Biomarkers of Airway Acidity and Oxidative Stress in ...

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Aug 28, 2008 - ammonium) and oxidative stress (8-isoprostane) in the EBC of grain workers. ... industry was associated with decreased 8-isoprostane.
AJRCCM Articles in Press. Published on August 28, 2008 as doi:10.1164/rccm.200711-1731OC

Biomarkers of Airway Acidity and Oxidative Stress in Exhaled Breath Condensate from Grain Workers Ron Do1, Karen H. Bartlett2, Helen Dimich-Ward3, Winnie Chu2, Susan M. Kennedy2,3 1 Experimental Medicine Program, University of British Columbia, Vancouver, BC, Canada 2 School of Environmental Health, University of British Columbia, Vancouver, BC, Canada 3 Division of Respiratory Medicine, University of British Columbia, Vancouver, BC, Canada. Correspondence and requests for reprints should be addressed to: Ron Do, MSc McGill University Health Centre Research Institute, Royal Victoria Hospital, H7.39, 687 Pine Avenue West, Montreal, Quebec, H3A 1A1 Tel: 514-934-1934 X35796 Fax: 514-843-2843 Email: [email protected] Input running title: Exhaled Breath Condensate of Grain Workers Journal Subject Head: [119] Epidemiology of environmental diseases Word count: 3340 words (excluding abstract, references, online supplement) This article has an online data supplement, which is accessible from this issue's table of content online at www.atsjournals.org At a Glance Commentary: Scientific Knowledge on the Subject Few occupational exposure studies have investigated the use of exhaled breath condensate to measure adverse changes in the airways. What this Study Adds to the Field We report smoking, obesity, and occupational factors are associated with biomarkers of acid and oxidative stress in EBC of grain workers. We conclude EBC can contribute to predicting the state of airways of workers exposed to pro-inflammatory agents. Financial Support This study was funded by a research operating grant from the Canadian Institutes for Health Research and by fellowship support from WorkSafeBC.

Copyright (C) 2008 by the American Thoracic Society.

1 Abstract (250 words) Rationale: Grain workers report adverse respiratory symptoms, due to exposures to grain dust and endotoxin. Studies have shown that biomarkers in exhaled breath condensate (EBC) vary with the severity of airway inflammation. Objectives: The purpose of the study was to evaluate biomarkers of airway acidity (pH and ammonium) and oxidative stress (8-isoprostane) in the EBC of grain workers. Methods: 75 workers from 5 terminal elevators participated. In addition to EBC sampling, exposure monitoring for inhalable grain dust and endotoxin was performed; spirometry, allergy testing, and a respiratory questionnaire derived from the American Thoracic Society were administered. Measurements and Main Results: Dust and endotoxin levels ranged from 0.010 to 13 mg/m3 (median: 1.0) and 8.1 to 11000 EU/m3 (median: 610) respectively. EBC pH values varied from 4.3 to 8.2 (median: 7.9); ammonium values from 22 to 2400 µM (median: 420) and 8-isoprostane values from 1.3 to 45 pg/ml (median: 11). Univariate and multivariable analyses revealed a consistent effect of cumulative smoking and obesity with decreased pH and ammonium, and intensity of grain dust and endotoxin with increased 8-isoprostane. Duration of work on the test day was associated with decreased pH and ammonium, while duration of employment in the industry was associated with decreased 8-isoprostane. Conclusions: We conclude that chronic exposures are associated with airway acidity while acute exposures are more closely associated with oxidative stress. These results suggest that the collection of EBC may contribute to predicting the pathological state of the airways of workers exposed to acute and chronic factors. Key words: Exhaled breath condensate, biomarkers, airway acidity, oxidative stress, grain dust and endotoxin

