INTRODUCTION

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The effect of ozone exposure on respiratory health has been demonstrated in animal and clinical human studies, as well as in epidemiological investigations conducted among children and adults (1). Among young adults, short-term exposure to ozone has been associated with increased airway inflammation, respiratory symptoms, and decrement in lung functions. These decrements varied according to the type of exposure and populations studied but were in the range of 0.2 to 4.5 ml/ ppb for FEV₁ and 0.5 to 4.7 ml/ppb for FVC (1, 2). The biological effect of ozone has been attributed to its ability to directly cause oxidation or peroxidation of biomolecules, via free radical reactions (2). Ozone would induce lipid peroxidation of polyunsaturated fatty acids (PUFAs) of lung cells, thus producing cyclooxygenase, a product of arachidonic acid (AA) (3), which in turn would stimulate the neural receptors of the upper airway, leading to acute pulmonary decrements (4). However, the mechanisms of the biological effects of ozone are still unclear (5).

Vitamin E is the principal defense against oxidant-induced membrane injury in human tissue (6). Ozone and vitamin E have opposite effects on the immune system, thus vitamin E could protect people against the adverse effects of photo-oxidant exposure by trapping and neutralizing free radicals (6). Elsayed and coworkers (7) observed that concentrations of vitamin E increased significantly in lungs of animals tested after exposure to ozone, suggesting a protective response. Beta carotene, which accumulates in tissue membranes, is known as a free-radical scavenger that can react directly with peroxyl free radicals, thereby serving as an additional lipid-soluble antioxidant (6). Vitamin C contributes to antioxidant activity through several mechanisms. It does so primarily by scavenging O₂, but it also contributes to the regeneration of membrane-bound oxidized vitamin E, allowing it to function again as a chain-breaking antioxidant (6). Dietary intake of these vitamins may therefore play a role in the host's defense against oxidative lung damage.

The metropolitan area of Mexico City experiences significant air pollution problems, particularly high ambient levels of ozone. Adverse effects of ozone exposure on the respiratory tract of residents of Mexico City have been observed in various epidemiological studies (8). We therefore conducted a study to determine if the acute effects of ozone on lung functions could be prevented by antioxidant supplementation among individuals with a high occupational exposure to ozone.

	METHODS

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Study Population

We enrolled 47 male shoe-cleaners working in the central area of Mexico City. These workers were recruited from the Workers' Union to participate in a randomized trial to determine the potential protective effect of antioxidant supplementation on respiratory health. Eligibility criteria were (1) being a male age 18 to 58 yr; (2) being a nonsmoker or a light smoker (fewer than 5 cigarettes per day); (3) working outdoors in the central area of Mexico City within 2 km of the monitoring station of Merced. The protocol was reviewed and approved by the ethics committee of the National Institute of Public Health (Mexico).

At baseline, interviewers applied a questionnaire to ascertain general information on participants' sociodemographic characteristics, household characteristics, and respiratory health. A semiquantitative food-frequency questionnaire was used to determine their usual dietary intake. Participants were asked to provide a blood sample, which was used to assess their antioxidant status. Additional blood samples were obtained at the end of the first and second phases of the study. Subjects were followed from mid March 1996 to mid August 1996. A double-blind crossover trial was conducted, and participants were randomly assigned to take a daily supplement (650 mg vitamin C, 75 mg vitamin E, and 15 mg beta carotene) or a placebo. During the entire study, participants, field workers, and investigators remained blinded to ozone levels and treatment groups.

Forty-one shoe cleaners participated in the first phase of the study (March 12 to May 30, 1996) and were randomly assigned to receive a supplement (n = 22) or a placebo (n = 19). Five participants dropped out after completing the first phase and six new participants were recruited at the end of the first phase. They only participated in the second phase. After a wash-out period (during which neither supplements nor placebos were given, May 31 to June 17, 1996), participants were enrolled in the second phase of the study (June 18 to August 9, 1996). The group that took supplements began taking placebos, and the group that took placebos began taking supplements. In addition, the new participants (n = 6) were randomly assigned to take either a supplement or a placebo. In total, 42 shoe-cleaners participated in this phase (23 received placebos and 19 received supplements). Overall, 34 (72%) participants completed both the first and second phases of the study.

