The Effects of Sequential Exposure to Acidic Fog and Ozone on Pulmonary Function in Exercising Subjects1 - 3

ROBERT ARIS, DOROTHY CHRISTIAN, DEAN SHEPPARD, and JOHN R. BALMES

Introduction

Acidic air pollution is a common occurrence in developed countries and is thought to have adverse respiratory health effects. Concern about the potential detrimental effects of acidic aerosols has led the Clean Air Scientific Advisory Committee of the U.S. Environmental Protection Agency to consider criteria pollutant status for such aerosols (1). Epidemiologic studies from London (2) and the Meuse Valley (3)provide substantial evidence that acidic fogs were associated with excess human morbidity and mortality. Unfortunately, epidemiologic studies cannot prove a cause-effect relationship primarily because of the covariation of atmospheric acidity with multiple, potential confounding factors such as ambient temperature, humidity, and elevated levelsof other pollutants including ozone (0 3 ) and sulfur dioxide. To date, human exposure studies with acidic aerosols alone have failed to demonstrate significant decrements in pulmonary function in normal subjects and have demonstrated only small decrements in subjects with asthma. One possible explanation for these conflicting data is that the toxicity of acidic pollution may not be manifest until interactions with important copollutants, particularly oxidants, are investigated. Support for this hypothesis comes from animal toxicologic studies demonstrating synergism between acidic sulfates and ozone on biochemical and histologic end points (4, 5). The purpose of this experiment was to test the hypothesis that a preexposure to an acidic fog would enhance the effects of ozone exposure on pulmonary function and methacholine responsiveness in exercising, healthy subjects. Nitric acid (HN03 ) , a photochemical product primarily of emissions from mobilesource polluters was used in this study because it is the most common acidic pollutant in many western regions of the United States (6), and because its effects on pulmonary function have not been

SUMMARY In Southern California coastal regions, morning fog Is often acidified by the presence of nitric acid (HN0 3). Pe.ak exposure to ozone (0 3) usually occurs In the afternoon and evening, after the fog has dissipated. Todetermine whether fog containing HN0 3might enhance pulmonary responses to 0 3,we studied a group of healthy, athletic subjects selected for lung function sensitivity to 0 3, On 3 separate days, the subjects exercised for 2 h In atmospheres containing HN0 3 fog (0.5 mg/ml), H20 fog, or clean, filtered air. After a t-h break, they exercised for an additional 3 h in an atmosphere containing 0.20 ppm 0 3, Surprisingly, the mean 03-lnduced decrements In FEY, and FYCwere smaller after exercise In each fog-containing atmosphere than they wereafter exercise in clean, filtered air. The mean (± SEM) 03-lnduced decrements In FEY, were 26.4 ± 5.3% after air,17.1 ± 3.7% after H20 fog, and 18.0 ± 4.3% after HN0 3fog, and In FVCthey were 19.9 ± 4.7% after air, 13.6 ± 2.8% after H20 fog, and 13.6 ± 4.2% after HN0 3 fog. Although the 03-sensltlve subjects studied were selected from a healthy group of volunteers solely on the basis of sensitivity to 0 3, as a group they were significantly more responsive to methacholine than was a group of subjects previously studied In our laboratory, and were also significantly more responsive to methacholine than was a group of similarly recruited subjects found not to be sensitive to 0 3(mean ± SEM PClOO of 2.95 ± 0.80 mg/ml for the 03-sensltlve subjects and 18.67 ± 4.54 mglml for the nonsensitive subjects, p < 0.005). These results Indicate that prior exposure to HN0 3 or H20 fog does not potentiate, but may attenuate, 03-lnduced decrements In pulmonary function. In addition, our finding of Increased methacholine responsiveness In 03-sensltlve subjects suggests that airway hyperresponslveness may be a risk factor for 0 3 sensitivity even among healthy, asymptomatic AM REV RESPIR DIS 1991; 143:85-91 athletes.

well studied. The sequential exposure protocol was designed to stimulate a worst-case scenario in a California coastal region in which morning acidic fogs are followed by sunny afternoons with high 0 3 levels. Methods The subjects were 39 healthy, nonsmoking volunteers who were informed of the risks of the experimental protocol and signed consent forms approved by the Committee on Human Research of the University of California, San Francisco. The subjects were 21 to 39 yr of age, and they specifically denied a history of pulmonary or cardiac diseases or respiratory infection within 6 wk of the study onset. No subject used theophylline preparations, inhaled beta-adrenergic agonists, antihistamines, prostaglandin inhibitors, or vitamins C and E within 1 wk, or consumed tea, coffee, or hot chocolate within 4 h, of any part of the experiment. All of the subjects received financial compensation for their participation.

Pulmonary Function Measurements Specific airway resistance (SRaw) was determined as the product of airway resistance and thoracic gas volume, the latter two having

been measured in a constant-volume body plethysmograph (Warren E. Collins, Braintree, MA). Specificairway resistancewas calculated as the average of five measurements taken 30 s apart. Spirometry was performed on a dry, rolling seal spirometer (S400; Spirotech Division, Anderson Instruments, Inc., Atlanta, GA). Mean values for FVC and FEV 1 were calculated from three acceptable FVC maneuvers obtained approximately 30 s apart. On the first visit to the laboratory, the mean was calculated from six (two sets of three, 5 min apart) FVC and FEV 1 values in an effort to minimize inaccuracy attributable to the subject's first-time performance on a spirometer. (Received in original form March 19, 1990 and in revised form July 10, 1990) 1 From the Lung Biology Center, Cardiovascular ResearchInstitute, Northern California Occupational Health Center, and the Medical Service, San Francisco General Hospital, University of California, San Francisco, California. 1 Supported in part by Contract No. A833-076 from the California Air Resources Board. 3 Correspondence and requests for reprints should be addressed to John R. Balmes,M. D.,Lung Biology Center, UC Box 0854, San Francisco, CA 94143.

