### 668 volume 117 | number 4 | April 2009 • 

### Environmental Health Perspectives 

# Research 

# | Children’s Health

Asthma is one of the most prevalent chronic health conditions in Canadian children. The 1998–1999 National Population Health Survey found that the prevalence of physi

cian-diagnosed asthma is 8.4% among the whole population and 10.7% among children and teenagers, corresponding to approxi

mately 845,000 children (Canadian Institute for Health Information et al. 2001). Asthma compromises the quality of life for patients and their families and imposes a burden on the nation’s health care expenditure. In 1990, the economic costs of asthma for Canada were estimated to fall between $504 and $648 million (Krahn et al. 1996). Epidemiologic studies have shown that air pollution is associated with asthma-related increases in hospital admissions (Burnett

et al. 1999; Wong et al. 2001), emergency department visits (Stieb et al. 2000; Sunyer

et al. 1997; Villeneuve et al. 2007), and respiratory symptoms (Brauer et al. 2002; Vichit-Vadakan et al. 2001), and decreases in pulmonary function (Delfino et al. 2004; Gielen et al. 1997). However, there is a dearth of information about whether these associa

tions can be attributed to lung tissue injury in an asthmatic population, partly because of the lack of appropriate non-invasive biomarkers indicating lung inflammation and tissue injury. In recent years, some researchers have used fractional exhaled nitric oxide (Fe NO 

### ) to investigate the relationship between exposure to air pollutants and airway inflammation (Delfino et al. 2006; Mar et al. 2005). Measurements of mediators in exhaled breath condensate have been used to better understand the pathological mechanisms of air

way diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis (Horvath

et al. 2005; Montuschi 2005). Recently, analy

sis of mediators in condensed breath has been extended to include lung diseases caused by occupational exposure (Lehtonen et al. 2007; Pelclová et al. 2007). Asthma is a chronic air

way inflammatory disease involving a variety of inflammatory cell types and proinflamma

tory mediators. Reactive oxygen species are also thought to play an active role in inducing airway inflammation and hyperresponsivness (Hulsmann et al. 2007). Reactive oxygen species result from an increased burden of exogenous oxidants, endogenous lipid peroxidation, as well as from inflammatory cells (Halliwell and Gutteridge 1989). The level of reactive oxygen species in the airways can be determined by assaying concentrations of oxidative stress mark

ers in breath condensate (Horvath et al. 2005). We designed this longitudinal study to test the hypothesis that an acute increase in ambient air pollutants is associated with increased levels of markers of oxidative stress and inflammation in breath condensate of children with asthma, and that changes of breath markers are consistent with changes in pulmonary function and Fe NO


### . 

## Materials and Methods Study subjects. The study protocol was approved by the Research Ethics Board of Health Canada. The parent of each child gave written informed consent before the child’s participation in this study. The study was conducted between October and December of 2005 in Windsor, Ontario, Canada. Asthmatic children, 9 to 14 years of age, from nonsmoking households, were identified from a questionnaire surveyof grades one to eight of elementary schoolchildren whose parents were asked whether or not the child had doctor-diagnosed asthma. The parents who gave a positive response were telephoned to confirm the child’s current sta

tus of doctor-diagnosed asthma and whether there was any smoking of tobacco products in the household. One hundred eighty two subjects agreed to participate in this longitudi

nal study. Information on family income and family asthma history was extracted from the survey questionnaire. For logistical purposes, children were studied in two test periods, either between 11 October and 11 November (95 children), or between 14 November and 9 December 2005 (87 children). We did not advise parents to alter their children’s daily activity and the use of asthma medications. Ambient air pollution monitoring. Hourly measures of particulate matter ≤ 2.5 μm in

Address correspondence to L. Liu, Health Canada. A.L. 4903B, 269 Laurier Ave., Ottawa, Ontario, Canada K1A 0K9. Telephone: (613) 954-4457. Fax: (613) 948-8482. E-mail: ling_liu@hc-sc.gc.ca

Supplemental Material is available online at http:// www.ehponline.org/members/2008/11813/suppl.pdf

We gratefully acknowledge S. De Pauw of Health Canada for coordinating the study, B. Nadeau and K.L. Ku for running laboratory assays, M . SmithDoiron for preparing the map, and D. Stieb and
P. Villeneuve for reviewing the manuscript.

This study was funded by the Canada-the United States Border Air Quality Strategy.

The authors declare they have no competing financial interests.

Received 17 June 2008; accepted 28 November 2008. 

# Acute Effects of Air Pollution on Pulmonary Function, Airway Inflammation, and Oxidative Stress in Asthmatic Children

Ling Liu, 1 

### Raymond Poon, 1 

### Li Chen, 1 

### Anna-Maria Frescura, 1 

### Paolo Montuschi, 2 

### Giovanni Ciabattoni, 2 

### Amanda Wheeler, 1 

### and Robert Dales 1

1 Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario, Canada; 2 Department of Pharmacology, Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy



### B

ac k gr

o und 

### : Air pollution is associated with respiratory symptoms, lung function decrements, and hospitalizations. However, there is little information about the influence of air pollution on lung injury.

