Lung Function and Inflammatory Responses in Healthy

Young Adults Exposed to 0.06 ppm Ozone for 6.6 Hours

Chong S. Kim1, Neil E. Alexis2, Ana G. Rappold1, Howard Kehrl1, Milan J. Hazucha2, John C. Lay2,

Mike T. Schmitt1, Martin Case1, Robert B. Devlin1, David B. Peden2, and David Diaz-Sanchez1

1

U.S. Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Research Triangle Park, North

Carolina; and 2Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina, Chapel Hill, North Carolina

Rationale: Exposure to ozone causes a decrease in spirometric lung

function and an increase in airway inflammation in healthy young

adults at concentrations as low as 0.08 ppm, close to the National

Ambient Air Quality Standard for ground level ozone.

Objectives: To test whether airway effects occur below the current

ozone standard and if they are more pronounced in potentially

susceptible individuals, such as those deficient in the antioxidant

gene glutathione S-transferase mu 1 (GSTM1).

Methods: Pulmonary function and subjective symptoms were measured in 59 healthy young adults (19–35 yr) immediately before and

after exposure to 0.0 (clean air, CA) and 0.06 ppm ozone for 6.6 hours

in a chamber while undergoing intermittent moderate exercise. The

polymorphonuclear neutrophil (PMN) influx was measured in 24

subjects 16 to 18 hours postexposure.

Measurements and Main Results: Subjects experienced a significantly

greater (P 5 0.008) change in FEV1 (6 SE) immediately after

exposure to 0.06 ppm ozone compared with CA (21.71 6 0.50%

vs. 20.002 6 0.46%). The decrement in FVC was also greater (P 5

0.02) after ozone versus CA (22.32 6 0.41% vs. 21.13 6 0.34%).

Similarly, changes in %PMN were greater after ozone (54.0 6 4.6%)

than CA (38.3 6 3.7%) exposure (P , 0.001). Symptom scores were

not different between ozone versus CA. There were no significant

differences in changes in FEV1, FVC, and %PMN between subjects

with GSTM1-positive and GSTM1-null genotypes.

Conclusions: Exposure of healthy young adults to 0.06 ppm ozone for

6.6 hours causes a significant decrement of FEV1 and an increase in

neutrophilic inflammation in the airways. GSTM1 genotype alone

appears to have no significant role in modifying the effects.

Keywords: pulmonary function; airway inflammation; polymorphism;

ozone exposure; exercise

Ozone is a major component of photochemical smog. Controlled human exposure studies have been critical in demonstrating that it can cause decrements in lung function (1–7) and

lung inflammation (8–11). Although the majority of these

studies involved exposures to relatively high (0.1–0.4 ppm)

concentrations for short periods of time (typically 2 h), prolonged exposure studies at lower levels were largely responsible

for the U.S. Environmental Protection Agency (EPA) lowering

the National Ambient Air Quality Standard for ground level

ozone in 2008. Several studies have now confirmed the initial

observation of Horstman and colleagues (4) that exposure of

healthy young adults to 0.08 ppm ozone for 6.6 hours while

undergoing intermittent moderate exercise will result in a significant drop (5–8%) in FEV1 (5, 6). Similarly, studies using this

exposure regime demonstrated that 0.08 ppm ozone can initiate

inflammatory responses in the lungs of healthy young adults (9),

characterized by increases in polymorphonuclear neutrophils

(PMN) in bronchoalveolar lavage fluid collected 24 hours

postexposure. Recently, we have confirmed and extended these

findings in 15 healthy young adults by showing a significant

increase in sputum neutrophilic inflammation 18 hours postexposure to 0.08 ppm ozone (12).

Recent community-based air pollution studies using emergency department records have reported associations for increased risk of pediatric asthma at very low ozone concentrations

(13). This has led some to question whether adverse responses

could occur in healthy adults at concentrations below the current standard. However, only two controlled human exposures

have investigated this and none to our knowledge have addressed inflammatory effects. Adams reported that healthy

young adults exposed to multiple ozone concentrations experienced a significant decrease in lung function at 0.08 ppm but not

at 0.06 ppm or 0.04 ppm (7). However, a secondary analysis by

others of the same data concluded that 0.06 ppm may have

induced a change in FEV1 (14). In 2009, Schelegle and colleagues (15) reported that healthy young volunteers exposed to

step-wise ozone concentrations experienced a significant decrement of FEV1 at concentrations as low as 0.07 ppm but not at

0.06 ppm.