2 Introduction (471 words) Workers in grain storage and transfer industries have been shown to have both acute and chronic respiratory impairment related to airborne exposure to components of grain dust (1, 2) and associated endotoxin (3). Adverse health outcomes have included asthma (4), acute crossshift reductions in airflow rates (2), and chronic airflow obstruction. Acute and chronic airflow obstruction are dose-related to both the level of grain dust (1, 4, 5) and endotoxin (3, 6). Epidemiological investigation of specific risk factors for airflow obstruction among workers in the grain industry is hampered by logistical challenges associated with conducting studies at the worksite. Traditional approaches to measuring pathological changes in the airways such as sputum induction, bronchoscopy and nasal lavage have limited applicability in occupational settings due to high degrees of invasiveness, specialized equipment and facilities, time commitments and associated risks. The collection of exhaled breath condensate (EBC) from workers may be a useful addition in occupational field studies to sample the epithelial fluid of the airways (7) due to the procedure being safe and simple to perform. Fluctuations in the acid-base equilibrium (pH/ammonium (NH4+)) of EBC can provide a quantitative measure of airway acidity in asthma (8-10), chronic obstructive pulmonary disease (COPD) (9, 11) and other respiratory disorders (12, 13). The regulation of airway pH is maintained by the production and release of acids and bases, such as ammonia in various buffer systems of the airways. Excessive or uncontrolled acidification of the airways can be caused by disturbance of the airway pH homeostatic regulatory system (14, 15), where acid stress can lead to adverse respiratory effects such as epithelial sloughing and mucous plugging (16). Oxidative stress, which is caused by an imbalance between the level of oxidants and antioxidants in the airway epithelium, has also been

3 linked to inflammation (17, 18). Elevated biomarkers of oxidative stress have been measured in the EBC of individuals with asthma (19-21) and COPD (22). In human airway epithelial cells, organic dust exposure (swine barn) causes airway acidification by the secretion of excess protons (23). In addition, endotoxin induces lipid peroxidation, resulting in the formation of reactive oxidant species (24) and airway hyperreactivity (25). Only a few studies have investigated the use of EBC to measure adverse changes in the airways of workers in occupational settings (7, 26, 27). Our study was conducted in 2003 as part of an ongoing project carried out by our research team involving a cross-section of workers employed at 5 terminal grain elevators in the port of Vancouver. Our objective was to evaluate the relationship, among grain elevator workers, between EBC biomarkers of airway acidity (pH and ammonium (NH4+)) and oxidative stress (8-isoprostane), on the one hand, and personal characteristics and work exposures, on the other. Some of the results of this study has been previously reported in the form of abstracts (28, 29).

Methods (500 words) An independent laboratory study was conducted previously to assess technical factors associated with biomarkers measured in the present study (30). In that study, six EBC samples were collected from each of five current smokers and five never smokers over one month. The current field study was nested in a larger cross-sectional survey of employees in five terminal grain elevators in Vancouver, Canada. Workers were chosen randomly from within specified job titles. The selected job titles represented the complete range of anticipated exposure levels to particulate matter and endotoxin (see online supplement for details). All participants gave informed written consent and confidentiality of personal identifiers was ensured.

4 EBC collection was performed using the R-Tube (Respiratory Research, Inc, Charlottesville, VA). It proved unworkable to require workers to refrain from eating or drinking throughout the workday. Hence, a 0.3 micron filter provided by Respiratory Research, Inc. was attached between the mouth valve and collecting tube to minimize contamination of food and drink. Filter effects were assessed in four repeated EBC samples taken from 2 individuals with and without a filter (n=16). No significant effect was found for all biomarker measurements (p>0.05, data not shown). EBC sample storage and pH/NH4+ measurements were performed as described by Do et al. (30). EBC collection was performed for 15 minutes at a tidal breathing rate and samples were subjected to argon deaeration at 350 µl/min for 10 minutes. The 8-isoprostane content of EBC samples was determined using an enzyme-linked immunoassay according to manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI) (see online supplement). Full shift (minimum 6 hours) personal exposure monitoring for dust and endotoxin was performed on the same day as EBC collection. Respiratory health assessment, lung function and atopy tests were performed within two weeks of EBC collection (see online supplement). Although exposure monitoring was conducted for the complete shift (as required for the full study), EBC collection was carried out at random times throughout the day. For data analysis purposes, exposure concentrations were adjusted to take into consideration the duration of work on the testing day prior to EBC collection, by multiplying the concentration value by the proportion of the full shift prior to EBC testing. Data were analyzed using SAS, version 9.1 (SAS Institute, Cary, NC) and statistical graphs were produced using R (R project, Vienna, Austria). In the laboratory study, the intraindividual variability was assessed using the coefficient of variation (CV) (see online