Participants were asked to come twice a week to the Union to perform a spirometric test at the end of their workday. For each participant, the tests were performed indoors at a similar time of the day (within 3 h) so as to minimize the effect of diurnal variation on pulmonary functions. At the time of the test, a trained technician asked each worker the hour of the day at which he started working, whether he had had some respiratory symptoms during the day, and whether he had smoked during the workday. Lung function tests were performed with a spirometer (Medifacts Pneumotachographs, San Clemente, CA) according to the specification of the American Thoracic Society (ATS) (11). Before testing, the volume reading of each spirometer was calibrated with a 3-L syringe. The spirometric tests were conducted in a room with stable temperature and relative humidity. All lung function tests were examined by the same pneumologist, and the best of three technically acceptable maneuvers was selected for each test.

To minimize noncompliance, a health worker visited each participant at his individual work site on workdays and provided either the supplement or the placebo, which the participant took in front of the health worker. For weekend days, workers were provided with supplements or placebos for 2 d and were asked to return the vials in which the pills were kept.

Exposure Assessment

We obtained measurements of nitrogen dioxide (NO₂), sulfur dioxide (SO₂), particulates (with a mass median diameter of less than 10 µm, PM₁₀), ambient ozone and climatic variables (relative humidity and minimum, maximum, and daily average temperature) from the Mexico City's air monitoring network, and used data from the Merced station located in the central part of Mexico City. SO₂ was measured with an ultraviolet fluorescent analyzer (analyzer model 100; Advanced Pollution Instrumentation [API], San Diego, CA), and NO₂ using chemiluminescence (analyzer model 200 MCA; API). Ozone levels were measured via ultraviolet photometry (analyzer model 400; API). For PM₁₀, the particle Mass Monitor (Series 1400 Sensor Unit; Rupprecht & Pata Schnick, Albany, NY) was used. For NO₂, SO₂, ozone, and PM₁₀ daily exposures were calculated by averaging the hourly air pollutant levels between the time participants started working until the spirometric tests were conducted. Given the low concentrations observed for SO₂ (0.0301 ppm maximum average for 24 h), we decided to drop SO₂ from further analyses.

Measurements of Beta Carotene and -Tocopherol Serum Levels

Blood specimens were collected in EDTA vacutainer tubes, immediately covered with aluminium foil, stored in an icebox, and centrifuged within 3 h to obtain plasma. Aliquots were then stored at 70° C and shipped on dry ice to the laboratory of Dr. Gerber (INSERM, Montpellier, France) for analysis. Concentrations of beta carotene were measured by high-pressure liquid chromatography (HPLC) (12) with a detection in the visible spectrum at 453 nM and automatic integration. -Tocopherol was determined with HPLC (13) with detection in UV at 292 nM and automatic integration. All serum samples of a specific participant were analyzed in the same HPLC run to minimize between-run variation.

Statistical Analysis

Of the 47 workers who agreed to participate in either phase of the study, four performed fewer than four spirometric tests in one of the study phases and were excluded from the analysis. Baseline data on lung functions, collected before supplementation started, were not used in the analysis in order to avoid bias due to learning effects.

We studied the effect of different measures of ozone exposure on the change of pulmonary function parameters, including the mean hourly ozone concentrations 1 h before the spirometry; the mean hourly ozone levels over the entire workday, estimated from the hour a specific participant started working until the time he underwent a spirometric test; and the mean hourly ozone levels over the concurrent and previous days, estimated as the mean hourly ozone levels over two entire workdays. If a participant started working in the first 30 min of an hour, the time period of exposure was calculated starting from the hour. If he started working in the last 30 min of an hour, the average concentration was calculated starting from the next hour. The same method was used for the hour the spirometric tests were performed. Similar exposure measures were estimated for NO₂ and PM₁₀. Pearson correlation was determined between concentrations of air contaminants and various climatic variables (14).

