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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Ozone is known to yield hydroxyl radical, which may contribute to ozone-mediated lung injury. In the presence of hydroxyl radical, salicylate is hydroxylated to form 2,3-dihydroxybenzoic acid (2,3-DHBA). There is no evidence of enzymatic formation of 2,3-DHBA. We hypothesized that salicylate hydroxylation might be used as a biomarker indicating human exposure to ozone. Healthy, nonsmoking volunteers, 18 to 34 yr of age, were given acetylsalicylic acid (975 mg) or placebo orally 0.5 h before an exposure. Subjects were exposed to ozone (0.12 or 0.4 ppm) or filtered air in an environmental chamber for 2 h, while performing intermittent exercise. Results indicate significant decrements in FVC, FEV1.0, forced expiratory flows at 50% and 75% of FVC, and peak expiratory flow rate, and an increase in airway resistance, after exposure to 0.4 ppm ozone in comparison with air control (p < 0.05). Exposure to 0.4 ppm ozone also resulted in increased symptom numbers and severity (p < 0.05). When subjects were exposed to 0.12 ppm ozone, changes of pulmonary function and symptoms reported were minimal. Plasma concentration of 2,3-DHBA was significantly increased after exposure to 0.12 and 0.4 ppm ozone in comparison with air control (p < 0.05). There was a significant correlation between ozone-induced changes of pulmonary function and normalized salicylate hydroxylation (p < 0.05). The results indicate that exposure to ozone can initiate in vivo production of hydroxyl radical, a potent reactive agent. Salicylate hydroxylation may then serve as a sensitive dosimetric biomarker for ozone exposure, even at subclinical ozone exposure levels.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Ozone, an important constituent of photochemical smog, has been demonstrated to pose a health concern. Epidemiologic studies conducted in Canada and in other countries have indicated that daily fluctuation of ambient ozone levels is associated with an increase in the incidence of respiratory symptoms (1) and hospital admissions due to respiratory diseases (2, 3). Controlled human clinical studies have demonstrated that short-term exposure to ozone can induce significant decrements in pulmonary function (4) and increases in inflammatory cell counts and mediators in bronchoalveolar lavage (5, 6) and nasal lavage (5), which may contribute to ozone-initiated respiratory problems. An estimate of the biologically effective dose of ozone is necessary for determining risks associated with ozone pollution, for assessing sensitive subpopulations, and for regulatory and policy issues. However, there has not yet been a practical biomarker validated in humans for assessment of the biologically effective dose of ozone in ambient air.
Many of the adverse effects caused by ozone may be attributed to its nature as a highly reactive oxidant. There is considerable evidence indicating that tissue damage caused by ozone involves free radical-mediated reactions (7), especially hydroxyl radical (10). Since hydroxyl radical readily reacts with macromolecules, it is considered a most potent free radical, initiating tissue injury and cell death. Detection of hydroxyl radical produced by ozone would be a useful biomarker in assessing the biologically effective dose of ozone at the target of the toxic action.
The hydroxyl radical can be detected in biologic systems by salicylate hydroxylation (11). Salicylate, a hydrolyzed form of acetylsalicylic acid (aspirin), is enzymatically metabolized by cytochrome P450 monooxygenases to form 2,5-dihydroxybenzoic acid (2,5-DHBA). Yet, its 2,3-dihydroxy metabolite (2,3-DHBA) is thought to be produced only when hydroxyl radical is present (16), a nonenzymatic reaction. We have previously used salicylate to detect ozone-mediated in vivo formation of hydroxyl radical in rats (17). Data from this study demonstrate that exposure to ozone caused significant increases in 2,3-DHBA in lungs and plasma of rats which were associated with acute pulmonary inflammatory injury by ozone exposure.
By means of using salicylate hydroxylation, a number of human clinical studies have shown an involvement of hydroxyl radical in the pathogenesis of several disease states (12). However, there has not been any report concerning ozone-initiated production of 2,3-DHBA in humans. Because the toxicity of salicylate is minor at its therapeutic range, and its 2,3-DHBA metabolite is not formed by cytochrome P450, we hypothesized that salicylate hydroxylation might be used as a biomarker for assessing the biologically effective dose of ozone in humans.