O bjective 

### : In this study we investigated acute effects of air pollution on pulmonary function and airway oxidative stress and inflammation in asthmatic children.

M eth o ds 

### : We studied 182 children with asthma, 9–14 years of age, for 4 weeks. Daily ambi

ent concentrations of sulfur dioxide, nitrogen dioxide, ozone, and particulate matter ≤ 2.5 μm in aerodynamic diameter (PM 2.5 

### ) were monitored from two stations. Once a week we measured spirometry and fractional exhaled nitric oxide (Fe NO 

### ), and determined thiobarbituric acid reactive substances (TBARS) and 8-isoprostane—two oxidative stress markers—and interleukin-6 (IL-6) in breath condensate. We tested associations using mixed-effects regression models, adjusting for confounding variables.

R

esults 

### : Interquartile-range increases in 3-day average SO 2 

### (5.4 ppb), NO 2 

### (6.8 ppb), and PM 2.5 

### (5.4 μg/m 3 

### ) were associated with decreases in forced expiratory flow between 25% and 75% of forced vital capacity, with changes being –3.1% [95% confidence interval (CI), –5.8 to –0.3], –2.8% (95% CI, –4.8 to –0.8), and –3.0% (95% CI, –4.7 to –1.2), respectively. SO 2 

### , NO 2 

### , and PM 2.5 

### were asso

ciated with increases in TBARS, with changes being 36.2% (95% CI, 15.7 to 57.2), 21.8% (95% CI, 8.2 to 36.0), and 24.8% (95% CI, 10.8 to 39.4), respectively. Risk estimates appear to be larger in children not taking corticosteroids than in children taking corticosteroids. O 3 

### (5.3 ppb) was not asso

ciated with health end points. Fe NO 

### , 8-isoprostane, and IL-6 were not associated with air pollutants.

C o nclusi o n 

### : Air pollution may increase airway oxidative stress and decrease small airway function of asthmatic children. Inhaled corticosteroids may reduce oxidative stress and improve airway function.

K

ey w o r ds 

### : air pollution, asthma, children, exhaled breath condensate, inflammation, oxidative stress, pulmonary function. Environ Health Perspect 117:668–674 (2009). doi:10.1289/ehp

. 11813 available via http://dx.doi.org/ [Online 28 November 2008]

### Air pollution is linked to lung injury in asthmatic children

Environmental Health Perspectives • volume 117 | number 4 | April 2009 

### 669 aerodynamic diameter (PM 2.5 

### ), sulfur dioxide, nitrogen dioxide, and ozone were available from the National Air Pollution Surveillance network of Environment Canada (http://www. etc-cte.ec.gc.ca/NapsData). There were two monitoring stations representing Windsor, located upwind from the prevailing winds. Of 182 participating children, 180 of them lived within a 10-km radius from the air monitoring sites, and one lived 12 km and another 17 km from the air sampling sites (Figure 1). Values from these two monitors were averaged. SO 2 

### was measured using ultraviolet fluorescence and the electrical conductivity from changes in chemical composition of a bromine solution, NO 2 

### measured using chemiluminescence, O 3 

### measured using ultraviolet absorbance, and
PM 2.5 

### measured using tapered element oscil

lating microbalance monitor. Pollutant con

centrations were averaged over a 24-hr period. Measurement of spirometry and Fe NO 

### . Once a week, registered respiratory tech

nologists visited the children’s schools, and conducted tests of spirometry and Fe NO 

### and collected exhaled breath condensate on study participants. These tests were run on the same day of the week for each child. Spirometry was performed according to American Thoracic Society criteria (American Thoracic Society 1995). KoKo Spirometers (Ferraris CardioRespiratory; Pulmonary Data Services, Inc. Louiseville, CO, USA) were cal

ibrated daily and results adjusted for tempera

ture and barometric pressure. A maximum of eight forced vital capacity (FVC) maneu

vers were carried out in an attempt to achieve three acceptable flow-volume loops and two being within 0.20 L for FVC and forced expi

ratory volume in one second (FEV 1 

### ), and within 0.20 L/sec for the mean forced expira

tory flow between 25% and 75% of the FVC (FEF 25–75% 

### ). The value assigned to the child was the largest acceptable value within 200 mL of a second value. Single-breath on-line measures of Fe NO 