Large heterogeneity in responses to ozone between individuals has been reported (16, 17). Ozone exerts oxidant stress and

results in airway inflammation, and therefore genes that modulate inflammation and antioxidant defense mechanisms have

been proposed as potential effect modifiers. In particular,

glutathione S-transferase mu 1 (GSTM1) has attracted attention

as it is present in the airways, and gene deletions (GSTM1-null

genotype) can be present in up to 50% of the population,

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

Inhalation of ozone causes decrements in lung function and

an increase in airway inflammation at concentrations near

the current Ambient Ozone Standard. It is not known what

the effects are at concentrations lower than this.

What This Study Adds to the Field

This study reports that acute exposure to ozone for 6.6

hours at a level of 0.06 ppm (a level below the current

Ambient Ozone Standard) causes significant effects on

pulmonary function and airway inflammation in healthy

young adults.

(Received in original form November 9, 2010; accepted in final form January 7, 2011)

Supported by U.S. Environmental Protection Agency Internal Fund, U.S. Environmental Protection Agency cooperative agreement CR83346301, and National

Institutes of Health grants RC1ES018417 and R01ES012706 (D.B.P.).

Correspondence and requests for reprints should be addressed to Chong S. Kim,

Ph.D., Environmental Public Health Division (MD-58B), National Health and

Environmental Effects Research Laboratory, U.S. Environmental Protection

Agency, Research Triangle Park, NC 27711. E-mail: kim.chong@epa.gov

This article has an online supplement, which is accessible from this issue’s table of

contents at www.atsjournals.org

Am J Respir Crit Care Med Vol 183. pp 1215–1221, 2011

Originally Published in Press as DOI: 10.1164/rccm.201011-1813OC on January 7, 2011

Internet address: www.atsjournals.org
resulting in complete absence of the enzyme. This polymorphism has been associated with reduced lung function (18, 19),

and numerous epidemiology and controlled exposure studies

have suggested that it may confer susceptibility to increased

airway inflammation to ozone as well as other oxidant air

pollutants (20, 21).

More than 100 million people in the United States now live

in the counties that do not meet the current ozone standard,

and public health consequences are enormous. In the present

study, we performed a randomized controlled human exposure

crossover study and investigated specifically if exposure of

healthy young adults to 0.06 ppm ozone for 6.6 hours with

exercise would cause measurable airway effects assessed by

pulmonary function (chosen a priori to be FEV1 and FVC) and

inflammatory markers (sputum PMN). We also examined if

responses were more pronounced in individuals with the

GSTM1-null genotype. Some of the preliminary results of

these studies have been previously reported in the form of

abstracts (22).

METHODS

Subjects

Fifty-nine healthy young adult subjects aged 19 to 35 years, with no

history of smoking in the past 2 years, completed the study. All subjects

underwent a screening procedure that included a complete medical

history and physical examination and a pregnancy test for females.

Exclusion criteria included respiratory illness or symptoms within 4

weeks or a positive pregnancy test. All subjects were genotyped for

GSTM1. The study protocol was approved by the Institutional Review

Board at the University of North Carolina Medical School in Chapel

Hill and the EPA, and informed consent was obtained from all subjects

before their participation in the study. Subject characteristics and

baseline lung function test values are shown in Table 1.

Study Design

The study design was similar to those used in previous studies (4, 7)

to facilitate comparison. Each subject was exposed to 0.00 ppm (clean

air, CA) and 0.06 ppm ozone for 6.6 hours with moderate exercise

in a stainless steel chamber (4 3 6 3 3.2 m). Exposures were randomized, double-blinded, and separated by at least 1 week. Minute

ventilation (VE) was measured hourly and exercise levels adjusted to

VE 5 20 L/min/m2 body surface area to ensure that subjects breathed

consistently throughout exposure. Spirometric lung function and

symptom scores were assessed immediately before and after the 6.6-

hour exposure period. Sputum was collected the next morning approximately 16 to 18 hours postexposure. Exposures were conducted only

during the cool weather season in Chapel Hill (November–March) to

minimize exposure to elevated ambient ozone.