The objectives of the present study were (1) to verify the use of salicylate hydroxylation as a biomarker indicating in vivo ozone exposure in humans in a controlled environment and (2) to verify the relationship between ozone-mediated salicylate hydroxylation and airway adverse health effects, including pulmonary function and symptom reports.
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METHODS |
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INTRODUCTION
METHODS
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DISCUSSION
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Materials
Acetylsalicylic acid (ASA) tablets (containing ASA 325 mg USP, starch [energy 1.4 kj (0.52 kcal)]; not containing alcohol, gluten, lactose, paraben, sodium, sulfite, and tartrazine) were obtained from Shoppers Drug Mart (Toronto, Ontario, Canada). Sodium bicarbonate tablets (as placebo) were obtained from Toronto General Hospital Central Pharmacy (Toronto, Ontario, Canada). Sodium salicylic acid, 2,3-DHBA, 2,5-DHBA, and resorcinol were obtained from the Sigma Chemical Co. (St. Louis, MO); the ADx salicylate assay kit was from Abbott Laboratories (Abbott Park, IL). All other materials were of reagent grade and obtained from common commercial suppliers.
Subjects
Sixteen healthy, nonsmoking volunteers, 18 to 34 yr of age, were recruited to participate in this study (Table 1). Potential participants
were excluded if they had a history of asthma, allergic rhinitis, cardiac
disease, chronic respiratory disease, recent acute respiratory illness, or
known extensive exposure to pollutants, or if their methacholine challenge response was positive (PC20 
8.0 mg/ml). Subjects must not
have taken ASA and vitamins in any prescription or over-the-counter
preparation prior to each exposure for at least 48 h. A brief review of
the diet and passive smoking on the testing day and the two previous
days was recorded. All studies were approved by the Ethics Committee of the University of Toronto on safeguarding the rights and welfare of human subjects. Informed consent forms approved by the Ethics Committee were signed by all subjects.
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Bronchial Responsiveness to Methacholine
Bronchial responsiveness to methacholine was measured before the
subjects entered the study. Methacholine concentration producing a
20% decrease in FEV1.0 (PC20) was measured using the method of
Juniper and colleagues (18). The PC20 was calculated by interpolation of the last two data points on the noncumulative concentration- response curve. Subjects were considered nonasthmatic if their PC20
was > 8.0 mg/ml. The maximum concentration of methacholine given
was 16 mg/ml. Subjects who had a PC20 8.0 mg/ml were excluded from the study.
Subject Treatments
Subjects were exposed in randomized fashion to ozone or filtered air in an environmental chamber constructed of transparent plexiglas and measuring 2.44 m × 2.44 m × 2.08 m (12.4 m3). There were six drug/ exposure conditions: (1) placebo + filtered air; (2) placebo + 0.12 ppm ozone; (3) placebo + 0.4 ppm ozone; (4) ASA + filtered air; (5) ASA + 0.12 ppm ozone; (6) ASA + 0.4 ppm ozone. Drug/exposure orders were blindly selected after a subject completed a preliminary visit, with 2 wk between treatments. The subject was blinded as to the drug (ASA/placebo) and the exposure (ozone/air). The technologist conducting the ozone/air exposure was blinded as to the drug only. The technologists who conducted analyses of pulmonary function, salicylate metabolism, cell counts, and biochemical parameters were blinded as to the drug and exposure.
ASA (975 mg) or placebo (sodium bicarbonate, 900 mg) was given orally 0.5 h before the exposure began. This ASA dose was chosen because it is within therapeutic range and reaches peak plasma value in approximately 2 h (19). Ozone was used at concentrations of 0.12 or 0.4 ppm for 2 h. These doses of ozone have been proved to be safe and effective concentrations for use in humans, even in asthmatics, while allowing for reversible mild symptoms, lung function changes, and bronchial hyperresponsiveness without clinically troublesome effects (20).