5 supplement). In the field study, normal probability plots were used to assess normality of the variables. To minimize the effect of outliers and to achieve univariate normality, variables were transformed (see online supplement). In univariate analyses, Student’s t-test (Satterthwaite if unequal variances) or analysis of variance were used to compare categorical distributions; simple linear regression was used to compare continuous variables. Multivariable analyses were performed using regression modeling for EBC marker parameters with marginally significant (p≤0.1) variables from univariate analyses offered using a manual stepwise procedure. A priori variables based on theory or previous studies were tested (see online supplement).

Results The intra-individual variability of pH, NH4+ and 8-isoprostane (over a one month period) was assessed in an independent laboratory study. Results for pH and NH4+ have been reported previously (30). Briefly, the CV was larger in current smokers than in never smokers for pH and NH4+ (intra-individual CV for never smokers: 2.2 % for pH, 10 % for NH4+; current smokers: 12 % for pH, 13 % for NH4+). The CV for 8-isoprostane measurements were similar between current smokers (32 %) and never smokers (32 %). Field Study participants: Demographic and Exposure Characteristics There were 82 eligible participants; four did not show up for testing and three were excluded from analysis because of missing exposure measurements, leaving 75 participants for this analysis (table 1). Most participants were Caucasian and male, with a mean age of 47 years, and an average weight of approximately 88 kilograms and 31% being obese (body mass index > 30 kg/m2). Personal and respiratory health characteristics of these participants were similar to

6 the rest of the full cross-sectional study cohort (table 1 and table E1 in the online supplement), suggesting no obvious selection bias. Work and exposure characteristics for participants are shown in table 2. Full shift grain dust concentrations ranged from 0.010 to 13 mg/m3 (median 0.99 mg/m3), with 10.7% of samples measuring over the American College of Governmental Industrial Hygienists threshold limit value of 4 mg/m3 (31). Full shift endotoxin concentrations ranged from 8.1 to 11000 EU/m3 (median 610 EU/m3), with 88% of samples measuring over the recommended health based exposure limit for endotoxin (50 EU/m3) (32). After adjustment for the duration of work prior to EBC collection, dust concentrations ranged from 0.0022 to 12 mg/m3 (median 0.34 mg/m3) and endotoxin from 1.3 to 4500 EU/m3 (median 270 EU/m3). Dust and endotoxin values were highly correlated (r = 0.68 for full-shift original values, r=0.87 for log transformed values (figure 1), both p 0.05). Exhaled Breath Condensate Biomarker Measurements EBC pH values ranged from 4.3 to 8.2 (median: 7.9); NH4+ from 22 to 2400 µM (median: 420) and 8-isoprostane values varied from 1.3 to 45 pg/ml (median: 11). The distribution of EBC pH was negatively skewed with the appearance of 2 modes: a minor mode from 4.5 - 5.5 and major mode from 7.5 - 8.5 (figure 2a). The distributions of both 8-isoprostane and NH4+ were positively skewed (figure 2b and 2c). EBC pH and NH4+ were highly correlated (r=0.53, p 20 42.6 (20.4), 6 former smokers 1–9 2.6 (2.9), 11 10 – 19 15.4 (2.6), 9 > 20 39.5 (20.5), 8 Age, years 46.8 (7.5), 75 FEV1, % predicted 98.1 (13.3), 75 FVC, % predicted 101.6 (12.1), 75 MMF, % predicted 91.2 (30.9), 75 BMI: body mass index; EBC: exhaled breath condensate; FEV1: forced expiratory volume in one second; FVC: forced vital capacity; MMF: maximum mid-expiratory flow; std: standard deviation