85

86

ARIS, CHRISTIAN, SHEPPARD, AND BALMES

Methacholine (MCh) responsiveness was tested by measuring the subject's SRaw before and after inhalation of 10 deep breaths of phosphate-buffered saline and doubling concentrations of MCh (0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, and 64.0 mg/rnl) delivered by a nebulizer (No. 646; Devilbiss Co., Somerset, PAl with a dose-metering device calibrated to deliver 0.01 ml/breath. The concentration of MCh that produced a 100% increase in SRaw from the postsaline baseline (PC 100 ) was calculated by log-linear interpolation.

Ozone-Sensitivity Determination Initially, subjects completed a medical history questionnaire and underwent screening for 0 3 sensitivity. Subject enrollment continued until there was a high degree of assurance that 10 would complete the sequential exposures. Each subject had his or her baseline SRaw, FEV 1, and FVC measured 5 to 10 min before undergoing exposure to 0 3 , The exposure protocol involved a 50-min exercise period followed by a lO-min measurement/rest period each hour for 3 or 4 h. The exposures took place in an exposure chamber filled with filtered air at 20° C and 50070 relative humidity, to which 0.20 ppm 0 3 was added. Each subject exercised on a treadmill (Model M9.l; Precor Co., Bothell, WA) and/or a cycle ergometer (No. 18070; Gould Godart, Bilthoven, The Netherlands), which were individually adjusted to produce a targeted ventilatory rate, measured with a pneumotachograph (No.3; A. Fleisch, Lausanne, Switzerland), of 40 L/min. The subjects chose either to cycle, jog, or alternate the two. The ventilatory rate, tidal volume, and respiratory rate were measured at the 10-min and 40min points of each 50-min exercise period, and the work load was readjusted as needed to maintain the targeted ventilatory rate. During each lO-min measurement/rest period, SRaw, FEV 1, and FVC were measured, in that order, immediately after exercise. Each subject was allowed to rest, drink water or juice, and eat ad libitum during the remainder of the measurement/rest period. The protocol was aborted if the subject became markedly symptomatic and felt unable to continue. Each subject who experienced an FEV 1 decrement of ~ 10070 of the baseline value after 3 h of 0 3 was considered "sensitive" to 0 3 and was asked to participate in the sequential exposures.

Sequential Exposures The sequential exposure protocol had a predetermined sample size of 10, with each subject acting as his or her own control. Subject characteristics are listed in table 1. Predicted values for the spirometric parameters are those of Knudson and coworkers (7). On the 3 exposure days, each separated from the others by 2 or more weeks, the subjects underwent double-blind, randomly ordered exposure to a fog containing 0.5 mg/m" HN03 , a fog of distilled water (H 20), or air for 2 h, followed by, after a l-h rest break, exposure

TABLE 1 SUBJECT CHARACTERISTICS

Subject No. 1 2 3 4 5 6 7 8 9 10

Sex

M M F

M M F

M F F

M

Age (yr)

Height (em)

FEV 1* (L)

FEV 1 (% pred)

FVC* (L)

FVC (% pred)

Specific Airway Resistance t (L x em H2 O/U s)

25 26 26 21 28 23 25 23 31 31

170 165 160 178 168 168 170 168 180 180

3.18 3.25 2.96 4.68 3.52 3.35 4.43 3.11 2.94 4.72

80 88 99 108 94 103 112 96 95 109

4.00 3.64 3.35 5.64 4.01 3.77 5.38 3.60 3.75 5.41

82 81 94 108 87 96 110 92 100 101

1.5 2.0 2.2 1.8 1.4 1.7 2.1 2.0 4.6 1.3

• Mean of 18 preexposure baseline values (6 on each of 3 separate days). Mean of 15 preexposure baseline values (5 on each of 3 separate days).

t

to 0.20 ppm 0 3 for 3 h. The subjects brushed their teeth and gargled with antiseptic mouthwash (pH, 4.0) before each fog or air exposure in an effort to reduce oropharyngeal ammonia. Each subject exercised at the previously determined work load that produced a targeted ventilatory rate of 40 L/min for 50 min of each hour of the 5-h sequential exposures. The subjects chose to either cycle, jog, or alternate the two. Ventilatory rate, tidal volume, and respiratory rate were measured twice hourly as previously described. Specific airway resistance, FEV 1, and FVC were measured before and at each measurement/rest period during the fog or air exposure and before and at each measurement/rest period during the 0 3 exposure for a total of seven measurement points for each sequential exposure. Symptom questionnaires, consisting of an Il-point rating scale (0 = least, 10 = most) for each of 12 symptoms (chest pain, chest tightness, shortness of breath, cough, sputum production, throat irritation, wheezing, back pain, headache, eye irritation, abnormal taste, and nausea) were completed before and after fog or air exposure and before and after 0 3 , Baseline MCh responsiveness was determined in the afternoon the day before each sequential exposure. Postexposure MCh was determined after each sequential exposure, either when the subject was at his or her baseline SRaw (i.e., ± 50070 of the mean of all previous preexposure SRaw measurements) or within 1 h of the completion of the exposure, whichever occurred first. The timing of the before and after exposure MCh responsiveness studies differed by 24 ± 3 h. During the sequential exposure protocol, it became evident that the subjects had hyperresponsiveness to MCh compared with a group of normal subjects (PC 100 > 4.0 mg/ml) previously studied in our laboratory. To verify this finding, we asked the nonsensitive subjects to return to our laboratory 1 to 2 months after completion of the sequential exposures to perform MCh challenge studies. Of the original 21 nonsensitive subjects, 10 were located and agreed to participate. The MCh responsiveness of the 10 Os-sensitive subjects were restudied during the same period.