### were carried out according to American Thoracic Society and European Respiratory Society Guidelines (American Thoracic Society 2005) using an Eco Physics CLD AL MED chemiluminescence analyzer (Eco Medics AG, Duernten, Switzerland). Before performing a slow vital capacity maneuver over at least 6 sec at 0.05 L/sec, subjects took three tidal volume breaths through a DENOX 88 (Eco Medics AG, Duernten, Switzerland) which contains an NO

x 

### absorber cartridge to scrub the ambient air to a value of between
0 and 1.5 ppb. The test was repeated a maxi

mum of eight times in an attempt to obtain at least two acceptable plateau Fe NO 

### values within 10%. The value assigned to the subject was the mean of the two values. We calculated
Fe NO 

### statistics using spiroWare 88 software (Eco Medics AG, Duernten, Switzerland). Collection of exhaled breath condensate, and measurement of amylase, oxidative stress, and inflammation. Exhaled breath condensate was collected using an RTube (Respiratory Research Inc., Charlottesville, VA, USA). Participants sat and breathed at tidal vol

umes orally into a mouthpiece attached to a cold condenser, for 10 min. Approximately
1 mL of breath condensate was collected. The condensed breath was then transferred into a microtube and stored at –20°C and then –80°C until use. To determine saliva contamination, we measured α -amylase activity in breath conden

sate using an assay kit (Pointe Scientific, MI, USA), which follows the increase in absorbance at 405 nm as the artificial substrate 2-chloro- p

nitrophenyl- α

- d -maltotrioside is hydrolyzed. Thiobarbituric acid reactive substances (TBARS) w ere determined in duplicate by a fluorescence method adapted from Yagi (1982). We used malonaldehyde bis-(dimethyl acetal) (Aldrich Chemical Co., Milwaukee, WI, USA) to construct a standard curve. To reduce the background interference with the very low levels of TBARS present in exhaled breath condensates, all glassware was washed in soap-free detergent followed by rinsing with distilled water and ethanol before use. We used butanol (OmniSolv, 99.95%; EM Science, Darmstadt, Germany) for extraction. The n -butanol extract was transferred to a Whatman glass bottom microplate (Whatman, NJ, USA), previously washed with ethanol, and the fluorescence measured at 530 nm/590 nm, excitation/emission, with a Cytofluor 2350 reader (Millipore, Billerica, MA, USA). The detection limit was 0.02 μM (three times the standard deviation above the blank). The within-run coefficient of variation was 8%. A colorimetric enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA) was used to measure 8-isoprostane concentra

tions in breath condensate. The assays were done in triplicate. A modified standard curve for 8-isoprostane was established for concen

trations between 0.975 and 62.5 pg/mL. We were able to detect 8-isoprostane at a concen

tration as low as 0.975 pg/mL (three times the standard deviation above the blank). The within-run coefficient of variation was 5%. We also measured 8-isoprostane on 8 random samples using radioimmunoassay, a proce

dure previously described by Montuschi et al. (2003), and compared the results from both methods [see Supplemental Material, Table 1 (available online at http://www.ehponline.org/ members/2008/11813/suppl.pdf)] . Pearson correlation coefficient for results from two methods was 0.744 ( p = 0.055), with a mean positive bias of approximately 35% in favor of the radioimmunoassay. Such a difference may be attributed to variations in the avidity and specificity of the antibodies used and the inherent variation during extrapolation from calibration curves at very low levels that were typical of exhaled breath condensates. We determined the level of interleukin-6 (IL-6) using an ELISA kit from R&D Systems (Minneapolis, MN, USA). We conducted the ELISA assays in duplicate by following manu

facturer’s instructions accompanying the kit. The detection limit was 0.48 pg/mL. Statistical analysis. We analyzed the corre

lations among environmental factors, daily use of inhaled corticosteroids (ICS) or short-acting

**Figure 1.** Map of Windsor, Ontario, Canada. **Detroit**

**Windsor** Wind direction 60204

60211 Ambassador Bridge Huron Church E.C. Row Expressway

Highway 401

NAPS monitors Children’s homes 0 2 1 4 6
Kilometers

### Liu et al.



### 670 volume 117 | number 4 | April 2009 • 

### Environmental Health Perspectives 

### β -agonist (SABA), and testing period, using Spearman rank order correlation, because some variables were not normally distributed. We used mixed-effects regression models to account for repeated measures and to analyze the associations between pollutants and health end points, assuming random slope and ran

dom intercepts, and accounting for intraindi

vidual correlation using S-PLUS (version 6.1; Insightful Corporation, Seattle, WA, USA). Data for Fe NO 

### , TBARS, and 8-isoprostane were log-transformed to fit a normal distribu

tion. Because > 99% of the samples gave a value > 0 for TBARS and 8-isoprostane, and most of them were above the detection limit, data points below the detection limit were not given special treatment. For exposure variables, we tested models for multiday moving aver

ages to examine potentially delayed response. Models were adjusted for time-dependent covariates that were statistically associated with either the exposure or response. We adjusted for testing period (as an indicator variable) to reduce the impact of seasonality. We also included product terms for the interaction of pollutant concentrations with sex and with the use of ICS to test potential modifying effects of sex and ICS. Since subjects served as their own control, we did not adjust for factors which remained constant during the study period such as age, race, family income, and family asthma history. Because health testing was kept on the same day of week for eachchild, we did not adjust for the effect of dayof-week in the models. When interactions were detected between pollutants and sex or the use of ICS, the par

ticipants were stratified for further analyses. We also ran two-pollutant models to adjust for potential confounding effects of co-pol

lutants. Results were expressed as percent change in FEV 1 

### , FEF 25–75% 

### , Fe NO 

### , TBARS

a nd 8-isoprostane per increase in inter-

nd 8-isoprostane per increase in interper increase in inter quartile-range concentration of a pollutant. A two-tailed value of p ≤ 0.05 was considered statistically significant.