23 Table 2. Work and exposure characteristics of the EBC study population Duration of work in grain industry (years) 0 – 9 (n=13) 10 – 19 (n=23) 20 – 29 (n=29) > 30 (n=10) in current job (years) Duration of work on study day prior to EBC collection (hours) Personal exposure on study day Grain dust Endotoxin Job title category

mean (std) 7.3 (1.7) 12.2 (1.9) 25.0 (2.0) 32.7 (2.2) 11.4 (8.4) 3.4 (1.7) 1,2

mean, std, median (min, max) 0.36, 5.8, 0.34 (0.0022, 12) 180, 6.9, 270 (1.3, 4500) 1,2, grain dust mean, std (endotoxin mean, std), n

Office based jobs Control room / quality control 0.030, 4.9 (10, 6.6), 9 Supervisor 0.088, 2.5 (45, 8.8), 4 Office Custodian 0.14, - (120, -), 1 Maintenance jobs Electrician 0.14, 2.3 (51, 2.4), 4 Millwright 0.67, 6.7 (290, 7.7), 6 Sheetmetal worker 1.1, 2.9 (580, 3.8), 7 Production jobs Trackshed (outside) 0.19, 6.6 (95, 4.5), 6 Pellet Plant Operator 0.34, 4.4 (170, 4.2), 7 Grain Cleaner Operator 0.39, 2.5 (230, 2.7), 9 General Labourer/ Sweeper 1.0, 3.3 (740, 2.9), 11 Basement worker 1.1, 1.7 (630, 1.1), 3 Gallery/ Distributor/ Bin Top/ 1.2, 4.6 (610, 4.6), 8 Annex worker Total 0.36, 5.78 (180, 6.9), 75 3 1 grain dust (mg/m ) and endotoxin (endotoxin units/m3), after adjustment for duration of work on the study day, see text for details 2 geometric mean and standard deviation std: standard deviation

24

Table 3. Factors associated with EBC Biomarker levels (Results from multivariable linear regression models) NH4+

pH

8-Isoprostane

coeff (std)

p

coeff (std)

p

coeff (std)

p

-0.5 (0.3)

0.05

-0.3 (0.3)

0.2

0.4 (0.2)

0.06

current smokers

-0.3 (0.1)

0.008

-0.3 (0.1)

0.04

-0.2 (0.1)

0.09

former smokers

-0.08 (0.1)

0.5

0.01 (0.1)

0.9

0.2 (0.1)

0.06

in the industry

-0.1 (0.1)

0.4

-0.2 (0.1)

0.07

-0.6 (0.1)

30) Pack-years of smoking

Duration of work

Intensity of2 grain dust (ln-mg/m3) or endotoxin (ln-EU/m3)

1 duration of work on test day, prior to testing (indicator of acute work duration) 2 due to co-linearity, only one or the other of grain dust and endotoxin were included; the model coefficients shown are from the model including grain dust, coefficients from the model including endotoxin were very similar and are shown in table E1 of the online supplement. BMI: body mass index; EU: endotoxin units; coeff (std): regression coefficient (standard deviation)

Figure 1.

Figure 2.

Figure 3.

1

Biomarkers of Airway Acidity and Oxidative Stress in Exhaled Breath Condensate from Grain Workers Ron Do, Karen H. Bartlett, Helen Dimich-Ward, Winnie Chu, Susan M. Kennedy Online Data Supplement