The Exposure Chamber and Atmospheric Monitoring All exposures took place in a 9 x 9 x 9 ft steel and glass chamber (Model W00327-3R; Nor-Lake, Inc., Hudson, WI), which was custom-built and designed to maintain chamber temperature and relative humidity within 0.5 0 C and 2070, respectively, ofthe set points (DSC 8500; Johnson Controls, Poteau, OK). Fogs were generated and monitored by methods previously described (8). Briefly, a stock solution of dilute HN0 3 (or distilled H 20) was atomized with a high-pressure, pump-nozzle system to generate a stable fog with a liquid water content (LWC) of NO.5 g/nr', Ninety percent of the chamber air was recirculated, 10070 was exhausted via a fog water collector (California Institute of Technology, Pasadena, CAl, and 10070 fresh air was introduced after filtration and humidification. Chamber air temperature was maintained at 22 ± 2 0 C. The fogs were monitored for LWC, volume medium diameter (VMD), and chemical constituency. Liquid water content was measured gravimetrically with 47-mm glass fiber filters (Type A/E; Gelman Sciences, Ann Arbor, MI) at the 10- and 40-min points during each hour. Fog droplet VMD was measured continuously with a phase/Doppler particle analyzer (Model 1100; Aerometrics, Mountain View, CAl. Fog water samples collected on filters (and eluted with 5 ml of H 20) at the to-min and 40-min points of each exposure hour were analyzed for HN03 with high performance ion chromatography, using a separator column (IonPac A 54A, PIN 37041; Dionex, Sunnyvale, CAl, a 2.5 ml/min flow rate, and eluent of 5.6 mM sodium bicarbonate and 4.8 mM sodium carbonate, and a 0.25 mM sulfuric acid regenerant. Fog water samples obtained at the 20- and 40-min points of each hour were analyzed for ammonium (NH 4 +) with a cation column (IonPac C53 PIN 37024; Dionex), a 1 ml/min flow rate, an eluent composed of 0.25 mM hydrochloric acid and 0.25 mM 2,3diaminopropionic acid, and a regenerant of 70 mM tetrabutylammoniumhydroxide. Initially, NH 4 + was measured from fog water collector samples; subsequently, nitrate cellulose

SEQUENTIAL EXPOSURE TO ACIDIC FOG AND OZONE

filters (0.45 urn pore size; Whatman, Inc., Clinton, NJ) were used because the former resulted in underestimation of the amount of NH 4 + present. Ozone was produced with a coronadischarge 0 3 generator (Model T 408; Polymetrics, Inc., San Jose, CA) and analyzed with an ultraviolet light photometer (Model 1008 PC; Dasibi, Glendale, CA). The 0 3 analyzer was calibrated biannually with a standard 0:; generator/analyzer instrument (Model 1009 IC; Dasibi) by the California Air Resources Board, and precision-checked on a monthly basis.

Statistical Analyses The mean changes in SRaw, FEV 1, and FVC over the 0 3 exposure were compared by twoway analysis of variance (ANOVA) to determine if preexposure to HN03 fog, H 20 fog, or air was associated with any statistically significant differences in these variables. Similarly, the mean changes in MCh responsiveness during the three sequential exposures were compared with twoway ANOVA. Changes in the respiratory rates and tidal volumes (calculated by subtracting the value obtained at the first measurement point from that at the last measurement point of each exposure period) during the sequential exposures werecompared with twowayANOVA. When a statistically significant p value was found in the twoway ANOVA calculations, a Newman-Keuls multiple range test was performed to determine which exposures were different. Differences between sensitive and nonsensitive groups of subjects wereanalyzed by the Mann-Whitney test. Symptom scores were categorized as: (1) lower respiratory (chest pain, chest tightness, shortness of breath, cough, sputum production, and wheezing); (2) upper respiratory (throat irritation, abnormal taste); and (3) nonrespiratory (back pain, headache, eyeirritation, nausea). Mean symptom scores for each of these categories and for total symptoms were compared by Friedman's test to determine ifthere were any significant differences among the three sequential exposures. Correlations among the changes in respiratory rate, tidal volume, FEV.. FVC, and SRaw were determined with linear regression analysis. In all of these analyses, a p value of ~ 0.05 was considered significant.

12 10

Fig. 1. Oa sensitivity determination. Frequency distribution of FEY, responses of 39 subjects after exposure to 0.20 ppm 0 3 for 3 h.