Study Protocol

Training day. On a training day, all subjects were trained for lung function

measurements. Settings for the treadmill and bicycle ergometer required

to produce a desired value of VE was determined for each subject.

Exposure day. On the day of each exposure, subjects were assessed

for vital signs, completed a symptom questionnaire, and performed

preexposure lung function spirometry and body plethysmography

maneuvers. They then entered the chamber set for the appropriate

condition (CA or 0.06 ppm ozone) and began exercising for a 50-minute

period at 20 L/min/m2 body surface area in VE followed by a 10-minute

rest period. The exercise session was repeated six times alternating between the treadmill and bicycle with VE maintained within 6

2 L/min of the subject’s target value. Subjects were also given 35

minutes to eat lunch. Thus, they remained in the chamber for a total of

6.6 hours, during which ventilatory parameters (VE, VT, breathing

frequency) were measured and electrocardiac signals, heart rate, and

blood oxygen saturation monitored continuously to ensure subject

safety. Subjects were exposed in pairs (of the same sex). At the end of

exposure, spirometric and plethysmographic lung function measurements were performed and a symptom questionnaire was obtained.

Follow-up day. On the next morning, subjects returned to the

laboratory and sputum samples were collected.

Measurements

Spirometry was performed on a 10.2-L dry seal digital spirometer

interfaced to a computer (SensorMedics Model 1022; SensorMedics;

Palm Springs, CA). At least three sets of qualified data were obtained

and the largest value selected for FEV1 and FVC as per American

Thoracic Society guidelines (23). Pulmonary function on all subjects

was measured using one dedicated spirometer and by one certified

pulmonary function technician to minimize variability. Specific airway

resistance (sRaw) was assessed by body plethysmography (SensorMedics Model V6200) and the average of two highest values out of

three to five measurements obtained. Measurements were performed

before and immediately after exposure for use in endpoint analysis.

Additional spirometry measurements were performed in the chamber

during 10-minute rest periods primarily for monitoring subject safety

and were not necessarily performed by the same technician.

Symptoms were assessed before and after 6.6-hour exposure. The

subjects were asked to rate the severity of cough, pain on deep inspiration

(PDI), shortness of breath (SOB), and throat irritation on a five-point

scale ranging from 0 (none) to 4 (most severe). Subjects recorded the

severity score directly on the computerized questionnaire. Total symptom

severity was obtained by adding scores of all four symptoms.

GSTM1 genotypes were determined using real-time polymerase

chain reaction as previously described by Gilliland and colleagues (24)

from DNA isolated from white blood cells using the QIAamp DNA

Mini Kit (Qiagen Inc., Valencia, CA).

Sputum Collection and Analysis

Sputum samples were obtained and processed by the method described

by Alexis and colleagues (25). Briefly, subjects underwent sputum

TABLE 1. SUBJECT CHARACTERISTICS AND BASELINE LUNG FUNCTION MEASURES

All Subjects Males Females GSTM1-p GSTM1-n

No. 59 27 32 30 29

Age, yr 25.0 (0.5) 26.1 (0.9) 24.0 (0.5) 24.9 (0.8) 25.1 (0.7)

Height, cm 171.2 (1.2) 178.9 (1.2) 164.7 (1.0) 172.2 (1.5) 170.2 (1.9)

Weight, kg 70.5 (1.8) 78.8 (2.3) 63.4 (2.1) 71.5 (2.5) 69.4 (2.6)

BSA, m2 1.8 (0.03) 2.0 (0.03) 1.7 (0.03) 1.8 (0.04) 1.8 (0.04)

BMI, kg/m2 23.8 (0.48) 24.5 (0.72) 23.3 (0.64) 23.9 (0.68) 23.8 (0.69)

FEV1, L 4.02 (0.10) 4.64 (0.12) 3.49 (0.07) 4.14 (0.12) 3.89 (0.16)

FVC, L 4.83 (0.13) 5.69 (0.14) 4.10 (0.11) 4.94 (0.17) 4.72 (0.21)