2 Methods Workers were chosen randomly from within specified job titles. The job titles were selected to represent the complete range of anticipated exposure levels to particulate matter and endotoxin. The selection of job titles covers those which involve grain production (ie. general labourer, sweeper, gallery, distributor, bin top, annex, grain cleaner operator, trackshed, basement, pellet plant operator), maintenance jobs inside the elevator (ie. electrician, millwright, sheetmetal) and office jobs (ie. panel control/ quality control operator, supervisor, office custodian). All participants gave informed written consent and confidentiality of all personal identifiers was ensured. Grain dust Full shift (minimum 6 h) personal exposure monitoring was performed on the same day as EBC collection. Personal air monitoring devices consisted of a constantflow sampling pump (SKC, Eighty-Four, PA, USA) attached to a 7-Hole inhalable aerosol sampler (JS Holdings Ltd., Stevanage, UK) and a 25 mm diameter, type A/E glass fiber filter (Pall Gelman Sciences, Ann Arbor, MI, USA) that were equilibrated to a stable temperature and a relative humidity. The pump was calibrated at a flow-rate of 2.0 L/min using a rotameter (Matheson Tri-gas, Montgomeryville, PA, USA) before and after each work shift. After sampling, filter samples were desiccated and re-equilibrated to the same stable temperature and relative humidity pre-weight. Grain dust particulate matter mass was quantified gravimetrically on a micro-balance (M3P, Sartorius, Germany) and samples were stored at 4 oC for endotoxin analysis. Endotoxin

3 Endotoxin analysis was performed on filter extracts using a kinetic limulus amoebocyte lysate assay according to manufacturer’s instructions (BioWhittaker Kinetic QCLTM, Cambrex Biomedicals, Walkersville, MD). Serial dilutions of endotoxin standard (EC O55:B5) were used to determine endotoxin concentration (50 – 0.049 EU/mL). Endotoxin concentration was calculated by fitting to a 4-parameter curve of the maximum rate of the colour reaction of the standards in a Spectromax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA). 8-isoprostane The 8-isoprostane content of EBC samples was analysed using an enzyme-linked immunoassay according to manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI). The concentration of 8-isoprostane was determined by comparison to a standard curve prepared for each assay (r2=0.95). The limit of detection (LOD) for this method was 2.5 pg/ml. Values below the LOD were calculated as 1.25 pg/ml (LOD / 2 = 2.5 / 2= 1.25 pg/ml). Respiratory health assessment, lung function, and atopy Each participant responded to an interviewer administered modified version of the standardized American Thoracic Society (ATS) questionnaire (1). Ever smokers (current and former) were defined by having smoked cigarettes (more than 20 packs of 20 cigarettes or more than one cigarette a day for one year), pipe (more than 12 oz. of tobacco in a lifetime), or cigars (more than 1 cigar a week for a year). Current smokers were defined by having smoked cigarettes, pipe or cigars as of one month ago. Packyears of smoking were calculated by number of years smoked multiplied by cigarette packs smoked per day. Smoking exposure equivalents of 4 cigars equal to 10 cigarettes,

4 and 1 gram of pipe tobacco equal to 1 cigarette were used for pipe and cigar smokers (n=10), based on Pechacek et al. (2). Lung function testing was performed by a trained pulmonary technician, using a dry rolling seal spirometer (model VRS 2000, S&M Instrument Co.) following the ATS standard protocol (3). FEV1, FVC and MMF were calculated as % predicted values, adjusting for age, sex, height and race (4). Atopy was defined by the presence of at least one positive skin reaction to any of four common aeroallergens (house dust mites: Dermatophagoides farinae and Dermatophagoides pteronyssinus, mixed grass pollen, and common weed mix), and a positive test was defined as having at least one wheal diameter 3 mm or more greater than that of the saline control. Data Analysis Data were analyzed using SAS, version 9.1 (SAS Institute, Cary NC) and statistical graphs were produced using R (R project for statistical computing, Vienna, Austria). In the laboratory study, the intra-individual variability was assessed using the coefficient of variation (CV), calculated as the intra-individual standard deviation (using a mixed effects regression model) as a percentage of the mean marker measurement. In the field study, normal probability plots were used to assess normality of the variables. To minimize the effect of outliers and to achieve univariate normality, ranked data were used for the three biomarker measurements by calculating inverse normal scores using the Blom method (proc rank). For independent variables, skewed distributions (ie. exposure concentrations) were natural log transformed to produce normal-like distributions, while other non-normal distributions were classified into categories as ordinal variables. In particular, smoking pack-years for current and former smokers were