The chamber was supplied with filtered air drawn from the building air, at an adjustable flow rate set at 17 air changes/h, via separate inlet and exhaust fans. Ozone was generated by passing medical-grade oxygen at a flow rate of 0.05 L/min through a high-intensity electrical field provided by a condenser discharge tube (60 cm × 4 cm outer diameter). Ozone concentration was monitored continuously using a Dasibi (Model 1008RS; Glendale, CA) ultraviolet photometric ozone analyzer. The relative humidity in the chamber was maintained at approximately 40%, and the temperature at approximately 21° C. Relative humidity and temperature were recorded every 15 min using a Kanomax Climomaster (Model 6511; Japan) anemometer/thermohygrometer. CO2 concentrations were monitored continuously using an ADC (Model PM2; Haddelson, UK) infrared gas analyzer.
Subjects exercised on a bicycle ergometer at a respiratory minute ventilation of 45 L/min for alternating 15-min rest/exercise intervals throughout each exposure. Workloads were calculated on an individual basis (for a respiratory minute ventilation of 45 L/min) according to the procedure described by Åstrand (21). For a given subject, the workload was kept the same for all visits and was monitored for consistency during exposure. Respiratory minute ventilation and breathing frequency (Parkinson Cowan Dry Gas Meter; New York, NY), and heart rate (Sport tester PE 3000; Kenpele, Finland), were monitored every 15 and 5 min, respectively. Subjects were asked to report any symptoms that occurred before and during chamber exposures, by recording the type of symptom, when it occurred, and the severity using a categorical scale.
Spirometry, Lung Volumes, Airway Resistance, and Diffusion
The procedures for measuring pulmonary function followed established principles and procedures described previously (22). Flow-volume curves during exposure were obtained using a MED Science 570 Wedge spirometer with a 7041A X-Y recorder (St. Louis, MO). Peak expiratory flow rates (PEFR) were obtained using a Collins DS computerized system. A whole-body pressure plethysmograph (Collins Body Plethysmograph System, Model 09001; Braintree, MA) was used to determine thoracic gas volume and airway resistance (Raw). The Collins DS computerized system was used to measure the single-breath diffusion test of carbon monoxide uptake.
Blood Samples
Three hours after the ASA/placebo ingestion, blood samples (5 ml)
were collected in tubes containing anticoagulant EDTA. The blood
samples were centrifuged at 600 × g for 10 min to collect plasma. The
plasma samples were stored at 
40° C for later analysis of salicylate
metabolism.
Nasal lavage and saline-induced sputum were also collected and processed for detecting salicylate hydroxylation and inflammatory response. Detailed methodology and results of these analyses will be reported in a separate article.
Detection of Salicylate and its DHBA Metabolites
The procedures for detection of salicylate and its DHBA metabolites followed procedures previously described (17). Proteins in plasma were precipitated with 1 vol of cold trichloroacetic acid (10%, vol/vol) and centrifuged for 10 min at 600 × g. The supernatant was added 1.0 nmol of resorcinol as an internal standard, and NaCl powder (0.1 g/ml sample). Samples were extracted with 5 ml of HPLC-grade ethyl acetate twice. The ethyl acetate layer was recovered and evaporated to dryness. The residue was dissolved in 400 µl of HPLC-grade water and 100 µl of HCl (1 N) and filtered through a 0.22-µm filter.
The resulting solution was injected into an HPLC system equipped with a BAS 400 solvent delivery system (West Lafayette, IN), a Gilson autosampler (Model 231XL; Middleton, MI), a Spherisorb 5 ODS reversed-phase column (25 cm × 4.6 mm; Jones Chromatography Ltd., Milford, MA), and an electrochemical detector equipped with a glassy carbon working electrode and an Ag/AgCl reference electrode (BAS amperometric detector, Model LC-4B). The mobile phase consisted of sodium citrate (30 mM) in acetate buffer (27.7 mM, pH 4.75), 97.2%, and methanol, 2.8%. The separation was performed at an oxidation potential of +0.96 V and a flow rate of 1.0 ml/min (17).