FEV1/FVC, % 84.1 (0.8) 82.0 (1.1) 85.8 (1.2) 84.6 (1.2) 83.5 (1.2)

FEF25-75%, L/s 4.22 (0.14) 4.64 (0.23) 3.87 (0.14 4.39 (0.21) 4.05 (0.18)

FEFmax, L/s 9.03 (0.25) 10.61 (0.28) 7.69 (0.17) 9.49 (0.35) 8.54 (0.34)

Definition of abbreviations: BMI 5 body mass index; BSA 5 body surface area; FEF25-75% 5 forced expiratory flow between 25

and 75% of FVC; FEFmax 5 maximum forced expiratory flow; GSTM1-n 5 glutathione S-transferase mu 1 null; GSTM1-p 5

glutathione S-transferase mu 1 positive.

Values are mean (6 SEM)

1216 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 183 2011
induction with hypertonic saline. Acquired samples were subjected

to plug selection and subsequent treatment with dithiothreitol. Cell

viability and total cell counts were evaluated and differential cell

counts examined (Hema-Stain3; Fisher Scientific). Acquired sputum

samples considered acceptable for processing had a minimum of 75 mg

of selected plug material, cell viability greater than 50%, and squamous

epithelial cells less than 40%. All sample processing and slide

preparations were performed on the same day of collection.

Ozone Generation and Monitoring

Ozone was generated by a silent electric discharge method (Model 502;

Meckenheim, Bonn, Germany) and introduced into the chamber that

was maintained at 22 6 1.08C and 40 6 5% relative humidity. The

concentration of ozone was continuously monitored using ultraviolet

photometric analyzers (TECO Model 49; Thermo Scientific, Franklin,

MA) that were periodically calibrated for 6 5% accuracy by NIST

traceable ozone calibrator (TECO Model 49PS).

Data and Statistical Analyses

The lung function endpoints were expressed as percent changes from

the preexposure (or baseline) values for each subject. Neutrophil

content in the sputum samples was expressed as percent of total cell

count (%PMN) and the measurements after each exposure were

compared. Linear mixed-effects models with a subject-specific random

intercept was used to test changes in response endpoints between clean

air and ozone exposures at the group level to account for subject-level

variability and repeated measures. The effect of GSTM1 and separately sex was examined using a two-factor mixed-effects model with

repeated measures on a single factor, exposure and subject-level

random effects. We report the magnitude and direction of the expected

change along with its associated 95% equal two-tail confidence intervals. R statistical software (Version 2.10.1) was used for the

analyses. a of 0.05 was used to determine statistical significance.

RESULTS

Exercise and Minute Ventilation

Means of six hourly measurements of VE, VT, breathing frequency, and heart rate during 6.6-hour exposure to CA and ozone

are summarized in Table 2. Overall, there was no difference in

both ventilation parameters and heart rates between CA and

0.06 ppm ozone exposure.

Exposure to 0.06 ppm Ozone Causes Decrements

in Lung Function

The primary hypothesis tested in this study was that exposure to

0.06 ppm ozone would decrease FEV1 and FVC after 6.6 hours.

The results are summarized in Table 3. Exposure to ozone

resulted in a 1.71 6 0.50% (mean 6 SEM) decrease in FEV1

compared with virtually no change (0.002 6 0.46%) after

exposure to CA (Figure 1A). Thus, relative to CA, exposure

to 0.06 ppm ozone for 6.6 hours resulted in a 1.71 6 0.64%

decrement in FEV1 (P 5 0.008). These decrements did not

appear to be driven by a small subset of subjects (Figure 1B). Of

the 59 individuals studied, only three subjects showed greater

than 10% drop after ozone exposure. Similarly, FVC decreased

by 2.32 6 0.41% after ozone exposure versus 1.13 6 0.34% after

CA (Figure 1C). Ozone exposure thus caused a relative decrement of 1.19 6 0.51% (P 5 0.02). Again, individual response to

ozone exposure was mostly within 6 5% change, as shown in

Figure 1D. Changes in other lung function parameters (forced

expiratory flow between 25% and 75% of FVC, maximum

forced expiratory flow, and sRaw) were not significant.

