relatively small number of subjects does not suggest that viral infections alter pulmonary function. In previous studies of both healthy (5) and asthmatic subjects, (10) we showed that quiet breathing of cold air through the nose elicited a vagal reflex bronchospasm mediated through the activation of trigeminal afferents. The airway response did not persist after the cold challenge ended, and there was no potentiation of the cold-induced bronchospasm when the cold tests were repeated 2 or 3 times. Bronchospasm, with excessive upper respiratory tract secretions, has been reported in divers who breathe a cold (+10[degrees]C) helium-oxygen mixture under high pressure. (3,4,16) Repeated episodes of vagal bronchospasm and associated hypersecretion of tracheobronchial mucus may limit airflow.

The mechanism of cold-induced bronchoconstriction in subjects working in cold stores may also be a reflex elicited by the activation of nerve endings in the face skin (17) and/or the stimulation of trigeminal afferents from the nose. (5,18) We do not suspect the activation of laryngeal (6,19) or tracheobronchial vagal afferents, (7,8) the roles of which in cold-induced reflex bronchospasm have been well documented. Indeed, the stimulation of these afferents requires lowering the temperature in the central airways--a condition realized during hyperventilation of freezing air, (20) but not in the present study.

In this study, the 12-mo exposure to cold did not significantly modify the bronchomotor response to nasal cold challenge, despite the occurrence of an enhanced airway responsiveness to a cholinergic agonist. We have previously reported--in sensitized rabbits (7) as well as in asthmatic individuals (10)--a marked increase in the cold-induced bronchoconstrictor reflex elicited by the activation of tracheal (7) or nasal afferents. (10) In these hyperreactive subjects, a nonspecific increase in airway smooth muscle response seemed to occur for any test agent that activated the respiratory afferents. The absence of changes in cold-induced bronchospasm in subjects who worked in cold stores corroborates our hypothesis that increased airway response to carbachol in these individuals did not indicate occupational asthma, but resulted, instead, from elevated airway resistance.

In conclusion, we maintain that altered pulmonary function in workers in cold stores should be assessed for reversibility.

Submitted for publication January 26, 2001; revised; accepted for publication June 29, 2001.

Requests for reprints should be sent to Pr. Y. Jammes, Laboratoire de Physiopathologie Respiratoire EA 2201, Universite de la Mediterranee, Institut Jean Roche, Faculte de Medecine, Bd. Pierre Dramard, 13916 Marseille cedex 20, France.

E-mail: jammes.y@jean-roche.univ-mrs.fr

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