Number 8 of 6 Subjects 4 2

o -10

+5 0

-20

-30

-40

-50

-60

Change in FEV1 (%)

the fourth hour of exposure. The frequency distribution of changes in FEV 1, after 3 h of 0 3 exposure at 0.20 ppm is shown in figure 1. The change in FEV 1 was unimodally distributed, with most subjects (95070) having some decrement in FEV 1. The frequency distribution of changes in FVC was similarly shaped (data not shown), with 37 of 39 subjects experiencing decreases in their FVC from baseline, with a range of -2 to -46%. The mean changes in FEV 1 and FVC for the 39 subjects after 3 h of 0.2 ppm 0 3 exposure were -15.6 and -11.8%, respectively, and were highly correlated, with an r value of 0.95 (p < 0.(05). The frequency distribution of the effect of 0 3 on SRaw is shown in figure 2, with 40070 having ~ a 100070 increase over baseline. For the 18 sensitive subjects, the mean ± SEM respiratory rate increased by 9.4 ± 0.5 breaths/min, and the mean tidal volume decreased 0.38 ± 0.02 L, whereas for the nonsensitive subjects these values were + 4.2 ± 0.5 breaths/min and - 0.22 ± 0.02 L. The mean changes in respiratory rate and tidal volume were significantly different (p < 0.05 for both measurements) between the Os-sensitive and nonsensitive subjects. For the Os-sensitive subjects, the changes in respiratory rate and tidal volume were inversely correlated, with an r value of 0.55 (p < 0.03).

Results

There was a weak inverse correlation between changes in FEV 1 and SRaw (r = - 0.25, p > 0.5). Ofthe 18 Os-sensitive subjects, 10 participated in the sequential exposures, and the remaining eight declined. Five of those eight cited severity of symptoms as the main reason for withdrawing from the study. One of these eight was unable to complete the 3-h 0 3 screening process because of severe symptoms. He stopped after 2.5 h, after having experienced decrements in FEV 1 and FVC of 61 and 46070, respectively. Of the remaining two subjects, both started the sequential exposures and either failed to finish (n = 1) or had inadequate exposures (n = 1). Surprisingly, the M Ch challenge tests indicated that the Os-sensitive subjects were significantly more sensitive to MCh than the Os-nonsensitive subjects. Methacholine challenge tests on 10 nonsensitive subjects demonstrated a mean ± SEM PC 100 of 18.67 ± 4.54 mg/ml, whereas concurrently performed MCh challenge tests on the 10 sensitive subjects demonstrated a significantly lower mean PC 100 of 2.95 ± 0.80 mg/ml (p = 0.(05). (Previous studies in our laboratory have established a PC 100 of 4 mg/ml as the cutoff between normal airway responsiveness and hyperresponsiveness based on studies in 50 subjects selected from

10

Ozone-Sensitivity Determination During the Os-sensitivity screening process, 24 (62%) of the 39 subjects demonstrated a ~ 10070 decrement in FEV 1 after exercising at 40 L/min in 0.20 ppm 0 3 for as long as 4 h. Eighteen (46070) subjects demonstrated such a decrement after 3 h of exposure and thus were considered "sensitive" to 0 3 • Of the remaining 21, three stopped after 3 h, and six of the remaining 18 demonstrated a ~ 10070 decrement in FEV 1 at the end of

8

Fig. 2. 0 3 sensitivity determination. Frequency distribution of changes in specific airway resistance (SRaw) in 36 subjects after exposure to 0.20 ppm 0 3 for 3 h (data not available for three subjects).

6

Number of 4 SUbjects 2

o

-25

0

25

75

125

175

Change in SRaw (%)

>225

ARIS. CHRISTIAN. SHEPPARD, AND BALMES

88

A.

TABLE 2

10

METHACHOLINE RESPONSIVENESS OF 03"SENSITIVE AND NONSENSITIVE SUBJECTS

PC100 *

Subjects

PC100 *

1 2 3 4 5 6 7 8 9 10

4.00 2.57 2.00 0.84 1.04 4.00 8.52 1.63 0.52 4.38

11 12 13 14 15 16 17 18 19 20

41.50 17.70 32.00 33.90 27.86 3.25 2.83 16.00 3.61 8.00

Mean SEM

2.95 0.80

Subjects

a

0

03-nonsensitive

°3"sensitive

a-wa in FEV1 (%)

a a

-10

-20

-30

-40

lunch

18.67 4.54

i

I

j

3

baseline 1 2 --fog or air--

4 5 6 -------ozone-------

Fig 3. Sequential exposures. Mean ± SEM % changes in FE~ (A) and FVC (B) at baseline, and at hourly intervals during fog or air exposure (Hours 1 and 2), and during 0.20 ppm 0 3 exposure (Hours 4 to 6). Open squares = H20 fog/0 3; closed diamonds = HN03 fog/0 3; open circles = air/03.