A second aim of the study was to determine the role of

GSTM1 in determining responses to ozone. Although both

genotypes had decrements in FEV1 after ozone exposure

relative to air, changes were only statistically significant for

GSTM1-positive subjects (Figure 2A). However, the difference

in FEV1 response between GSTM1-null and -positive subjects

was not statistically significant (P 5 0.72). Similarly, females

had a significant decrement in CA-adjusted FEV1 (1.93 6

0.88%, P 5 0.02), whereas males did not (1.45 6 0.95%, P 5

0.14), but the difference between sexes was not significant (P 5

0.66). No differences between GSTM1-null versus -positive and

males versus females were seen for FVC (Figure 2B).

Exposure to 0.06 ppm Ozone Causes

Pulmonary Inflammation

This study is the first to examine ozone concentrations below the

current standard to cause pulmonary inflammation. The results

are summarized in Table 4. Graphic illustration in Figure 3A

shows that ozone exposure alters the airway milieu as evidenced

by increases in %PMN in induced sputum samples. After air

exposure, %PMN averaged 38.3 6 3.7%. In contrast, ozoneexposed samples averaged 54.0 6 4.6%. Thus, relative to clean

air, ozone exposure resulted in a 15.7 6 3.1% increase in %PMN

for the whole group (P , 0.002). Of the 24 subjects studied, all

but 5 subjects showed an ozone-induced increase in %PMNs and

10 showed greater than or equal to 20% increase (Figure 3B).

Figure 4 shows a significant increase in ozone-induced

%PMN for both GSTM1-null (20.0%; 95% CI, 11.0–29.0; P 5

0.001) and GSTM1-positive subjects (11.3%; 95% CI, 2.3–20.3;

P 5 0.02). Those carrying the null allele had a stronger response

(P 5 0.001) than those carrying the positive allele (P 5 0.02);

however, the estimate of the modifying effect of GSTM1 did not

reach significance (P 5 0.17; also see Table 4). Both males

(24.2%; 95% CI, 15.8–32.6; P 5 0.001) and females (8.5%; 95%

CI, 0.79–16.20; P 5 0.03) had statistically significant increases in

ozone-induced %PMNs. The modifying effect of sex was

significant (P 5 0.009). The changes in %PMN were not

accompanied by changes in total cell numbers for the whole

group or any subgroup after ozone versus air. Total cell counts

in sputum samples were 5.05 (6 0.82) 3 106 after CA and 6.93

(6 1.52) 3 106 after ozone (P 5 not significant [NS] vs. CA) for

the whole group.

Symptom Questionnaire

Of 56 subjects who had no symptoms at baseline, 20 subjects

reported symptoms after either CA or 0.06 ppm ozone exposure.

The most commonly reported symptom was throat irritation

followed by shortness of breath, pain on deep inspiration, and

cough. The mean (6 SEM) total symptom score was 0.43 6 0.11

for CA and 0.41 6 0.11 for ozone (P 5 NS versus CA). For

genotype subgroups, total symptom score was 0.40 6 0.16 for CA

and 0.47 6 0.17 for ozone in GSTM1-positive subjects and 0.46 6

0.16 for CA and 0.35 6 0.13 for ozone in GSTM1-null subjects

(P 5 NS versus CA for both groups). The score and nature of

the symptoms were similar between CA and ozone exposures.

DISCUSSION

In 2008 the EPA revised National Ambient Air Quality Standard

for ground level ozone down to 0.075 ppm (3-yr average of the

TABLE 2. BREATHING PARAMETERS AND HEART RATE

OF SUBJECTS DURING 6.6-HOUR EXPOSURE

VE (L/min) VT (L) F(min-1) Heart Rate (min-1)

Clean air 37.1 (0.2) 1.38 (0.05) 29.1 (0.9) 128.4 (1.1)

Ozone 36.5 (0.2) 1.37 (0.05) 29.0 (0.9) 127.0 (1.1)

Definition of abbreviations: F 5 breathing frequency.

Values are mean (6 SEM). Note that the target value of VE 5 20 L/min/m2. BSA

corresponds to unadjusted mean value of 36.5 L/min.