We analyzed data using generalized estimating equation models, (GEEs). These models, which have been described by Liang and Zeger (15), are specially developed to account for the autocorrelation in the data and to allow for the use of time-dependent covariates. A key feature of this approach is that it models the marginal distribution with the autocorrelation in the variance. This gives the coefficients the usual interpretation. We first ran bivariate GEE models studying the association between air pollutant exposure and results of pulmonary function tests: FEV₁, FVC, peak expiratory flow rate (PEFR), and FEF_25-75. We adjusted models for potential confounding factors including participants' age, age², height, height², and smoking status; temperature (mean temperature or temperature 1 h before the test); relative humidity; and levels of other air pollutants (PM₁₀ and NO₂). In the final models, we only adjusted for participants' age and height and for mean temperature because the inclusion of other variables did not improve the fit of the models and the estimates of the ozone effects remained similar. We analyzed the first and the second study phases separately, including all participants and comparing the changes in pulmonary function related to ozone exposure in the supplement group with those in the placebo group. Results were similar to those obtained when we included only the 34 workers who completed both phases of the study. We compared regression coefficients using a t test (14) to estimate the effect of the supplementation and conducted all analyses using Stata software (16). We present results from the 34 workers who completed both phases of the study.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Population

The 34 participants who completed both phases of the study had a mean age of 38.9 yr (SD = 10). Most of the participants had resided in Mexico City since their birth. The mean time of residence in the city was 28.7 years (SD = 10). Participants worked on average 9 h a day (SD = 1) and had been working as shoe-cleaners for an average of 13 yr (SD = 11). Forty-one percent (n = 14) reported smoking but none smoked more than 5 cigarettes per day, and 79% of the smokers (n = 11) smoked fewer than 4 cigarettes per day. Three participants reported wheezing in the previous 12 mo, and close to half of the participants reported cough (n = 16) or phlegm (n = 21) episodes during the previous 12 mo. None of the participants had a history of asthma. Baseline mean lung function values were 4.29 L (SD = 0.67) for FVC, 3.44 L (SD = 0.58) for FEV₁, 10.40 L/s (SD = 1.87) for peak expiratory flow (PEF), and 3.49 L/s (SD = 1.38) for FEF_25-75. Participants underwent 14 to 44 tests each (mean = 33; SD = 7). Participants were exposed an average of 7.4 h (SD = 1.9) per day during working hours prior to the spirometric test, and only 5% of the spirometric tests were performed after less than 5 working hours.

At baseline, participants' mean reported caloric intake was 2,008 kcal/d (SD = 856). Their mean reported dietary intakes of vitamin C, vitamin E, and beta carotene were 98.6 mg/d (SD = 59), 5.46 mg/d (SD = 2.6), and 978.4 µg/d (SD = 491.1), respectively. Intakes of these nutrients were similar in the supplement and placebo groups.

Exposure Data

Figure 1 presents the daily 1-h maximum ozone ambient concentrations and the daily maximum temperature during the study. The maximum daily 1-h average concentrations for ozone exceeded the Mexican standard of 110 ppb (SD = 16) on 55% of the days. The hourly average pollutant concentrations during workday prior to the spirometric tests were 67.3 ppb (SD = 24, 5th to 95th percentiles = 28.7 to 105.8 ppb) for ozone, 52.3 ppb (SD = 19.8, 5th to 95th percentiles = 24.0 to 89.4 ppb) for NO₂, and 76.5 µg/m³ (SD = 26.1, 5th to 95th percentiles = 34.6 to 149.8 µg/m³) for PM₁₀. Neither NO₂ nor PM₁₀ levels exceeded the Mexican standards (17) during the study period. Average ozone concentrations during workdays prior to the spirometric tests were positively correlated with the average concentrations of NO₂ and PM₁₀ (r = 0.53, p < 0.001; r = 0.26, p < 0.001, respectively). The mean temperature during workdays was 18.0° C (SD = 2.0). Daily ozone and NO₂ levels were positively correlated with mean daily temperature (r = 0.26, p < 0.001 and r = 0.27, p < 0.001, respectively).