B. 10

• The concentration in milligrams per milliliter of methacholine required to produce a 100% increase in specific airway resistance above baseline.

the general population.) The individual PC lOO values of the sensitive and nonsensitive subjects are displayed in table 2. The Os-sensitive and nonsensitive subjects did not differ with regard to age, sex, or health status.

a

0

CI-W\IGE in FVC -10

at

i5

(%) -20

-30

baseline 1 2 --fog or air--

Sequential Exposures The preexposure and postexposure mean values for SRaw, FEV1 FVC, MCh responsiveness, and symptom scores are displayed in table 3. The mean FEV l and FVC were unchanged from baseline during the fog and air exposures, and then decreased progressively during 0 3 exposure for all three exposure arms (figure 3). Although both H 20 fog/O, and HN03 fog/O, exposures were associated with smaller (mean ± SEM) decrements in FEV 1 than the air/O, exposure (-17.1 ± 3.7070 and -18.0 ± 4.3070, respectively, compared with - 26.4 ± 5.3070), only the difference between the H 20 fog/O, and the air/O, were significantly different (using a two-tailed alpha of 0.05). Although both fog/O, ex-

lunch i i i 3 4 5 6 -------ozone-------

TIME (hrs)

posures were also associated with a smaller mean decrement in FVC than the air/O, exposure (-13.6 ± 2.8070 and -13.6 ± 4.2070, respectively, compared with -19.9 ± 4.7070), the differences did not achieve statistical significance (p = 0.09). There were no significant differences in the mean changes in SRaw (figure 4A), MCh responsiveness, and symptom scores among the three exposures. The mean ± SEM changes in FEVland FVC for the 10 sequential exposure subjects in the Os-sensitivity determination (-25.9 ± 9.6070, -17.3 ± 8.6070) were

similar to the mean changes after the air/O, exposure. There were no significant differences in preexposure SRaw, FEV., FVC, MCh responsiveness, or symptom scores among the three sequential exposures (table 3). There was an increase in the mean respiratory rate and decrease in the mean tidal volume for each of the sequential exposures, mainly because of changes in these parameters during 0 3 exposure. The mean ± SEM changes in respiratory rate for the 10 subjects for the H 20 fog/Or, HN03 fog/Oj, and air/O, exposures were

TABLE 3 SEQUENTIAL EXPOSURE: PULMONARY FUNCTION RESULTS AND SYMPTOM SCORES· HN03 Fog/03 Before FEV1 , L FVC, L SRaw, L x cm H20/Us PC100 , mg/ml Symptom scores

3.62 4.20 1.8 1.43 2.0

(0.26) (0.33) (0.3) (0.30) (0.8)

H20 Fog/03

After

2.91 3.57 2.6 1.50 18.1

(0.18) (0.24) (0.4) (0.47) (5.8)

Definition of abbreviations: SRaw = specific resistance of the airways; PC100 increase in specific airway resistance above baseline. • Values are means with SEM shown in parentheses.

Before

3.67 4.21 2.0 1.13 2.3

(0.25) (0.30) (0.4) (0.25) (1.0)

= the concentration

After

2.99 3.62 2.6 1.10 22.2

(0.17) (0.22) (0.4) (0.33) (5.5)

Before

3.66 4.27 1.8 1.37 1.8

(0.23) (0.31) (0.2) (0.41) (1.0)

After

2.62 3.33 2.7 1.56 22.3

(0.15) (0.19) (0.5) (0.53) (6.5)

in mg/ml of methacholine required to produce a 100%

89

SEQUENTIAL EXPOSURE TO ACIDIC FOG AND OZONE

A.

50

25

a-w-.G: in

SRAW

0

(%)

-25

lunch i 3

baseline 1 2 --fog or air--

i i

4

5

i

6

-------Olone-------

B. 50

Fig. 4. Sequential exposures. A. Mean % changes in specific airway resistance (SRaw) at baseline, and at hourly intervals during fog or air exposure (Hours 1 and 2), and during 0.20 ppm 0 3 exposure (Hours 4 to 6). Mean respiratory rates (B) and tidal volumes (C)at the 20min and the 40-min points of each hour during fog or air exposure (Hours 1 and 2) and during 0.2 ppm 0 3 exposure (Hours 4 to 6). Open squares = H20 fog/0 3 ; closed diamonds = HN03 fog/0 3 ; open circles = air/0 3 .

40

RESPIRATORY RATE (breaths/min) 30

lunch ~I--'-~---'-~--T----'

20

o

2

···fog or

3

---olone-----·····

air----

c. 1.5

TIDAL VOLUM: 1.0 (L)

0.5 +-~~~""Iunch -I--.--,...-----.----i i 4 6 o 1 3 ···log or air··· ·········ozone········

TIME (hrs)

+14.0 ± 1.8, +6.4 ± 1.0, and +16.3 ± 2.8 breaths/min, respectively (figure 4B). The mean ± SEM changes in tidal volume for the H 2 0 fog/Os, HNQ3 fog/Os, and air/O, exposures were - 0.27 ± 0.07L, - 0.32 ± 0.07 L, and - 0.36 ±

0.10 L, respectively (figure 4C). These mean changes in respiratory rate and tidal volume were not significantly different among the three sequential exposures. There were no significant differences in the mean ventilatory rates among the

TABLE 4 EXPOSURE CHARACTERISTICS DATA *

Fog or air exposures LWC, g/m 3 VMD, J.1m HN03 , mg/m 3 RH, % Temp, °C Ozone exposure Ozone, ppm RH, % Temp,oC

H20 Fog/0 3

HN03 Fog/0 3

0.54 ± 0.1 6.47 ± 0.4 f\.I100 t 21.9 ± 0.7

0.55 ± 0.1 6.00 ± 0.2 0.43 ± 0.04 f\.I100t 22.0 ± 1.1

49.8 ± 0.3 21.8 ± 1.8

0.201 ± 0.005 46.0 ± 1.5 21.3 ± 0.4

0.201 ± 0.004 46.1 ± 2.5 21.5 ± 0.4

0.201 ± 0.005 49.7 ± 0.4 22.4 ± 0.7

Definftion of abbreviations: LWC = liquid water content; VMD = volume median diameter; RH • Data are mean ± SEM. t These exposures were characterized by visible tog; RH was not measured.