Kim, Alexis, Rappold, et al.: Effects of Ozone at 0.06 ppm Concentration 1217
fourth highest daily maximum 8-h average) (26). This was based

primarily on controlled human chamber studies of FEV1

changes postexposure after 6.6 hours. Inflammation of the

airway, as well as toxicology and epidemiology studies, were

used as supportive evidence. In this study, we demonstrate,

using the same exposure approach, decrements in FEV1 and

FVC at a concentration (0.06 ppm) below the current standard.

Furthermore, we demonstrate increased neutrophil airways

inflammation at this low ozone concentration.

To date, two previous studies have investigated lung function

at 0.06 ppm ozone using a 6.6-hour protocol; neither found

statistically significant effects (7, 15) even though their observed

effect size was greater than in our study. For example, they

reported clean air–adjusted FEV1 after 0.06 ppm ozone decrements of 3.52 and 2.86%, whereas we observed a smaller drop

of 1.71%, yet our results were highly statistically significance

(P 5 0.008). Similarly, our reported change in FVC of 1.18%

was statistically significant (P 5 0.02), although in the range of

the 3.16 and 0.45% reported previously (7, 15). The key difference between the previous and the current study is that

although their studies were designed to compare multiple concentrations at multiple time points, ours was specifically designed to limit the need for multiple comparisons.

To accomplish this, we used several strategies to increase the

statistical power and to minimize experimental variability. First,

our analysis was focused on only one ozone concentration and

TABLE 3. LUNG FUNCTION RESPONSES TO 6.6-HOUR EXPOSURE TO CLEAN AIR AND 0.06 PPM OZONE IN HEALTHY

YOUNG ADULTS

All Subjects Males Females GSTM1-p GSTM1-n

(n 5 59) (n 5 27) (n 5 32) (n 5 30) (n 5 29)

%D 95% CI %D 95% CI %D 95% CI %D 95% CI %D 95% CI

FEV1

CA 20.002 (0.46) 20.9, 0.9 0.59 (0.53) 20.5, 1.6 20.51 (0.72) 21.9, 0.9 20.08 (0.46) 21.0, 0.8 0.078 (0.82) 21.5, 1.7

Ozone 21.71 (0.50) 22.7, 20.8 20.86 (0.62) 22.1, 0.4 22.43 (0.74) 24.0, 21.1 22.07 (0.71) 23.5, 20.7 21.34 (0.69) 22.8, 20.1

Ozone-CA 21.71 (0.64)* 23.0, 20.5 21.45 (0.95) 23.4, 0.5 21.93 (0.88)* 23.8, 20.3 21.99 (0.90)* 24.0, 20.2 21.42 (0.92) 23.4, 0.3

FVC

CA 21.13 (0.34) 21.8, 20.5 20.77 (0.33) 21.4, 20.1 21.44 (0.56) 22.5, 20.3 21.11 (0.49) 22.1, 20.2 21.15 (0.48) 22.1, 20.2

Ozone 22.32 (0.41) 23.1, 21.5 21.26 (0.52) 22.3, 20.2 23.22 (0.57) 24.3, 22.1 22.41 (0.52) 23.4, 21.4 22.23 (0.65) 23.5, 21.0

Ozone-CA 1.19 (0.51)* 22.2, 20.2 20.49 (0.74) 22.0, 1.0 21.78 (0.68)* 23.1, 20.4 21.30 (0.73) 22.7, 0.1 21.07 (0.71) 22.5, 0.4

Definition of abbreviations: CA 5 clean air; CI 5 confidence interval; GSTM1-n 5 glutathione S-transferase mu 1 null; GSTM1-p 5 glutathione S-transferase mu 1

positive.

%D indicates change relative to preexposure, mean (6 SEM).

* P , 0.05.

Clean Air Ozone

% FEV1 Change

-20

-15

-10

-5

0

5

10 (B)

Clean Air Ozone

% FEV1 Change

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

*

* p=0.008

(A)

Clean Air Ozone

% FVC Change

-20

-15

-10

-5

0

5

10

Clean Air Ozone

% FVC Change

-4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

*

* p=0.02 (C) (D)

Figure 1. Percent changes in FEV1 and FVC after 6.6-hour

exposure to clean air and 0.06 ppm ozone. (A, B) Group

means (6 SEM) and individual changes, respectively, for

FEV1. (C, D) Group means (6 SEM) and individual changes,

respectively, for FVC.

