Human Biological Responses to Low Level Ozone (SNOZ)

Air pollutants including ozone have been implicated in affecting health outcomes. In particular, high level ozone exposure has been shown to affect pulmonary function and cause pulmonary inflammation. Troubling community-based work has implicated high ozone levels as being correlated with increased pediatric asthma emergency room visits. Because of adverse health effects, EPA standards for safe ozone levels have been set, currently at 0.07 ppm. Still, it is estimated that 100 million Americans live in areas where ozone levels periodically remain above the EPA standard. And while this EPA standard had been set based on available data, it remained unclear at the time whether naturalistic low-level ozone exposure, such as fluctuations between 0.06-0.08 ppm throughout the day, might affect health as well. This group previously examined lung function and inflammatory response in adults exposed to low-level ozone, 0.06 ppm exposure for 6.6 hours, while undergoing intermittent moderate exercise. The investigators found that in response to low-level ozone exposure (0.06 ppm) with exercise, lung function declines and neutrophilic airway inflammation is observed. What remains unclear, is whether low-level ozone alone - without exercise - will cause similar health effects. To mimic exposure to ozone on a typical summer day in a polluted city, the investigators will expose subjects to a varying level of ozone, form 0.06 ppm to 0.08 ppm, rather than a constant 0.07ppm. The variation from 0.06ppm to 0.08ppm, then back to 0.06ppm will occur each hour.

Participants will be exposed to a concentration of ozone for 6.5 hours at a concentration that varies 0.06 to 0.08

Change in % Polymorphonuclear Leukocytes (PMN) in Nasal Lavage Fluid Immediately Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Nasal lavage fluid (NLF) will be collected from participants at a baseline visit within two weeks prior to each exposure, and at the following time points for each exposure: immediately after exiting the exposure chamber, and at 24 hours after the exposure. The % PMN as a percentage of total inflammatory cells (total of monocytes and macrophages, PMN, eosinophils, basophils, lymphocytes, and bronchial epithelial cells) will be determined in each NLF collection by differential counts of cells on cytospin slides. The change in the values from baseline to immediately Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3.

Change in % Polymorphonuclear Leukocytes (PMN) in Nasal Lavage Fluid 24 Hours Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Nasal lavage fluid (NLF) will be collected from participants at a baseline visit within two weeks prior to each exposure, and at the following time points for each exposure: immediately after exiting the exposure chamber, and at 24 hours after the exposure. The % PMN as a percentage of total inflammatory cells (total of monocytes and macrophages, PMN, eosinophils, basophils, lymphocytes, and bronchial epithelial cells) will be determined in each NLF collection by differential counts of cells on cytospin slides. The change in the values from baseline to 24 hours Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3.

Interleukin-6 (IL-6) Concentrations in Nasal Epithelial Lining Fluid (ELF): Change Immediately Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Nasal epithelial lining (ELF) will be collected from participants at a baseline visit within two weeks prior to each exposure, and at the following time points for each exposure: immediately after exiting the exposure chamber, and at 24 hours after the exposure. The IL-6 concentration will be determined in each ELF sample by immunoassay. The change in IL-6 concentrations from baseline to immediately Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3 to determine the effect of ozone on the production of nasal IL-6.

% Polymorphonuclear Leukocytes (PMN) in Induced Sputum: Change 24 Hours Post-Exposure From Screening Visit

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Induced sputum will be collected from participants after inhaled hypertonic saline. Induced sputum will be collected at the screening visit within six weeks prior to the first exposure, and at 24 hours after each exposure. The % PMN as a percentage of total inflammatory cells (total of monocytes and macrophages, PMN, eosinophils, basophils, lymphocytes, and bronchial epithelial cells) will be determined in each induced sputum collection by differential counts of cells on cytospin slides. The change in the values from baseline to 24 hours Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3.

The Percentage of Predicted Forced Expiratory Volume in One Second (% Predicted FEV1): Change Immediately Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Standard spirometry to obtain FEV1 and forced vital capacity (FVC) measurements will be done at baseline within two weeks prior to first exposure, and immediately after exiting the exposure chamber for each exposure. The % Predicted FEV1 will be calculated from measured versus expected values. The change in % Predicted FEV1 from baseline to immediately Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3 to determine the effect of ozone on the % Predicted FEV1.

Interleukin-6 (IL-6) Concentrations in Nasal Epithelial Lining Fluid (ELF): Change 24 Hours Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Nasal epithelial lining (ELF) will be collected from participants at baseline within two weeks prior to first exposure, and at the following time points for each exposure: immediately after exiting the exposure chamber, and at 24 hours after the exposure. The IL-6 concentration will be determined in each ELF sample by immunoassay. The change in IL-6 concentrations from baseline to 24 hours Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3 to determine the effect of ozone on the production of nasal IL-6.

Interleukin-8 (IL-8) Concentrations in Nasal Epithelial Lining Fluid (ELF): Change Immediately Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Nasal epithelial lining (ELF) will be collected from participants at baseline within two weeks prior to first exposure, and at the following time points for each exposure: immediately after exiting the exposure chamber, and at 24 hours after the exposure. The IL-8 concentration will be determined in each ELF sample by immunoassay. The change in IL-8 concentrations from baseline to immediately Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3 to determine the effect of ozone on the production of nasal IL-8.