= relative humidity.

three exposures. There were no significant differences in the mean changes in symptom scores among the three sequential exposures (table 3). Changes in FVC and FEV 1 were closely correlated for all three sequential exposures, with r values between 0.82 and 0.97 (p < 0.(05). There was a high inverse correlation (r = 0.88, p < 0.(02) between the change in respiratory rate and change in tidal volume in the air/O, exposure, but these variables were not significantly correlated during the HN03 fog/O, and H 2 0 fog/O, exposures. There were weak inverse correlations between changes in FEV 1 and SRaw: r == - 0.66 (p = 0.09) for the H 2 0 fog/O, exposures, r = - 0.19 (P > 0.5) for the HN03 fog/O, exposures, and r = - 0.57 (p = 0.18)for the air/O, exposures. There were no other statistically significant correlations between changes in FVC, FEV 1, SRaw, respiratory rate, and tidal volume. The environmental characteristics of the three sequential exposures are listed in table 4. The H 2 0 fog and HN03 fog differed only in the concentration of HN03 • The 0 3 exposures were not different with regard to 0 3 concentration, temperature, or relative humidity.

Fog and Filtered Air Exposures Analysis of the data from the 2-h H 2 0 fog, HN03 fog, and air exposures revealed no significant changes in FEV 1, FVC, or SRaw. The mean ± SEM percent changes in FEV h after 2 h of exposure to H 2 0 fog, HN03 fog, and air were + 0.8 ± 1.3010, + 1.3 ± 2.0010, and + 1.2 ± 0.9070, respectively. The corresponding mean ± SEM percent changes in FVC were -1.0 ± 1.2010, -1.0 ± 1.7010, and - 0.6 ± 0.8070, and in SRaw were - 7 ± 9.2010, -15 ± 9.2010, and +6 ± 11.7070. The data from the air exposures, illustrated in figures 3 and 4, show no evidence of exercise-induced bronchospasm in our subjects. The mean changes in symptom scores for the H 20 fog, HN03 fog, and air exposures were + 0.4, + 1.9, and 0, respectively, and were not significantly different. Although respiratory rates increased during fog and air inhalation, the mean changes in respiratory rate werenot significantly different among the three exposures. Tidal volume changed variably during fog and air inhalation, with no significant differences among the three exposures. Ammonium concentrations in fog water rose with time across exposures, with the highest level seen during the last half of the second exposure hour when two

90

ARIS, CHRISTIAN, SHEPPARD, AND BALMES

subjects were exercising together. The mean ammonium concentration during HN0 3 fog exposures resulted in an approximately 15% (range,S to 450/0, with only one value> 30%) neutralization of the acid present. Discussion

The results of this study indicate that a sequential exposure to a HN0 3-containing fog and 0 3 does not cause greater decrements in pulmonary function than an exposure to 0 3 alone in "Os-sensitive" healthy, exercising subjects. We hypothesized that a "preexposure" to a HN03 containing fog would have an additive or synergistic effect on pulmonary function responses to an 0 3 exposure. However, our results suggest that both HN0 3 containing and H 20 fogs ameliorate 0 3 induced decrements in FEVland FVC. Our experiments were designed to stimulate a worst-case scenario by using HN03 concentrations greater than the highest ambient measurements and by performing prolonged exposures with vigorous exercise. In keeping with this approach, Os-sensitive subjects were selected as subjects who would be most likely to develop enhancement of Os-induced pulmonary function responses after preexposure to an acidic fog. Because we studied a selected segment of the population, we cannot be certain that HN03 fog would not potentiate 0 3 induced decrements in lung function in persons less sensitive to 0 3 , This is unlikely, however, since the subjects we selected had a wide range of 0 3 sensitivity (i.e., the decrements in FEV 1 varied from 15 to 490/0), yet no subject demonstrated convincing potentiation of this effect after exposure to HN03 fog. Sequential exposure to air and 0 3 resulted in greater decreases in FEVland FVC than did sequential exposure to HN0 3 fog and 0 3 or to H 20 fog and 0 3 , In an attempt to determine whether previous exercise in air worsens, or fog mitigates against, Os-induced pulmonary function changes, we compared the 0 3 sensitivity screening results with the sequential exposure data for the 10 subjects. On the Os-sensitivity determination day, the subjects, who had no previous exposure that would have led to airway drying, experienced decrements in FEV 1 (-25.9 ± 9.6070) and FVC (-17.3 ± 8.6070) that were similar to those on the air/O, day (changes in FEV l and FVC of -26.4 ± 5.3070 and -19.9 ± 4.7%, respectively). Thus, airway drying is an unlikely explanation for the larger deere-