1218 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 183 2011
a comparison to clean air. Second, we enrolled nearly twice as

many subjects (n 5 59) as previous studies, as the study was

powered to examine the differences between two GSTM1

polymorphisms. Third, our analysis was determined a priori to

focus only on changes observed immediately postexposure for

lung function. Similarly, analysis of airway inflammation was

limited to %PMN 16 to 18 hours after exposure. Fourth, we

used only one dedicated pulmonary technician and spirometer

for primary lung function measurements. Last, our studies were

performed exclusively in the winter season, during which ambient ozone level was lower than 0.06 ppm (see Figure E1 in the

online supplement), and thus potential influence of prior

exposure to ambient ozone was minimized.

Our results may have significant public health implications.

Although most subjects (. 60%) showed less than 10% decrease in lung function after 0.06 ppm ozone, 3 of 59 subjects in

the present and 2 of 30 subjects in the Adams study (7) showed

a lung function decrement greater than 10%. This suggests

that 0.06 ppm ozone may cause lung function decrements in

the majority of young individuals with some 6% (e.g., 5 of 89

subjects) of them having a greater response. In addition, this is

the first study to examine and observe airway inflammatory

effects for ozone at concentration levels below 0.08 ppm.

Chronic airway inflammation may cause airway damage and

thereby bronchoconstriction and bronchial hyperresponsiveness. An increase in neutrophilic inflammation has been shown

to be strongly associated with exacerbation of airway disease

in people with asthma. Thus, ozone is uniquely associated with

worsening of asthma and increased hospitalization in children

with asthma (13). Although the current study is limited to

healthy subjects, the health consequences may be more severe

in individuals with preexisting diseases, particularly in asthma.

Effects of sex on response to ozone exposure have been reported variably. Some studies found a greater response in FEV1

in female than in male subjects, whereas others found a comparable response between them, mostly after a short-term exposure

to high-concentration ozone (27, 28). For a prolonged exposure

to low-concentration ozone, most studies report combined results

of both males and females with no specific analysis for sex effects

(6, 7, 15). The studies, however, appear to have assumed or found

no sex effects in pulmonary function response. In our study,

FEV1 and FVC decrement was not significant in males but was

significant in females; however, we found no significant difference

between males and females. In contrast, %PMN was increased

significantly in both sexes, with males showing a greater response

than females (P 5 0.009). Thus, it appears that ozone sensitivity

may differ between sexes depending on endpoints.

A two-factor mixed-effects model with repeated measures

was used to test if there was a difference in FEV1 responses

between GSTM1-positive and GSTM1-null subjects. We did not

find such an association in either FEV1 or FVC. This was

consistent with earlier studies (20, 29) that have shown no

independent role of GSTM1-null genotype on lung function

decrement. The role of GSTM1-null, however, may become

evident when it presents with other genotypes (NQO1 and

GSTP1) (19, 29). Regarding airway neutrophilia, although the

All M F GSTM1-p GSTM1-n

CA-adjusted FEV1 % Change

-6

-4

-2

0

2

4

* * *

p=NS p=NS

All M F GSTM1-p GSTM1-n

CA-adjusted FVC % Change

-6

-4

-2

0

2

4

* †

*

p=NS p=NS

(A)

(B)

Figure 2. Clean air (CA) adjusted % changes (mean and 95%

confidence interval) in (A) FEV1 and (B) FVC after ozone exposure for

all subjects, and by sex and GSTM1 genotype. *P , 0.05; †0.05 , P ,

0.1. F 5 females; GSTM1-n 5 glutathione S-transferase mu 1 null;

GSTM1-p 5 glutathione S-transferase mu 1 positive; M 5 males; NS 5

not significant.