The retention times for 2,3-DHBA and 2,5-DHBA were approximately at 6.2 and 7.2 min, respectively. The linear range used for quantification of DHBAs was 20 to 100 pmol, and the detection limit
was < 1 pmol. Acidified aqueous samples of 2,3-DHBA, 2,5-DHBA, and salicylate were relatively stable for 24 h at room temperature or
for 1 wk at 4° C, i.e., about 80% of initial values for DHBAs. The yield
of the extraction step for aqueous standards was 80% for DHBAs.
Based on the report by Grootveld and Halliwell (12), who developed
this method, storage of aqueous samples at 20° C until use did not
affect the results. According to the User's Instruction for the ADx salicylate assay kit from Abbott Laboratories, salicylate in serum/plasma
is stable for 2 d at 4° C and samples should be stored frozen (20° C
or colder) if not analyzed within 24 h. Therefore, we believe that salicylate and its DHBA metabolites in samples stored at 40° C should
be rather stable.
Plasma salicylate concentration was measured using an ADx salicylate assay kit based on the fluorescence polarization immunoassay technology. The sensitivity of this assay is 0.2 µmol/ml.
Data Analysis and Statistics
Results are expressed as mean ± SE. Data were transformed when necessary to meet criteria of normality and homogeneity of variance. Data of pulmonary function and symptoms were tested for statistically significant differences (p < 0.05) by repeated-measures two-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test. Data on salicylate hydroxylation were tested for statistically significant differences (p < 0.05) by repeated-measures one-way ANOVA, and by repeated measures ANOVA on rank, followed by the Student-Newman-Keuls test. Linear correlation coefficients were calculated using the Pearson Product Moment Correlation test. All the statistical tests were performed using the SigmaState software (Jandel Corp., San Rafael, CA).
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RESULTS |
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DISCUSSION
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Ozone-induced Pulmonary Function Alterations
Exposure of subjects to 0.12 ppm ozone resulted in a significant decrease in PEFR and an increase in Raw compared with exposure to filtered air (Table 2) but did not cause significant changes in other pulmonary function indices with either placebo or ASA (Figure 1, Table 2). Exposure to 0.4 ppm ozone resulted in significant time-dependent decreases in FVC (Figure 1A), FEV1.0 (Figure 1B), FEF50 (Figure 1C ), and FEF75 (Figure 1D). Exposure to 0.4 ppm ozone also caused a significant increase in Raw and decrease in PEFR (Table 2) in comparison with exposure to filtered air. Diffusing capacity for carbon monoxide (DLCO) did not change significantly following ozone exposure (Table 2).
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When subjects were pretreated with ASA, there was a significant protection against a decrement in PEFR and increase in Raw induced by 0.4 ppm ozone in comparison with placebo treatment (Table 2). ASA treatment appears to have some impact on other ozone-induced pulmonary function changes, too; however, the impact was not statistically significant, presumably because of marked interindividual variation (Table 2).
Report of Symptoms following Exposure to Ozone
When subjects were pretreated with placebo and exposed to 0.12 ppm ozone, there was a significant increase in the total number of symptoms in comparison with air control (Table 3). The severity of symptoms was not significantly changed following exposure to 0.12 ppm ozone (Table 3). When subjects inhaled 0.4 ppm ozone, a significant increase in total number of symptoms was observed (Table 3). The severity of cough and tightness in chest was also significantly increased after exposure to 0.4 ppm ozone when compared with air control. Ozone inhalation had no significant effect on other symptoms.
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When subjects were pretreated with ASA, there was a significant reduction of total number of symptoms induced by 0.4 ppm ozone in comparison with placebo treatment (Table 3). ASA treatment did not have any significant effect on other ozone-induced symptom outcomes.
Salicylate Hydroxylation Induced by Ozone
The diet and passive smoking on the testing day and the two previous days were recorded and did not reveal a significant difference among visits (data not shown). This suggests that day-to-day variations of intake of oxidants and antioxidants, and likely salicylate, via food would be minimal.