## Results A total of 182 asthmatic children participated in the study, for a total of 691 spirometry test results, 672 Fe NO 

### test results, and 616 breath samples. Seven children had 5 measurement days, 142 had 4 days, 27 had 3 days, and 6 had 2 days. In total, there were 39 meas urement days during the study period, and no missing day for air monitoring to match health testing days. The characteristics of this cohort are pre

sented in Table 1. We meas ured the concen

tration of amylase in 25% of breath samples to determine saliva contamination. The median amylase concentration was 0.60 unit/L, with the 95th percentile being 1.46 unit/L. None of the samples had amylase concentrations near the reported salivary amylase concentra

tion of 1,250 unit/L (Brand et al. 2006). We estimated that saliva contamination was likely

< 0.1%, and therefore interference of breath analysis by saliva was negligible. In 616 breath samples, only 2 samples had detectable IL-6 levels. We therefore did not conduct further analysis on IL-6. The environmental conditions and daily concentrations of air pollutants measured dur

ing the study period are presented in Table 2. Because Spearman rank order correlation analyses showed that daily temperature, rela

tive humidity and testing period were signifi

cantly correlated with some of the pollutants (Table 3), we included these variables in mod

els to adjust for any confounding/modifying effects. We also included daily use of SABA and ICS to adjust for their potential confound

ing effects. Because pollutants were correlatedwith each other (Table 3), we also ran twopollutant models to adjust for confounding. Results from single-pollutant regression models. Table 4 presents percentage changes of health end points in relation to an increase in interquartile range concentrations of pol

lutants in a single-pollutant model which also included interaction terms of ICS and sex with pollutants. FEV 1 

### and FEF 25–75% 

### exhib

ited a consistent trend of negative associa

tions with all four pollutants, with FEF 25–75% **Table 1.** Selected characteristics of 182 asthmatic children from Windsor, Ontario, Canada.

Characteristic Value
Age (years) [median (range)] 11 (9–14) Sex [no. (%)]

Female 68 (37.4) Male 114 (62.6) Race [no. (%)] Caucasian 160 (87.9) Other 22 (12.1) Parental asthma [no. (%)]

No 11 (6.0) Yes 70 (38.5) Unknown 101 (55.5) Self-reported medication use a [no. (%)] ICS 68 (37.4) SABA 64 (35.2) Other asthma medications 33 (18.1) None 77 (42.3) FEV 1 (L) [median (5th– 95th percentile)] 2.4 (1.7–3.3) Percent predicted FEV 1 (%) [median (5th– 95th percentile)] b 90.5 (68.6–107.1) FEF 25–75% (L/sec) [median (5th– 95th percentile)] 2.5 (1.3–3.9) Percent predicted FEF 25–75% (%) [median (5th– 95th percentile)] b 78.2 (45.3–118.0) Fe NO (ppb) [median (5th– 95th percentile)] 19.4 (4.5–81.6) TBARS (μM) [median (5th– 95th percentile)] 0.26 (0.03–1.53) 8-Isoprostane (pg/mL) [median (5th– 95th percentile)] 1.43 (0.30–4.20)

a More than one medication could be used. b Predicted normal values were calculated based on a given height, age, sex, and race/ethnicity (Hankinson et al. 1999).

**Table 2.** Values for selected environmental conditions during study period.
Median [interquartile range (5th–95th percentile)]
Environmental variable 1-day average 2-day average 3-day average

SO 2 (ppb) 4.5 [6.5 (0.5 to 15.5)] 5.0 [5.6 (1.0 to 13.0)] 5.6 [5.4 (1.3 to 13.8)] NO 2 (ppb) 19.8 [9.8 (9.5 to 29.5)] 18.3 [9.0 (11.3 to 28.0)] 18.3 [6.8 (12.3 to 27.0)] O 3 (ppb) 13.0 [9.0 (6.5 to 26.5)] 14.1 [6.4 (6.8 to 23.3)] 14.0 [5.3 (7.5 to 21.0)] PM 2.5 (μg/m 3 ) 6.5 [6.0 (2.0 to 19.0)] 6.6 [6.4 (2.0 to 15.0)] 6.9 [5.4 (2.7 to 14.3)] Temperature (°C) 6.6 [13.0 (–7.7 to 15.4)] 7.4 [12.0 (–5.5 to 14.7)] 7.4 [11.2 (–5.9 to 14.5)] Relative humidity (%) 67.4 [11.6 (53.3 to 87.9)] 65.6 [9.7 (54.4 to 75.6)] 65.7 [7.3 (54.9 to 74.2)]

Air quality was monitored between 8 October and 9 December 2005, 62 sampling days.