TABLE 4. POLYMORPHONUCLEAR NEUTROPHIL RESPONSES TO 6.6-HOUR EXPOSURE TO CLEAN AIR AND 0.06 PPM OZONE

IN HEALTHY YOUNG ADULTS

All Subjects Males Females GSTM1-p GSTM1-n

(n 5 24) (n 5 11) (n 5 13) (n 5 12) (n 5 12)

Mean (6 SEM) 95% CI Mean (6 SEM) 95% CI Mean (6 SEM) 95% CI Mean (6 SEM) 95% CI Mean (6 SEM) 95% CI

% Polymorphonuclear neutrophils

CA 38.3 (3.7) 31.1, 45.6 33.4 (5.3) 23.0, 43.8 42.4 (5.1) 32.4, 52.4 34.7 (5.6) 23.7, 45.7 41.9 (4.8) 32.5, 51.3

Ozone 54.0 (4.6) 45.0, 63.0 57.6 (6.4) 45.1, 70.1 50.9 (6.6) 38.0, 63.8 46.1 (6.7) 33.0, 59.2 61.9 (5.5) 51.1, 72.7

Ozone-CA 15.7 (3.1)* 9.6, 21.8 24.2 (4.3)* 15.8, 32.6 8.5 (3.7)* 0.79, 16.2 11.3 (4.5)* 2.3, 20.3 20.0 (4.6)* 11.0, 29.0

Total cell count

CA 5.05 3 106 (0.82) 4.08 3 106 (0.98) 5.86 3 106 (1.26) 3.97 3 106 (0.90) 6.13 3 106 (1.34)

Ozone 6.93 3 106 (1.52) 9.42 3 106 (3.03) 4.83 3 106 (0.96) 5.58 3 106 (2.23) 8.28 3 106 (2.10)

Definition of abbreviations: CA 5 clean air; CI 5 confidence interval; GSTM1-n 5 glutathione S-transferase mu 1 null; GSTM1-p 5 glutathione S-transferase mu 1

positive; PMN 5 polymorphonuclear neutrophils.

%PMN indicates polymorphonuclear neutrophils as % of total cell counts. SEM of total cell count is for the base number of 106.

* P , 0.05.

Kim, Alexis, Rappold, et al.: Effects of Ozone at 0.06 ppm Concentration 1219
difference was not significant, GSTM1-null subjects appeared to

have a greater neutrophilic inflammatory response than

GSTM1-positive subjects. A sample size estimate, however,

revealed that the nonsignificant increase would likely become

significant with a larger cohort of approximately 47 subjects,

which would be consistent with our earlier report on response of

GSTM1-null genotype to 0.4 ppm ozone (20). This seemingly

contradicting trend versus lung function response may be due to

differences in the putative modes of action of ozone. Although

changes in lung function are believed to occur via activation of

a subset of airway C-fibers (30), inflammation is believed to

originate from activation of nuclear factor-kB induced by

reactive oxygen species generation (31). Thus, corticosteroids

can blunt ozone-induced neutrophilia but not lung function

responses (32). GSTM1 is believed to act by reducing oxidative

stress, including detoxification of byproducts generated by

inflammation. In line with previous studies (33), we found no

relationship between lung function and inflammation.

In summary, our study shows ozone effects on two independent markers of airway health at a level as low as 0.06 ppm in

healthy young adults. We did not find a significant role of GSTM1

genotype alone on the ozone-induced airway effects, but there

may be individuals or subpopulations with enhanced sensitivity

(such as in asthma) at these levels. We therefore conclude that

exposure to ozone levels below the current standard can cause

changes in lung function and initiate an airway inflammatory

response in a young adult population and pose a health risk, particularly to susceptible populations.

Author Disclosure: None of the authors has a financial relationship with a

commercial entity that has an interest in the subject of this manuscript.

Disclaimer: Although the research described in this article has been supported by

the U.S. Environmental Protection Agency, it has not been subjected to Agency

review and therefore does not necessarily reflect the views of the Agency, and no

official endorsement should be inferred. Mention of trade names or commercial

products does not constitute endorsement or recommendation for use.

Acknowledgment: The authors thank the nurses and the technical staff of the U.S.

Environmental Protection agency, the University of North Carolina–Chapel Hill,

and TRC Environmental Corp for their invaluable assistance, and Brent Coull of

Harvard University for reviewing statistical analyses.

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