The formation of plasma 2,3-DHBA and 2,5-DHBA was normalized by plasma salicylate concentration in order to adjust interindividual and day-to-day variations in ASA pharmacokinetics. There was a significant increase in mean plasma 2,3-DHBA concentration after exposure to 0.12 or 0.4 ppm ozone (Table 4). While the means of plasma 2,3-DHBA concentrations were not proportional to ozone doses, the medians of 2,3-DHBA levels reveal a trend of ozone dose-dependent pattern, although the difference between 0.12 and 0.4 ppm ozone was not statistically significant. The 2,3-DHBA data were not normally distributed (p < 0.05).
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Exposure to ozone did not significantly change the mean plasma 2,5-DHBA level (Table 4), suggesting that enzymatic metabolism of salicylate was not modified by ozone. When plasma salicylate hydroxylation was expressed as the ratio of 2,3-DHBA to 2,5-DHBA (2,3-DHBA/2,5-DHBA), inhalation of ozone was associated with a significant enhancement in the mean plasma 2,3-DHBA/2,5-DHBA (Table 4), with a 1.8-fold increase after exposure to 0.12 ppm ozone and a 2.5-fold increase after exposure to 0.4 ppm ozone.
Correlations between Salicylate Hydroxylation and Changes in Pulmonary Function and Symptom Report
Data from exposure to air and to 0.12 and 0.4 ppm ozone with ASA treatment were pooled to calculate the correlations between salicylate hydroxylation and pulmonary function and symptom report. Plasma 2,3-DHBA level was marginally correlated with changes of FEF50, FEF75, and FEV1.0 but not with other pulmonary function indices (Table 5). However, 2,3-DHBA/2,5-DHBA was significantly correlated with most pulmonary function changes (Table 5). There was no significant correlation between salicylate hydroxylation and symptom reports.
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Correlations between plasma salicylate hydroxylation and pulmonary function and symptom report were also analyzed within the 0.4 ppm ozone exposure group (Table 6). The correlations between ozone-induced pulmonary function decrements and 2,3-DHBA/2,5-DHBA were substantially improved. No significant correlation was observed between salicylate hydroxylation and symptom reports.
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DISCUSSION |
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INTRODUCTION
METHODS
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DISCUSSION
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The present study was designed to investigate the usage of plasma 2,3-DHBA formation as a tool to address the issue of the biologically effective dose received by human subjects during an acute exposure to ozone. This is partly because bronchoalveolar lavage, the investigation of choice for studying ozone exposure and lower airway damage, requires specialized procedures and local anesthesia and is considered more invasive. On the other hand, although the pulmonary function parameters are less invasive and are widely used for assessing the adverse health effects of ozone exposure, they may not be sensitive enough to reflect low-level ozone exposure. Therefore, studies are required to develop a less invasive technique for evaluating the effective dose of ozone for risk assessment purposes, particularly one that may eventually be used in epidemiologic field surveys.
The results of this study reveal an approximately 2-fold increase in plasma 2,3-DHBA after ozone exposure of human subjects (Table 4), even at 0.12 ppm, which is the current American ambient air quality standard for ozone. We attribute the excess formation of 2,3-DHBA to the hydroxyl radical generated via ozone-induced lipid peroxidation (7) and the subsequent Fenton reaction with transition metals within pulmonary tissues (8, 11). Ozone may also yield hydroxyl radical directly during the reaction with physiologic fluid on the airway surfaces (9). Plasma 2,5-DHBA level was not significantly increased after ozone exposure, indicating that enzymatic metabolism of salicylate was not altered by ozone exposure. Although the increase of plasma 2,3-DHBA concentrations did not reveal an ozone dose-dependent pattern, the ratio of 2,3-DHBA to 2,5-DHBA (2,3-DHBA/2,5-DHBA) was elevated with the increase of ozone concentrations. The ratio of 2,3-DHBA to 2,5-DHBA should remove some variations due to potential day-to-day differences in salicylate pharmacokinetics, diet, exercise, and potential exposure to other oxidants, since the variations of basal salicylate metabolism generally affect both isomers in the same direction. These data are consistent with our previous observations using rats as a model (17), although human subjects demonstrated a basal level of plasma 2,3-DHBA/2,5-DHBA 20-fold higher than that in rat plasma, presumably because of the differences in the enzymatic metabolic rates and possibly the influence of exercises.