Interleukin-8 (IL-8) Concentrations in Nasal Epithelial Lining Fluid (ELF): Change 24 Hours Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Nasal epithelial lining (ELF) will be collected from participants at baseline within two weeks prior to first exposure, and at the following time points for each exposure: immediately after exiting the exposure chamber, and at 24 hours after the exposure. The IL-8 concentration will be determined in each ELF sample by immunoassay. The change in IL-8 concentrations from baseline to 24 hours Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3 to determine the effect of ozone on the production of nasal IL-8.

Left ventricular strain will be assessed at baseline prior to exposure (within two weeks), and immediately after the exposure by measuring global longitudinal strain (GLS), an echocardiographic measure of myocardial mechanics. GLS is measured using speckle tracking, a technique by which small myocardial footprints, or speckles, are tracked over the cardiac cycle to enable quantification of left ventricular systolic function. GLS is more sensitive than traditional measures of ventricular function, such as ejection fraction, in detecting clinically inapparent but prognostically important decrements in contractility. The change in global longitudinal strain will be calculated to determine the effect of ozone on left ventricular strain.

Nasal Epithelial Cell IL-1 Beta Gene Expression: Relative mRNA Counts Immediately Post-Exposure (Baseline-corrected), Ozone vs Filtered Air

RNA isolated from nasal epithelial cell biopsies collected at the screening visit (baseline) within six weeks prior to the first exposure and immediately after each exposure. RNA will be analyzed via real-time quantitative polymerase chain reaction (qPCR) to determine the impact of O3 on inflammatory gene expression.

Participants will undergo assesment of flow-mediated brachial artery dilation by brachial ultrasound at baseline prior to exposure (within two weeks), and immediately after exposure to either FA or O3 to assess the impact of O3 exposure on endothelial function. Flow mediated dilation of the brachial artery (FMD) is a noninvasive index of vascular endothelial function. FMD is measured using high-frequency ultrasound, and is expressed as the percent change in arterial diameter in response to the reactive hyperemia induced by 5 minutes of forearm ischemia. Impaired endothelial function leads to atherosclerosis and is associated with an increased risk of cardiovascular events.

Measure was not performed as the equipment (ECG leads and monitor recording heart rate and rhythm) belonged to the EPA and was not made available for this study

Nasal Epithelial Cell IL-6R Gene Expression: Relative mRNA Counts Immediately Post-Exposure (Baseline-corrected), Ozone vs Filtered Air

RNA isolated from nasal epithelial cell biopsies collected at the screening visit (baseline) within six weeks prior to the first exposure and immediately after each exposure. RNA will be analyzed via real-time quantitative polymerase chain reaction (qPCR) to determine the impact of O3 on inflammatory gene expression.

Nasal Epithelial Cell IL-8 Gene Expression: Relative mRNA Counts Immediately Post-Exposure (Baseline-corrected), Ozone vs Filtered Air

RNA isolated from nasal epithelial cell biopsies collected at the screening visit (baseline) within six weeks prior to the first exposure and immediately after each exposure. RNA will be analyzed via real-time quantitative polymerase chain reaction (qPCR) to determine the impact of O3 on inflammatory gene expression.

The Percentage of Predicted Forced Vital Capacity (% Predicted FVC): Change Immediately Post-Exposure From Baseline

Participants will be exposed to either filtered air (FA), then ozone (O3), or O3, then FA. Standard spirometry to obtain FEV1 and forced vital capacity (FVC) measurements will be done at baseline within two weeks prior to first exposure, and immediately after exiting the exposure chamber for each exposure. The % Predicted FVC will be calculated from measured versus expected values. The change in % Predicted FVC from baseline to immediately Post-Exposure will be calculated for each participant for each exposure. Values will be compared between FA and O3 to determine the effect of ozone on the % Predicted FVC.

Inclusion Criteria: Males and females between 18 and 50 years of age. Vital signs within normal limits on admission to the study: Peripheral oxygen saturation (SpO2) > 94%, systolic blood pressure between 150-90 mm Hg, diastolic blood pressure between 100-60 mm Hg, afebrile. Forced Expiratory Volume (FEV1) of at least 80% of predicted. Exclusion Criteria: Any chronic medical condition considered by the PI as a contraindication to the exposure study, including significant cardiovascular disease, diabetes requiring medication, chronic renal disease, chronic thyroid disease, or kidney disease. Use of systemic or inhaled steroids. Use of NSAID or aspirin within 7days of each study visit, and inability to withhold these medications prior to each session of the study. Pregnant or nursing women Use of cigarettes or other inhaled nicotine products within the past year, or more than a lifetime 5 pack year history of cigarette smoking.

Peden DB, Setzer RW Jr, Devlin RB. Ozone exposure has both a priming effect on allergen-induced responses and an intrinsic inflammatory action in the nasal airways of perennially allergic asthmatics. Am J Respir Crit Care Med. 1995 May;151(5):1336-45. doi: 10.1164/ajrccm.151.5.7735583.