ments in the air/O, exposures. Rather, exposure to fog appears to ameliorate the effects of a subsequent 0 3 exposure on FEVland FVC. Changes in SRaw and MCh responsiveness over the three sequential exposures were not significantly different. Interestingly, there was a poor correlation between FEV 1 and SRaw, indicating that changes in these variables may be due to different mechanisms. We speculate that the relatively larger decrements in FEV 1 are probably due to Os-induced substernal discomfort, which hinders taking a deep breath (and thus results in a restrictive ventilatory defect), and the increases in SRaw are probably due to an element of bronchospasm and/or enhanced airway secretions (9). Despite animal toxicologic and epidemiologic data which suggest that acidic pollutants may enhance the toxicity of 0 3 , our results indicate that preexposure to a HN0 3-containing fog decreases the pulmonary toxicity of a subsequent 0 3 exposure when spirometric end points are studied. The H 20 fog and HN0 3 containing fog exposures produced very similar results, suggesting that it is fog itself that is responsible for this effect rather than HN0 3 • The exact reason for this fog-induced "protection" is not known. It is possible that fog inhalation produces a thin aqueous coating over the bronchial epithelium that shelters it from Os-induced oxidative injury. However, a tOO-min exposure to a fog with a 0.5 g/m" LWC would result in the deposition of only approximately 2 g of H 20 even if every inhaled droplet impacted on the epithelium. A more plausible explanation of that fog is a secretagogue acting to enhance mucous and aqueous secretions from the epithelial layer. In this way, the natural secretions produced by fog exposure might protect against a secondary 0 3 exposure. Another possible mechanism is the induction of antioxidant pathways that could lessen the effects of 0 3 , To date, this experiment is the only systematic study of a HN0 3-containing fog in humans. The 2-h HN03 fog exposure in which the concentration of HN03 inhaled was at least an order of magnitude greater than the highest ambient levels resulted in no changes in pulmonary function in exercising healthy subjects. As a caveat, however, our ability to detect small changes in pulmonary function was limited by our small sample size. We calculated that our study had almost 100070 power to detect a difference of 10070 in the change in FEV 1 between the con-

trol and HN0 3 fog exposure groups with 10 subjects serving as their own controls (using a two-tailed alpha of 0.05). Our results are consistent with previous studies of sulfuric acid fog by Avol and coworkers (10), sulfuric acid aerosols by Utell and colleagues (11, 12), and sulfuric and hydroxymethanesulfonic acid fogs by our laboratory (8). Small decrements in pulmonary function have been found in allergic adolescents (13) and in subjects with asthma (14)after near-ambient level acidic aerosol exposures, but not in healthy subjects. One of the major confounding variables in acidic pollutant exposures in humans is the level of oropharyngeal NH3 • Although we made an effort to decrease oropharyngeal NH 3 with oral hygiene before each exposure, we did not measure oral NH 3 levels to verify this. We did measure fog water ammonium concentrations and found a wide range, with the mean concentration resulting in a 15070 neutralization of inhaled HN0 3 • In an ambient exposure, the percentage neutralization of an acidic pollutant might well be higher. Furthermore, oropharyngeal concentrations of ammonia are probably higher than fog water concentrations, which would result in a greater percentage neutralization of inhaled acidic droplets (15). It is possible that our failure to find HNOrcontaining fog-induced pulmonary function decrements was due to high oropharyngeal NH 31eveis in our subjects, especially since they were allowed to eat and drink during the exposures. The results of the Os-sensitivity screening demonstrated that the distribution of pulmonary function responses was unimodal and negatively skewed, with almost all subjects experiencing decrements in FEV 1 and increases in SRaw (figures l and 2). This information does not support the concept that the general population is easily divided into 0 3 responders and nonresponders. Our data suggest that the entire population probably experiences some degree of 0 3 induced oxidant injury and that the severity of the injury pattern is dose- and individual-dependent. The mean decrements in FEVland FVC reported here are similar to those in studies by Schelegle and Adams (16), McDonnell and coworkers (17), Folinsbee and colleagues (18), and Gong and coworkers (19), all of whom used similar doses of 0 3 (0.09 to 0.21 ppm) in young, healthy, exercising subjects. Most importantly, our results suggest that subjects with increased airway re-

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SEQUENTIAL EXPOSURE TO ACIDIC FOG AND OZONE

sponsiveness may be a group of individuals who are susceptible to greater 0 3 induced pulmonary dysfunction. These results are contrary to those reported by McDonnell and coworkers (20), who found no association between nonspecific airway responsiveness to histamine and 0 3 sensitivity. Because McDonnell and coworkers anticipated that the magnitude of an 0 3 response would be associated with baseline airway responsiveness, they postulated that both the size of their sample and the range of PCso values (2.7 to 20.0 mg/ml) in their subjects may have been too small to detect this relationship. Our study design, which selected young, healthy, athletic subjects, provided a broader range of PC 100 values (0.5 to 41.5 mg/ml) and 0 3 responses. Our results are supported by a recent study by Hackney and colleagues (21), which indicated that 8 of 12 subjects ("responders"), chosen because of 0 3 sensitivity based on FEV 1 changes, demonstrated enhanced responsiveness to MCh. Furthermore, Eschenbacher and coworkers (22) exposed both normal subjects and thosewith asthma to 0.4 ppm 0 3 and found greater decrements in FEV 1 in the subjects with asthma. These studies, taken together, indicate that persons with MCh hyperresponsiveness, whether clinically asthmatic, atopic, or healthy, appear to be a subgroup that has a greater sensitivity to 0 3 • In conclusion, the results of these sequential exposures with fogs and 0 3 suggest that preexposure to fog lessens the effects of a subsequent 0 3 exposure on pulmonary function. Nitric-acid-containing fog alone produced no significant changes in pulmonary function or symptoms. As is often the case in controlled human exposure studies, we had insufficient power to detect small effects caused by HN0 3 fog exposures. Because the sequential fog/O, exposures actually produced findings in a direction opposite to that anticipated (i.e., protection, rather than enhancement), failure to find en-