The results show that the salicylate hydroxylation was significantly enhanced at 0.12 ppm ozone, an exposure that did not cause appreciable symptoms and alterations of pulmonary function (Tables 2 and 3). However, we did find a significant increase in numbers of inflammatory cells in nasal lavage fluid and a trend of increase in sputum inflammatory cells (data will be presented in a separate report). In view of the above information, it is conceivable that pulmonary function parameters and symptom reports may not be sensitive enough to indicate ozone-induced injury. Contrastingly, plasma salicylate hydroxylation would be rather sensitive with respect to indicating ozone uptake and effects in humans, even at doses lower than symptomatic or pulmonary function decrements can be shown.
It may, however, be noted that the increase in plasma 2,3-DHBA was not proportional to exposure concentrations of ozone. One interpretation for this observation may be that 0.4 ppm ozone caused a closure of small airways as a consequence of pronounced pulmonary function decrements in comparison with 0.12 ppm ozone (Figure, Table 2). We observed a significant modification of the breathing pattern following exposure to 0.4 ppm ozone. Exposure to 0.4 ppm ozone had a significantly faster breathing frequency (31.5 ± 0.82 breaths/min for 0.4 ppm ozone versus 29.9 ± 1.37 breaths/min for air control and 28.4 ± 1.22 breaths/min for 0.12 ppm ozone at 108 min of exposure; p < 0.05) and a lower tidal volume (1.36 ± 0.07 L/ breath for 0.4 ppm ozone versus 1.50 ± 0.09 L/breath for air control and 1.55 ± 0.10 L/breath for 0.12 ppm ozone at 78 min of exposure; p < 0.05) than did exposure to filtered air and 0.12 ppm ozone. These data suggest that exposure to 0.4 ppm ozone resulted in a reduction in the amount of air in each breath and a decrease in retention time of air in airways when compared with exposure to 0.12 ppm ozone. Thus, the actual amount of ozone delivered to small airways and reacting with cellular components might not be proportional to exposure concentrations. Alternatively, the nonlinear increase of 2,3-DHBA could be due to the marked interindividual variations. The medians of 2,3-DHBA levels (Table 4) reveal a trend of ozone dose-dependent pattern, although the difference between 0.12 and 0.4 ppm ozone was not statistically significant. Another explanation may be that salicylate was ozonized by the higher concentration of ozone to produce other products. However, in the chromatograms, we did not find other breakdown product(s) of salicylate which are likely to occur during ozonization processes, although the products were limited to those that could be electrochemically detected.
Since pulmonary tissue is the primary target for ozone, a question arises of whether plasma salicylate hydroxylation level can be indicative or predictive of lung injury. While we recognize that salicylate hydroxylation may take place in other organs as well as in lungs, ozone-induced production of 2,3-DHBA is likely to occur in lungs. This is because ozone does not readily pass through cell membranes because of its high reactivity (23). It is true that a cascade of reactive intermediates produced in the reactions of ozone with primary target molecules have a lower reactivity and longer life span than ozone itself. Nevertheless, hydrogen peroxide will be quickly transformed to hydroxyl radical because of its marked reactivity and an abundant pulmonary supply of iron. When subjects are pretreated with ASA, hydroxyl radical in pulmonary tissue can be trapped by salicylate to produce 2,3-DHBA, which then diffuses to the capillary bed.
Another possible mechanism for salicylate hydroxylation is by sequelae of ozone-stimulated neutrophils which may produce the reactive oxygen species. However, based on the evidence reported by Kettle and Winterbourn (24), the hydroxylated salicylate metabolite produced by activated neutrophils is mainly 2,5-DHBA. The reaction was dependent on superoxide and myeloperoxidase activity but independent of hydrogen peroxide and hydroxyl radical and was not affected by hydroxyl radical scavengers. Their data suggest that nonspecific sequelae of the neutrophilic inflammation may not contribute significantly to the production of 2,3-DHBA, when compared with the impact of hydroxyl radical generated from ozone.