**Table 3.** Spearman rank order correlation coefficients of variables.

Relative Variable Temperature humidity SO 2 NO 2 O 3 PM 2.5 SABA ICS
Testing period –0.73* 0.01 0.35* –0.30* 0.06 –0.07 0.10* –0.07 Temperature 0.18* –0.17* 0.00 0.18* 0.10* –0.10* –0.02 Relative humidity –0.04 0.03 –0.51* 0.20* 0.01 –0.02 SO 2 0.18* –0.02 0.56* 0.04 0.05 NO 2 –0.51* 0.71* –0.04 0.07 O 3 –0.41* 0.00 –0.10* PM 2.5 –0.05 0.07 SABA 0.27*

* p

< 0.05.

### Air pollution is linked to lung injury in asthmatic children

Environmental Health Perspectives • volume 117 | number 4 | April 2009 

### 671 having a statistically significant association with NO 2 

### and PM 2.5 

### . The significant asso

ciations for FEF 25–75% 

### persisted for at least 3 days. Fe NO 

### had a trend of positive asso

ciations with SO 2 

### , NO 2 

### , and PM 2.5 

### , but the associations were not statistically significant.
Fe NO 

### had a statistically significant negative association with O 3 

### . TBARS was significantly positively associated with SO 2 

### , NO 2 

### , and
PM 2.5 

### , but not with O 3 

### . The associations for TBARS persisted for at least 3 days. 8-Iso

prostane was significantly positively associated with same-day SO 2 

### , but not associated with other pollutants. Modifying effects by the use of ICS and

sex. Product terms between pollutants and the use of ICS showed significant interactions for some of the health end points. For example, the product term for SO 2 

### and ICS was posi

tively associated with TBARS ( p = 0.04), but negatively associated with FEV 1 

### ( p < 0.01),
FEV 25–75% 

### ( p = 0.02), and 8-isoprostane ( p = 0.01), indicating that the use of ICS was a significant effect modifier. The participants were then stratified by the use of ICS dur

ing the study period to adjust for modifying effects of use of ICS (Figure 2). Children who did not use ICS appeared to have larger risk estimates for pollutants than did those who used ICS. Among children not taking ICS, risk estimates for pollutants were statistically significant with respect to FEV 1 

### , FEF 25–75% 

### , and TBARS. Risk estimates for Fe NO 

### and 8-isoprostane were not very different between the two groups of children (data not shown). Product terms for pollutants and sex showed no significant interaction ( p > 0.05), suggesting that sex was not a significant modi

fier for the associations between air pollutants and health end points. We therefore did not run further analysis on boys and girls separately. Results from two-pollutant models. Because we found consistent associations with pollutants for FEV 1 

### , FEF 25–75% 

### , Fe NO 

### , and TBARS, we ran two-pollutant models on these end points to adjust for confounding effects of co-pollutants (Figure 3). For FEV 1 

### and FEF 25–75% 

### , although two-pollutant mod

els did not result in marked changes in risk estimates for PM 2.5 

### and O 3 

### , including PM 2.5 

### in models did somewhat change risk esti

mates for SO 2 

### and NO 2 

### , suggesting that the effects of SO 2 

### and NO 2 

### on pulmonary func

tion were influenced by PM 2.5 

### . For Fe NO 

### and TBARS, including two pollutants in models did not markedly change the regression results,

**Table 4.** Percent change (95% confidence interval) in health end points of 182 asthmatic children, in association with an interquartile-range increase of a pollut

ant, in single–pollutant models with various lag times.