hancement is unlikely to be due to our relative lack of statistical power. Most importantly, our results also suggest that healthy persons with increased MCh responsiveness may have enhanced sensitivity to 0 3 • Because asymptomatic nonspecific airway hyperresponsiveness is common, the issue of whether such persons are at increased risk of Os-induced pulmonary toxicity is of obvious importance for public health. Acknowledgment The writers thank Theresa Klevenfor her valuable help in preparing the manuscript, and Dane Westerdahl for his advice regarding the design of the experimental protocol. References 1. Sun M. Acid aerosols called health hazard. Science 1988; 240:1727. 2. Thurston GD, Ito K, Lippman M, Hayes C. Reexamination of London, England mortality in relation to exposure to acidic aerosols during 19631972 winters. Environ Health Perspect 1989; 79: 73-82. 3. Firket M. The causes of accidents which occurred in the Meuse Valley during the fogs of December, 1930. Bull Acad R Med (Belg) 1931; 11: 683-741. 4. Last JA, Cross CEo A new model for health effects of air pollutants: evidence for synergistic effects of mixtures of ozone and sulfuric acid aerosols on rat lungs. J Lab Clin Med 1978; 91:328-39. 5. Warren DL, Last JA. Synergistic interaction of ozone and respirable aerosols on rat lungs: III. ozone and sulfuric acid aerosol. ToxicolAppl Pharmacol 1987; 88:203-6. 6. Hering SV.The nitric acid shootout: field comparison of measurement methods. Los Angeles: University of California at Los Angeles, Chemical Engineering Department, 1986 (CARB contracts A4-164-32 and A5-068-32). 7. Knudson RJ, Lebowitz MD, Holberg CJ, Burrows B. Changes in the maximum expiratory flowvolume curve with growing and aging. Am Rev Respir Dis 1983; 127:725-34. 8. Aris R, Christian D, Sheppard D, Balmes JR. Acid fog-induced bronchoconstriction: the role of hydroxymethanesulfonic acid. Am Rev Respir Dis 1990; 141:546-51. 9. Lippman M. Health effects of ozone: a critical review. J Air Pollut Control Assoc 1989:39:672-94. 10. Avol EL, Linn WS, Wightman LH, Whynot JD, Anderson KR, Hackney JD. Short-term respiratory effects of sulfuric acid in fog: a laboratory

study of healthy and asthmatic volunteers. J Air Pollut Control Assoc 1988; 28:258-63. 11. Utell MJ, Morrow PE, Hyde RW. Latent development of airway hyperreaetivity in human subjects after sulfuric acid aerosol exposure. J Air Pollut Control Assoc 1984; 34:931-5. 12. Utell MJ, Morrow PE, Hyde RW. Airway reactivity to sulfate and sulfuric acid aerosols in normal and asthmatic subjects. J Air Pollut Control Assoc 1987; 34:431-5. 13. Koenig JQ, Pierson WE, Horike M. The effects of inhaled sulfuric acid on pulmonary function in adolescent asthmatics. Am Rev Respir Dis 1983; 128:221-5. 14. UtellMJ, MorrowPE, MariglioJA, etal. Exercise, age and the route of inhalation influence airway response to sulfuric acid aerosols in exercising asthmatics (abstract). Am Rev Respir Dis 1984; 129:AI75. 15. Utell MJ, Mariglio JA, Morrow PE, Gibb FR, Speers DM. Effects of inhaled acid aerosols on respiratory function: the role of endogenous ammonia. J Aerosol Med 1987; 2:141-7. 16. Schelegle ES, Adams WC. Reduced exercise time in competitive simulations consequent to low level ozone exposure. Med Sci Sports Exer 1986; 18:408-14. 17. McDonnell WF, HorstmanDH, HazuchaMJ, et al. Pulmonary effects of ozone exposure during exercise: dose-response characteristics. J Appl Physiol 1983; 54:1345-52. 18. Folinsbee LJ, Bedi JF, Horvath SM. Pulmonary function changes after l-hour continuous heavy exercise in 0.21 ppm ozone. J Appl Physiol 1984; 57:984-8. 19. Gong H, Bradley PW, Simmons MS, Tashkin DP. Impaired exerciseperformance and pulmonary function in elite cyclists during low-level ozone exposure in a hot environment. Am Rev Respir Dis 1986; 134:726-33. 20. McDonnell WF, Horstman DH, Abdul-Salaam S, Raggio LJ, Green JA. The respiratory responses of subjects with allergic rhinitis to ozone exposure and their relationship to nonspecific airway activity. Toxicol Ind Health 1987; 3:507-17. 21. Hackney JD, Linn Ws, Shamoo DA, Avol EL. Responses of selected reactive and nonreactive volunteers to ozone exposure in high and low pollution seasons. In: Schneider T, et al. eds. Atmospheric ozone research and its policy implications. Amsterdam: Elsevier Science Publishers BY., 1989; 311-8. 22. Eschenbacher WL, Ting RL, Kreit JW, Gross KB. Ozone-induced lung function changes in normal and asthmatic subjects and the effect of indomethicin. In: Schneider T, et al eds. Atmospheric ozone research and its policy implications. Amsterdam: Elsevier Science Publishers BY., 1989;439-9.

The effects of sequential exposure to acidic fog and ozone on pulmonary function in exercising subjects.

In Southern California coastal regions, morning fog is often acidified by the presence of nitric acid (HNO3). Peak exposure to ozone (O3) usually occu...
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