5 ordered as 0, 1 - 9, 10 -19 and ≥ 20, which has been shown to reduce misclassification bias (5). In addition, duration of work in the industry were ordered as 0 – 9, 10 – 19, 20 – 29, and ≥ 30. Boundaries were chosen such that there were relatively equal and adequate numbers in each category, while still retaining equally spaced cut-offs (6). In univariate analyses, Student’s t-test (Satterthwaite t-test if unequal variances) or analysis of variance were used to compare categorical distributions; simple linear regression was used to compare continuous variables. Multivariable analyses were performed using regression modeling for each EBC marker parameter with marginally significant (p≤0.1) variables from univariate analyses offered using a manual stepwise procedure. A priori variables based on theory or previous studies were also tested in each model (atopy, reported asthma status, smoking, spirometry measures (forced expiratory volume in one second, forced vital capacity, maximum mid-expiratory flow, % predicted), personal exposure to grain dust and endotoxin). The variables that were included in the final model were: obesity (body mass index > 30 kg/m2), pack years for current and former smokers, years worked in the industry, duration of work before performing the EBC test (hours), and intensity of grain dust (ln-mg/m3) or endotoxin (ln-EU/m3). All tests were two-sided and considered statistically significant at the 0.05 level.

6

E1.

Ferris BG. Epidemiology standardization project (american thoracic society). Am

Rev Respir Dis 1978;118:1-120. E2.

Pechacek TF, Folsom AR, de Gaudermaris R, Jacobs DR, Jr., Luepker RV,

Gillum RF, Blackburn H. Smoke exposure in pipe and cigar smokers. Serum thiocyanate measures. JAMA 1985;254:3330-3332. E3.

ATS. Standardization of spirometry, 1994 update. American thoracic society. Am

J Respir Crit Care Med 1995;152:1107-1136. E4.

Crapo RO, Morris AH, Gardner RM. Reference spirometric values using

techniques and equipment that meet ats recommendations. Am Rev Respir Dis 1981;123:659-664. E5.

Bernaards CM, Twisk JW, Snel J, Van Mechelen W, Kemper HC. Is calculating

pack-years retrospectively a valid method to estimate life-time tobacco smoking? A comparison between prospectively calculated pack-years and retrospectively calculated pack-years. Addiction 2001;96:1653-1661. E6.

Rothman KJ, Greenland S. Modern epidemiology. Philadelphia, PA: Lippincott-

Raven Publishers; 1998.

7 Figure Legend

Figure E1. Partial residual plot of obesity on transformed pH, adjusted for pack-years of current and former smokers, duration of work in the industry, duration of work on the test day, and intensity of grain dust concentration. Inverse normal scores were used to transform pH.

Figure E2. Partial residual plot of pack-years of current smoking on transformed pH, adjusted for obesity, pack-years of former smokers, duration of work in the industry, duration of work on the test day, and intensity of grain dust concentration.

Figure E3. Partial residual plot of duration of work on test day prior to EBC collection on transformed pH, adjusted for obesity, pack-years of current and former smokers, duration of work in the industry and intensity of grain dust concentration.

Figure E4. Partial residual plot of pack-years of current smoking on transformed NH4+, adjusted for obesity, pack-years of former smokers, duration of work in the industry, duration of work on the test day, and intensity of grain dust concentration. Inverse normal scores were used to transform NH4+.

Figure E5. Partial residual plot of duration of work on test day prior to EBC collection on transformed NH4+, adjusted for obesity, pack-years of current and former smokers, duration of work in the industry and intensity of grain dust concentration.