Air pollutant FEV 1 FEF 25–75% Fe NO TBARS 8–Isoprostane

SO 2 Same day –0.3 (–1.3 to 0.7) –1.0 (–3.1 to 1.3) 5.8 (–5.0 to 17.8) 17.4 (0.3 to 37.4)* 14.1 (2.5 to 26.9)* Lag 1 day 0.1 (–1.1 to 1.2) –0.9 (–3.1 to 1.4) 1.6 (–9.3 to 13.9) 14.1 (–1.8 to 32.5) –2.4 (–11.7 to 7.9) 2-day average –0.1 (–1.4 to 1.3) –1.8 (–4.7 to 1.1) 5.9 (–9.3 to 23.7) 35.1 (9.5 to 66.8)* 9.4 (–5.2 to 26.2) 3-day average –0.3 (–1.9 to 1.4) –2.3 (–5.5 to 1.0) 1.7 (–14.1 to 20.3) 61.8 (24.9 to 109.5)* –0.3 (–16.4 to 18.9 NO 2 Same day –0.6 (–1.6 to 0.3) –2.4 (–4.3 to –0.4)* 8.2 (–2.9 to 20.6) 21.2 (1.9 to 44.2)* 0.0 (–10.7 to 12.1) Lag 1 day –0.3 (–1.1 to 0.6) –1.4 (–3.1 to 0.3) 3.7 (–6.1 to 14.6) 10.2 (–5.6 to 28.7) –5.6 (–14.8 to 4.5) 2-day average –0.6 (–1.6 to 0.4) –2.4 (–4.3 to –0.3)* 7.4 (–4.6 to 21.0) 20.3 (–0.4 to 45.3) –4.7 (–1.1 to 8.1) 3-day average –0.8 (–1.9 to 0.3) –2.8 (–5.0 to –0.5)* 0.5 (–12.1 to 14.9) 32.9 (7.2 to 64.6)* –4.0 (–16.3 to 10.2) O 3 Same day –0.4 (–1.6 to 0.7) 0.2 (–2.1 to 2.5) –12.2 (–22.3 to –0.8)* 5.0 (–13.2 to 27.0) 7.0 (–6.5 to 22.4) Lag 1 day –0.2 (–1.1 to 0.7) 0.1 (–1.7 to 1.9) –8.3 (–16.0 to 0.2) 7.3 (–6.7 to 23.4) 6.6 (–3.4 to 17.6) 2-day average –0.4 (–1.6 to 0.9) 0.4 (–2.2 to 3.0) –16.0 (–26.4 to –4.1)* 14.4 (–8.5 to 43.0) 12.0 (–3.8 to 30.5) 3-day average –0.5 (–2.0 to 1.0) –0.4 (–3.5 to 2.7) –1.6 (–16.7 to 16.1) 8.8 (–17.4 to 43.4) 15.9 (–3.5 to 39.1) PM 2.5 Same day –0.5 (–1.3 to 0.3) –1.9 (–3.5 to –0.3)* 5.3 (–3.6 to 15) 16.9 (2.2 to 33.6)* 5.1 (–3.6 to 14.5) Lag 1 day –0.3 (–1.1 to 0.5) –1.2 (–2.8 to 0.3) 1.7 (–6.3 to 10.4) 14.6 (0.8 to 30.4)* –3.8 (–12.1 to 5.3) 2-day average –0.6 (–1.5 to 0.4) –2.0 (–3.8 to –0.2)* 4.3 (–5.4 to 15.1) 22.0 (4.8 to 42.1)* 0.1 (–9.8 to 11.1) 3-day average –1.1 (–3.1 to 0.9) –3.3 (–7.2 to 0.8) –17.3 (–33.5 to 2.9) 69.1 (20.1 to 138.2)* 5.8 (–15.8 to 33.0)

2-day average: the median concentration of a pollutant collected on the day and 1 day before the clinical testing was done. 3-day average: the median concentration of a pollutant col

lected on the day and 2 days before the clinical testing was done. Models were adjusted for testing period, daily temperature, relative humidity, and use of SABA and ICS. Interaction terms were included in the models to test modifying effects of taking ICS (a binary indicator) and sex (binary indicator) on air pollutants.

* p

< 0.05. **Figure 2.** Percent changes and 95% confidence intervals in FEV 1 ( A ), FEF 25–75% ( B )

, Fe NO ( C ), and breath TBARS ( D ) of asthmatic children taking or not taking ICS during the study period, in association with an interquartile-range increase in a pollutant. Sixty-eight asthmatic children took (Yes), and 114 children did not take (No) ICS. Pollutant concentrations were averaged over 3 days including the same day and 2 days before the clinical testing was done. Models were adjusted for testing period, daily temperature, relative humidity, and use of SABA. 8

6

4

2

0

–2

–4

–6**Percent change in FEV 1** 

No No No No Ye s Y es Ye s Y es SO 2 NO 2 PM 2.5

No No No No Ye s Y es Ye s Y es**Percent change in FEF 25–75%**  12

8

4

0

–4

–8 SO 2 NO 2 O 3 PM 2.5

No No No No

Ye s Y es Ye s Y es No No No No Ye s Y es Ye s Y es **Use of IC S U**  **se of ICS**

**Use of IC S U**  **se of ICS Percent change in Fe** **NO Percent change in TBARS**  SO 2 NO 2 O 3 PM 2.5 SO 2 NO 2 O 3 PM 2.5 80

60

40

20

0

–20

–40 120

100

80

60

40

20

0

–20

–40 **A B** 

**C D**  O 3

### Liu et al.



### 672 volume 117 | number 4 | April 2009 • 

### Environmental Health Perspectives 

### suggesting that the results of each pollutant were independent of other pollutants.



## Discussion Principal findings of this study are that SO 2 

### , NO 2 

### , and PM 2.5 

### in ambient air were signifi

cantly associated with decrements in FEV 1 

### and
FEF 25–75% 

### , and increases in TBARS in breath condensate among children with asthma. The risk estimates were larger for pollutant con

centrations averaged over 3 days than over a shorter term. Clinically FEF 25–75% 

### has been considered to be sensitive in detecting small airway dysfunction (Lebecque et al. 1993). These associations were robust after inclusion of daily temperature, relative humidity, study period, and the use of asthma medications SABA and ICS in models. The associations for O 3 