View larger version (34K): [in this window] [in a new window]   Figure 1.   Daily 1-h maximum ozone ambient concentrations and daily maximum temperature during the study, Mexico 1996.

Blood Data

At baseline, blood samples were collected from the 34 subjects, at the end of the first phase from 26 subjects, and at the end of the second phase from 20 subjects. At baseline, the supplement group had beta carotene and -tocopherol plasma levels similar to those of the placebo group (Table 1). At the end of the first phase, beta carotene and vitamin E plasma levels were significantly higher in the supplement group than in the placebo group. At the end of the second phase, only beta carotene levels were significantly higher in the supplement group.

Between baseline and the end of the first phase, we observed a significant increase in the mean plasma levels of beta carotene for the supplement group (p = 0.0002) but not for the placebo group. Over the same period, plasma -tocopherol levels increased significantly in the supplement group (p = 0.05), and decreased in the placebo group (p = 0.06). After the crossover, we observed a significant increase in plasma beta carotene and -tocopherol levels among subjects who received the supplement (p = 0.001 and p = 0.03, respectively) and a significant decrease among subjects who received the placebo (p = 0.02 and 0.005, respectively).

Lung Function Measurements and Air Pollutant Exposure

Table 2 presents the results of the regression analyses of lung function parameters and hourly average ozone concentrations during workdays for the supplement and placebo groups during the first phase of the study. Ozone levels were negatively associated with lung function parameters in the placebo group. Significant decrements were observed for FVC, FEV₁, and FEF_25-75. In the supplement group, none of the lung function parameters were associated with ozone concentrations. The difference between the two groups was statistically significant for all parameters except for PEF. Adjustments for the age and height of participants and for mean temperature did not materially change the estimated regression coefficients. Hourly average NO₂ concentrations during workdays were inversely related to lung functions in the placebo group, but not in the supplement group. Significant differences were observed between the two groups for all parameters. In contrast, hourly average PM₁₀ concentrations during workdays were not significantly associated with lung function either in the supplement or the placebo groups. When we evaluated simultaneously the effect of ozone, NO₂ and PM₁₀ using multipollutants models, we observed significant decrements in lung function parameters in the placebo group but not in the supplement group for ozone. The difference between the two groups was statistically significant for FVC, FEV₁, and FEF_25-75 (0.85 ml/ppb, 1.34 ml/ppb, and 3.18 ml/ppb, respectively). NO₂ concentrations were inversely related to FVC in the placebo group but not in the supplement group; the difference between the two groups was statistically significant (2.75 ml/ppb). PM₁₀ concentrations were not significantly related to lung function parameters either in the placebo or in the supplement groups. However, due to the small sample size and the multicolinearity between pollutants, these models should be interpreted with caution.

During the second phase of the study, we also observed that ozone concentrations were related to lung function decrements in the placebo group, except for FVC; however, the magnitude of the change was in general lower than that observed during the first phase of the study (Table 3). NO₂ and PM₁₀ concentrations were not significantly related to lung function parameters either in the placebo or in the supplement groups.

Because of the high ozone levels registered in Mexico on subsequent days, it was important to determine the effect of several days of ozone exposure on lung functions. We therefore investigated the impact of the hourly mean ozone levels over two workdays (the day prior to and the day of the spirometric tests) on the lung parameters of the participants (Tables 4 and 5). Decrements in lung function parameters in the placebo group were larger than those observed when only 1-d exposure was considered. These decrements were significant for all parameters except PEF. In contrast, ozone exposure was not related to lung parameters in the supplement group. During the second phase of the study, we observed lower lung function results in the placebo group than in the supplement group, although the magnitude of the lung function decrements in the placebo group was lower than that observed during the first phase of the study.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of this study suggest that vitamin supplementation might protect against the acute effects of ozone on lung functions. We estimated that for a daily mean ozone exposure of 70 ppb, the supplement effect would correspond to an attenuation of lung function decrements of 3.4% for FEV₁ and 6.1% for FEF_25-75. For a daily mean ozone exposure of 70 ppb during two consecutive days, this effect would reach 7.4% and 16.6% for FEV₁ and FEF_25-75, respectively. Adjustment for temperature and other pollutants (NO₂ and particulate matter [PM₁₀]) did not materially change the results.