8

Figure E6. Partial residual plot of duration of work in the industry on transformed 8isoprostane, adjusted for obesity, pack-years of current and former smokers, duration of work on the test day prior to EBC collection and intensity of grain dust concentration. Inverse normal scores were used to transform 8-isoprostane.

Figure E7. Partial residual plot of adjusted grain dust concentration on transformed 8isoprostane, adjusted for obesity, pack-years of current and former smokers, duration of work in the industry, and duration of work on the test day prior to EBC collection.

9 Results Table E1. Personal and Respiratory Health Characteristics of the EBC study subset, grain study subset, and overall grain study.

Female Non-Caucasian Obese (BMI > 30) Current asthma diagnosis Atopic Smoking status current former never Smoked in hour prior to EBC test

EBC Study Subset (n=75) n (%) 6 (8) 11 (15) 23 (31) 6 (8) 33 (44)

Rest of Grain Study (n=254) n (%) 25 (10) 25 (10) 56 (22) 11 (4) 84 (33)

Full Grain Study (n=329) n (%) 31 (9) 36 (11) 79 (24) 17 (5) 117 (36)

13 (17) 28 (37) 34 (45) 11 (15)

62 (24) 93 (37) 99 (39) -

75 (23) 121 (37) 133 (40) -

0.4

mean (std), n

mean (std), n

mean (std)

p1

Pack-years of smoking current smokers 1–9 8.3 (1.1), 2 5.0 (3.3), 10 5.5 (3.3), 12 10 – 19 15.0 (4.0), 5 14.8 (3.1), 17 14.8 (3.2), 22 > 20 42.6 (20.4), 6 41.3 (27.8), 33 41.5 (26.5), 39 former smokers 1–9 2.6 (2.9), 11 4.0 (2.5), 38 3.6 (2.6). 49 10 – 19 15.4 (2.6), 9 14.1 (3.1), 23 14.5 (3.0), 32 > 20 39.5 (20.5), 8 33.5 (15.9), 30 34.8 (16.8), 38 Age (years) 46.8 (7.5), 75 46.5 (7.7), 254 46.6 (7.6), 329 FEV1, % predicted 98.1 (13.3), 75 97.6 (14.0), 254 97.7 (13.8), 329 FVC, % predicted 101.6 (12.1), 75 102.8 (12.9), 254 102.5 (12.7), 329 MMF, % predicted 91.2 (30.9), 75 87.5 (28.6), 254 88.3 (29.1), 329 1 p-value for comparisons of EBC study subset (n=75) and rest of grain study (n=254). Chi-squared test used to compare proportions, analysis of variance or simple linear regression used to compare continuous distributions. BMI: body mass index; EBC: exhaled breath condensate; std: standard deviation; FEV1: forced expiratory volume in one second; FVC: forced vital capacity; MMF: maximum mid-expiratory flow

1

p 0.6 0.2 0.2 0.2 0.08

-

0.2

0.9 0.8 0.8 0.5 0.4

10

Table E2. Factors associated with EBC Biomarker levels: Endotoxin model (Results from multivariable linear regression models) NH4+

pH

8-Isoprostane

coeff (std)

p

coeff (std)

p

coeff (std)

p

-0.5 (0.3)

0.05

-0.3 (0.3)

0.3

0.5 (0.2)

0.05

current smokers

-0.3 (0.1)

0.008

-0.3 (0.1)

0.03

-0.2 (0.1)

0.07

former smokers

-0.08 (0.1)

0.5

0.02 (0.1)

0.9

0.2 (0.1)

0.05

in industry

-0.1 (0.1)

0.4

-0.2 (0.1)

0.07

-0.6 (0.1)

30) Pack-years of smoking

Duration of work

Intensity of endotoxin (ln-EU/m3)

1 duration of work on test day, prior to testing (indicator of acute work duration) BMI: body mass index; EU: endotoxin units; coeff (std): regression coefficient (standard deviation)

11 Figure E1.

12 Figure E2.

13 Figure E3.

14

Figure E4.

15 Figure E5.

16 Figure E6.

17 Figure E7.