### and PM 2.5 

### were not sensitive to inclusion of co-pollutants, as adjusting for other pol

lutants in the model did not markedly change the strength of the risk estimates for O 3 

### and
PM 2.5 

### . The associations between pulmonary function decrements and SO 2 

### and NO 2 

### were sensitive to the inclusion of PM 2.5 

### in models, suggesting that PM 2.5 

### may have confounded some of the effects of SO 2 

### and NO 2 

### . Adverse changes in FEF 25–75% 

### were consistent with those of TBARS in response to SO 2 

### , NO 2 

### , and PM 2.5 

### with a similar lag structure, sug

gesting a coherent outcome for small airway function and oxidative stress. Our results on pulmonary function changes were similar to those published previously (Delfino et al. 2004; Epton et al. 2008; Gielen et al. 1997;
Romieu et al. 2008), although air pollutant levels in the present study were low compared to those studies. Exhaled breath condensate has been used in clinical settings to study pathologi

cal mechanisms of respiratory illness such as asthma, chronic obstructive pulmonary dis

ease, and cystic fibrosis (Baraldi et al. 2003; Horvath et al. 2005). Elevated concentra

tions of oxidant nitrate (Balint et al. 2001), IL-6 and leukotriene B4 (Carpagnano et al. 2003), and 8-isoprostane (Montuschi et al. 2000), have been found in breath condensate of adult smokers. Markers of inflammation and oxidative stress in breath were associated with occupational exposure to cobalt and tungsten (Goldoni et al. 2004), chromium (Caglieri et al. 2006), silica (Pelclová et al. 2007), and asbestos (Lehtonen et al. 2007). Montuschi et al. (2002) exposed healthy nonsmoking adults to high concentration of O 3 

### (400 ppb) and clean air for 2 hr on sep

arate occasions in a laboratory setting, and reported that exposure to O 3 

### led to a signifi

cant increase in 8-isoprostane concentrations in breath condensate. Barregard et al. (2008) exposed adult human subjects to wood smoke in a controlled environment and reported an increase in malondialdehyde levels in breath for at least 20 hr postexposure. However, lit

tle work has been done to use biomarkers in breath condensate to examine the effects of ambient air pollutants on airway inflamma

tion and oxidative stress among asthmatic children. Very recently, Epton et al. (2008) reported a cohort study on 12- to 18-year-old male healthy and asthmatic students in a New Zealand boarding school, where they found small effects of PM 10 

### (PM with aerodynamic diameter < 10 μm) in ambient air on FEV 1 

### , but no effect on pH and hydrogen peroxide in exhaled breath. However, with a panel of asthmatic children living in Mexico City,
Romieu et al. found an association between malondialdehyde in exhaled breath and PM 2.5 

### and O 3 

### in ambient air (Romieu et al. 2008). A 14.2-μg/m 3 

### (interquartile-range) increase in 8-hr moving average PM 2.5 

### was signifi

cantly associated with a 1.12-nmol increase in malondialdehyde, while a 15.9-ppb (inter

quartile-range) increase in 8-hr moving aver

age O 3 

### was significantly associated with a 1.16-nmol increase in malondialdehyde. Our results are consistent with these findings in that inhaled ambient air pollutants were sig

nificantly associated with an increase in an oxidative stress marker in breath condensate. Oxidative stress–inducing reactive oxygen species are physiologically active mediators that can be induced by air pollutants. PM 2.5 

### , SO 2 

### , NO 2 

### , and O 3 

### have been demonstrated to cause formation of excessive amount of reactive oxygen species in airways and in the cardiovascular system in experimental animals, leading to tissue inflammation and cell death (Dye et al. 1997; Meng et al. 2003; Persinger

et al. 2002). Oxidative stress has been linked to clinical phenotypes such as asthma and atherosclerosis (Chuang et al. 2007; Walter

et al. 2004). TBARS are a group of lowmolecular-weight chemicals that are formed during the decomposition of lipid peroxida

tion products (Halliwell and Chirico 1993), and thus are often used as an index of lipid peroxidation and oxidative stress (Kodavanti

et al. 2000; Rhoden et al. 2004; Walter et al. 2004). 8-Isoprostane is a prostaglandin F2 α

– like compound produced by free radicals via the nonenzymatic peroxidation of arachidonic acid in membrane phospholipids (Morrow

et al. 1990). Because TBARS are derived from oxidative degradation of a broader range of biological substances including lipids and sugars than is 8-isoprostane (Halliwell and Gutteridge 1981; Janero 1990), and because we observed an increase in TBARS but not 8-isoprostane in breath samples in relation to elevated air pollutants, it appears that TBARS may be a more sensitive biomarker than 8-iso

prostane in breath condensate as a useful tool for investigating air pollution-related oxidative stress among asthmatic children, especially for acute exposures. It remains to be seen whether immunoassays with significantly higher sensi

tivity and specificity can improve the efficacy of 8-isoprostane as a biomarker of oxidative stress in exhaled breath condensate. ICS are thought to reduce inflamma

tion and oxidative stress in airways (Baraldi

et al. 2003; Khor et al. 2007). Children with asthma exacerbation have reduced 8-isopros

tane in breath condensate after ICS treatment (Baraldi et al. 2003). In the present study, we found that ICS seems to be able to reduce some of the adverse effects of air pollutants, **Figure 3.** Percent changes and 95% confidence intervals in FEV 1 ( A ), FEF 25–75% ( B )