The role of antioxidant supplementation in reducing the acute effects of ozone exposure has been investigated in animal and human controlled studies (18); however, there is little information on the impact of antioxidant supplementation on the acute effects of photo-oxidant exposure in free-living populations. Studies of animals have shown that exposure to a high ozone concentration (1 ppm) during 1 h led to lipid peroxidation as reflected by an increase in the amount of ethane and penthane exhaled and by an increase in levels of antioxidative enzymes such as glutathione (GSH) peroxidase, GSH reductase, and glucose-6-phosphatase deshydrogenase (G6PD). These increases were higher in animals that did not receive vitamin E supplements. Elsayed and coworkers (7) have also shown that concentrations of vitamin E in the lung tissue of animals receiving vitamin E supplements increased significantly after the animals were exposed to ozone (0.5 ppm for 5 d), whereas vitamin E levels in the lung tissue of nonsupplemented animals decreased. This suggests that vitamin E is mobilized toward the lung tissue in response to oxidative stress. In a human controlled study, supplementation with vitamin C (1 g before ozone exposure) and vitamin E (800 IU daily) protected against the acute effect of ozone (600 µg/m³ equivalent to 300 ppb) on FEV₁ and FVC (19). In another study conducted among asthmatics, the results suggest that vitamin C (500 mg) and vitamin E (400 IU) had a protective effect against PEF decrements after exposure to ozone (240 µg/m³ equivalent to 120 ppb) (20). Two studies conducted in The Netherlands suggest that antioxidant supplementation protects against the acute effect of ozone on lung functions of free-living individuals. In the first study, 26 cyclists exposed to ambient ozone (8-h mean levels of 101 µg/m³) received a similar dose of vitamin supplementation as in our study (vitamin C [650 mg], vitamin E [75 mg or 75 IU], and beta carotene [15 mg]) for 3 mo, and as in our study, larger decrements in lung functions (FEV₁, FVC, PEF) were observed in the group that did not receive supplements (21). A similar study was conducted in a larger group of cyclists (n = 36) who received vitamin C (500 mg) and vitamin E (100 mg) for 15 wk and who were exposed on average to 77 µg/m³ of ozone during exercise. A partial protective effect of supplementation was observed for FEV₁ and FVC (22). In the present study, we also observed that antioxidant supplements protected against lung function decrements after ozone exposure. The strongest protective effects were observed for FEV₁ and FEF_25-75. Participants' exposure to ozone was high, as mentioned in the RESULTS section. These relatively high levels of ozone exposure probably led to larger lung function decrements than those observed in The Netherlands' studies. However, the effects of differences in air pollution mixture and nutritional status cannot be excluded.

Several factors needed to be considered in the interpretation of our results. At baseline, the major characteristics of the two groups participating in our randomized trial were similar. By using a placebo, we were able to conduct a double-blind intervention in which neither the participants nor the spirometer technicians knew to which group participants were assigned. Therefore information bias could not explain our results. Forty-one percent of the participants reported smoking; however, they were all light smokers (none smoked more than 5 cigarettes per day, and 79% smoked fewer than 4 cigarettes per day) and we believe that this level of smoking had no significant effect on participants' antioxidant status. This was supported by the fact that, at baseline, plasma beta carotene levels were similar among smokers and nonsmokers (mean = 12.26 µmol/L, SD = 8.76 among smokers; mean = 12.25 µmol/L, SD = 6.67 among nonsmokers). Futhermore, the randomization produced a quite even distribution of smokers in the supplement and the placebo groups (n = 8 and 6, respectively), and after we stratified participants by smoking status, results were similar to those before such stratification.