A correlation analysis was conducted in an attempt to assess the potential utility of plasma salicylate hydroxylation as a predictor of acute pulmonary adverse response. The correlations between 2,3-DHBA/2,5-DHBA and pulmonary function changes were modest using data pooled from air control and 0.12 and 0.4 ppm ozone exposure groups (Table 5). The correlation was substantially improved when analyzed within the 0.4 ppm ozone exposure group, where pronounced pulmonary physiologic changes were observed (Table 6). This is probably because the latter analysis eliminated the dilution effects of data from air control and 0.12 ppm ozone exposure groups. The correlations between salicylate hydroxylation and the decrements in pulmonary function support the notion that salicylate hydroxylation may serve as a useful marker to indicate the biologically effective dose of ozone.
Interestingly, we noticed that some of the subjects (subjects A15 and A18 from placebo treatment; subject A15 from ASA treatment) had extraordinarily larger decrements of pulmonary function than other subjects (> mean + 2 SD) following exposure to 0.4 ppm ozone. When exposed to 0.12 ppm ozone, the FEV1.0, FEF50, and FEF75 in Subjects A15 decreased, too. Concomitantly, these subjects had higher plasma levels of the 2,3-DHBA/2,5-DHBA ratio than other subjects (> mean + 3 SD). This implies that the high sensitivity of some subjects to ozone observed in the present and previous studies (25) might be due to a higher level of ozone uptake in their airways.
It is noteworthy that ASA pretreatment significantly reduced the total number of symptoms (Table 3) and the deterioration of lung function (Table 2) induced by ozone exposure. This observation is consistent with previous reports that demonstrate that indomethacin (26, 27) and ibuprofen (28), analogues of ASA as a nonsteroid anti-inflammatory drug, can reduce ozone-induced pulmonary function decrements and alleviate symptoms. It is well known that ASA and indomethacin are potent inhibitors of prostaglandin synthesis in macrophages (29) and airway epithelial cells (30). Ozone has been found to stimulate synthesis of prostaglandins and thromboxanes in human airways (6). These findings suggest that cyclooxygenase products of arachidonic acid play a prominent role in the development of pulmonary function decrements and airway inflammatory response consequent to acute ozone exposure. To place these findings in perspective, there exists the possibility that ASA may serve as a protective agent to block ozone-mediated acute lung injury, by both inhibiting prostaglandin synthesis and scavenging hydroxyl radical that attacks cell integrity. The benefit of ASA would be more significant when taking into consideration that ozone-initiated syntheses of endogenous antioxidants such as superoxide dismutase, catalase, and glutathione occur between 12 and 24 h after exposure (31).
In conclusion, the present study demonstrates that enhanced plasma salicylate hydroxylation is associated with human exposure to ozone, even at an ozone concentration (0.12 ppm) that causes minimal changes in pulmonary function and symptoms reported. Elevated plasma salicylate hydroxylation is also correlated with decrements in pulmonary function. Our results add support to the view that exposure to ozone can initiate in vivo production of hydroxyl radical, a potent reactive agent that may contribute substantially to the impairment of lung function. The study suggests a potential use of plasma salicylate hydroxylation to indicate the biologically effective dose of ozone in humans.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Ling Liu, Postal Locator 0301A1, Environmental Health Directorate, Health Canada, Tunney's Pasture, Ottawa, ON, K1A 0L2 Canada.
(Received in original form October 28, 1996 and in revised form July 7, 1997).
Acknowledgments: The writers thank Ms. Lilyanna Trpeski for her technical assistance in conducting human chamber exposures, performing pulmonary function measurements, collecting blood samples, and performing data entry; Ms. Ruth Zhang and Ms. Joanne M. Kubay for processing blood samples; Dr. Irvin Broder for conducting physical examinations on subjects; and Drs. Renaud Vincent and David Stieb for their critical review of the manuscript.
Supported by Health Canada Project K281218.
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References |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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