, Fe NO ( C ), and breath TBARS ( D ) of asthmatic children, in association with an interquartile-range increase in a pollutant, in oneand two-pollutant models. Pollutant concentrations were averaged over 3 days including the same day and 2 days before the clinical testing was done. Models were adjusted for testing period, daily tempera

ture, relative humidity, and use of SABA and ICS. 6

4

2

0

–2

–4**Percent change in FEV 1** 

Single SO 2 NO 2 O 3 PM 2.5**Percent change in FEF 25–75%** 
12

8

4

0

–4

–8 SO 2 NO 2 O 3 PM 2.5 **A B** 

**C D** + NO2+ O3+ PM2.5Single+ SO2+ O3+ PM2.5Single+ SO2+ NO2+ PM2.5Single+ SO2+ NO2+ O3Single+ NO2+ O3+ PM2.5Single+ SO2+ O3+ PM2.5Single+ SO2+ NO2+ PM2.5Single+ SO2+ NO2+ O3Single+ NO2+ O3+ PM2.5Single+ SO2+ O3+ PM2.5Single+ SO2+ NO2+ PM2.5Single+ SO2+ NO2+ O3Single+ NO2+ O3+ PM2.5Single+ SO2+ O3+ PM2.5Single+ SO2+ NO2+ PM2.5Single+ SO2+ NO2+ O3 40

30

20

10

0

–10

–20

–30

–40**Percent change in Fe NO**  SO 2 NO 2 O 3 PM 2.5 100

80

60

40

20

0

–20

–40**Percent change in TBARS**
SO 2 NO 2 O 3 PM 2.5

### Air pollution is linked to lung injury in asthmatic children

Environmental Health Perspectives • volume 117 | number 4 | April 2009 

### 673 as the risk estimates appear to be larger for the children who did not take ICS during the study period. Consistent with our findings, previous studies have also reported stronger associations between exposure to air pollutants and decrements in pulmonary function and
increases in Fe NO 

### for children not taking antiinflammatory medications than for children taking the medications (Delfino et al. 2006, 2008; Mar et al. 2005). In the present study only 37% of children used ICS, which could result in a reduced statistical power to detect associations with pollutants in this group. Oxidative stress often occurs along with tissue inflammation, which induces the release of reactive oxygen species to cause tis

sue injury (Chuang et al. 2007; Dye et al. 1997; Kodavanti et al. 2000). Unlike previ

ously published studies (Delfino et al. 2006;
Mar et al. 2005), in our study we did not find statistically significant adverse changes in inflammatory marker Fe NO 

### associated with air pollution, and the level of IL-6 in breath condensate was too low to be detectable. One interpretation for the results of inflamma

tion markers was that we might need a larger sample size to detect significant changes in airway inflammation. Another possible expla

nation might be that the inflammation in lower airways of the children was not captured by Fe NO 

### measured at a flow rate of 0.05 L/ sec; Barregard et al. (2008) reported that exposure to wood smoke particles resulted in significant increase in Fe NO 

### measured at a flow rate of 0.27 L/sec, but not at 0.05 L/sec. Alternatively, this group of asthmatic chil

dren might have been in such a state of airway inflammation that it overwhelmed the effects of air pollution, particularly when exposure concentrations were lower than those in other studies (Delfino et al. 2006; Mar et al. 2005). We did not find a consistent adverse effect of O 3 

### . O 3 

### exhibited a significantly negative association with Fe NO 

### (Table 4), but a positive association with TBARS (Figure 3). The nega

tive association between O 3 

### concentration and
Fe NO 

### is counterintuitive, because laboratory studies have shown that O 3 

### at high concentra

tions (200–400 ppb) caused inflammation in the airways of human subjects (Balmes et al. 1996; Frampton et al. 1997). In the present study, adjusting for co-pollutants and study periods in the models did not change the results. A sound interpretation for this result remains to be found at this point in time. When a large number of regression mod

els are run on a data set, some statistically significant associations may occur by chance. To reduce the possibility of spurious relation

ships due to multiple comparisons, we limited sensitivity analyses to those that consistently demonstrated statistical significance in base models, and considered only results showing a consistent pattern as actual effects. 

## Conclusion Results from our study demonstrate a sig

nificant decrement in small airway function and an increase in airway oxidative stress in asthmatic children in association with SO 2 

### , NO 2 

### , and PM 2.5 

### in ambient air. TBARS in breath condensate may be a useful tool for investigating air pollution-related oxidative stress among asthmatic children. ICS seems to be able to reduce some of the adverse respira

tory response to air pollutants.

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