At baseline, antioxidant intake and plasma concentrations of beta carotene and -tocopherol were similar in the supplement and placebo groups. Participants' reported intake of vitamin C was close to the recommended dietary daily allowance (RDA); however, beta carotene and vitamin E intakes were below the RDA (23). Plasma beta carotene levels of participants in our study were also lower than those observed in other populations (21, 24), as would be expected given the high sensitivity of plasma levels to dietary intakes for this nutriment. In contrast, vitamin E plasma levels of our study participants were similar to those observed in other populations (21, 24), although the vitamin E intake of our participants was low. This may be related to the inaccuracy of nutrient composition data and the fact that the tocopherol content of specific foods varies greatly with the procedures used in harvesting, processing, storing, and preparing those foods (25). During the course of the study, average plasma levels of beta carotene in the supplement group rose by 254% (range, 55% to 1,032%) in the first phase and by 292% (range, 27.8% to 749%) in the second phase. For -tocopherol the increase was 44% (range, 67 to 204%) in the first phase and 53% (range, 16% to 118%) in the second phase. These increases were comparable with those found in other supplementation studies (21, 26, 27), in particular with changes observed in The Netherlands study in which similar doses of supplementation were used (21). Two men who received supplements during the first phase had lower plasma beta carotene levels at the end of this phase. We do not have a clear explanation for this reduction, other than that it may be related to individual variation in absorption of beta-carotene. In contrast, these two men had higher plasma -tocopherol levels, which confirmed that they took the supplement. When comparing the two phases of the study, we observed that the lung function decrements of the placebo group during the first phase were larger than those of the placebo group during the second phase. This suggests a potential remaining protective effect linked to antioxidant supplementation. Our washout period may have been too short in particular for vitamin E which tends to accumulate slowly in tissues, and plasma levels may not reflect concentrations in the lung adequately.

The mechanism by which ozone induces acute pulmonary function decrements has been under discussion. The dominant hypothesis is that lung function decrements are related to a reduction of maximal inspiratory capacity caused by stimulation of the neural receptors in the upper airways. These receptors are stimulated by cyclooxygenase products of arachidonic acid (AA), which are released upon ozone exposure (3). Lipid peroxidation of the polyunsaturated fatty acids of lung cells is thought to be a mechanism by which ozone could induce pulmonary function decrements (4) and an inflammatory response (28). Menzel (29) has observed a change in the amount of fatty acids in lung tissue subsequent to ozone exposure (1 ppm ozone during 9 d), including a decrease in oleic and linoleic acids and an increase in AA. Free AA will subsequently be converted in prostaglandin E₂ (PGE₂) and prostaglandin F₂ (PG F₂), which have been linked to the lung function decrements observed after ozone exposure (30). PGE₂ in bronchoalveolar lavage recovered from ozone-exposed subjects has been reported to have a positive correlation with decrements of FVC and FEV₁ in these subjects (4). Airway C fibers can also be stimulated by PGE₂, suggesting that stimulation of bronchial C fibers (in part by AA products) may account for changes in lung function (FVC and FEV₁) after ozone exposure (31). However, other mechanisms may be involved since the nonsteroidal anti-inflammatory drug ibuprofen does not affect the increase in specific airway resistance provoked by ozone exposure (5). Vitamin C and vitamin E have been shown to affect arachidonic metabolism, but the role of antioxidants in this mechanism is not fully understood. Ascorbate can regenerate vitamin E from the tocopheroxyl radical (32) and act in synergism with vitamin E to inhibit lipid peroxidation (33). Beta carotene could act through its scavenger effect and therefore neutralize free radicals generated by ozone exposure (6). There are no clear data on the amount of antioxidant supplements that should be used to attenuate the impact of ozone exposure on the lung functions of humans. Animal studies suggest a plateau effect in enzyme activation with increasing vitamin E intake (34). In our study, plasma values indicated that our population was not deficient in vitamin E but was slightly deficient in beta carotene. Our results suggest that supplementation above the recommended allowance may provide additional protection against the acute effect of high ozone exposure on lung functions. Further epidemiological studies should be carried out to confirm these results, particularly in areas where populations are chronically exposed to ozone.