Effects of short-term increases in personal and ambient pollutant concentrations on pulmonary and cardiovascular function: A panel study analysis of the Multicenter Ozone Study in oldEr subjects (MOSES 2).

BACKGROUND: The cardiovascular effects of ozone exposure are unclear. Using measurements from the 87 participants in the Multicenter Ozone Study of oldEr Subjects (MOSES), we examined whether personal and ambient pollutant exposures before the controlled exposure sessions would be associated with adverse changes in pulmonary and cardiovascular function. METHODS: We used mixed effects linear regression to evaluate associations between increased personal exposures and ambient pollutant concentrations in the 96 h before the pre-exposure visit, and 1) biomarkers measured at pre-exposure, and 2) changes in biomarkers from pre-to post-exposure. RESULTS: Decreases in pre-exposure forced expiratory volume in 1 s (FEV1) were associated with interquartile-range increases in concentrations of particulate matter ≤2.5 µm (PM2.5) 1 h before the pre-exposure visit (-0.022 L; 95% CI -0.037 to -0.006; p = 0.007), carbon monoxide (CO) in the prior 3 h (-0.046 L; 95% CI -0.076 to -0.016; p = 0.003), and nitrogen dioxide (NO2) in the prior 72 h (-0.030 L; 95% CI -0.052 to -0.008; p = 0.007). From pre-to post-exposure, increases in FEV1 were marginally significantly associated with increases in personal ozone exposure (0.010 L; 95% CI 0.004 to 0.026; p = 0.010), and ambient PM2.5 and CO at all lag times. Ambient ozone concentrations in the prior 96 h were associated with both decreased pre-exposure high frequency (HF) heart rate variability (HRV) and increases in HF HRV from pre-to post-exposure. CONCLUSIONS: We observed associations between increased ambient PM2.5, NO2, and CO levels and reduced pulmonary function, and increased ambient ozone concentrations and reduced HRV. Pulmonary function and HRV increased across the exposure sessions in association with these same pollutant increases, suggesting a "recovery" during the exposure sessions. These findings support an association between short term increases in ambient PM2.5, NO2, and CO and decreased pulmonary function, and increased ambient ozone and decreased HRV.

Multicenter Ozone Study in oldEr Subjects (MOSES): Part 2. Effects of Personal and Ambient Concentrations of Ozone and Other Pollutants on Cardiovascular and Pulmonary Function.

INTRODUCTION: The Multicenter Ozone Study of oldEr Subjects (MOSES) was a multi-center study evaluating whether short-term controlled exposure of older, healthy individuals to low levels of ozone (O3) induced acute changes in cardiovascular biomarkers. In MOSES Part 1 (MOSES 1), controlled O3 exposure caused concentration-related reductions in lung function with evidence of airway inflammation and injury, but without convincing evidence of effects on cardiovascular function. However, subjects' prior exposures to indoor and outdoor air pollution in the few hours and days before each MOSES controlled O3 exposure may have independently affected the study biomarkers and/or modified biomarker responses to the MOSES controlled O3 exposures. METHODS: MOSES 1 was conducted at three clinical centers (University of California San Francisco, University of North Carolina, and University of Rochester Medical Center) and included healthy volunteers 55 to 70 years of age. Consented participants who successfully completed the screening and training sessions were enrolled in the study. All three clinical centers adhered to common standard operating procedures and used common tracking and data forms. Each subject was scheduled to participate in a total of 11 visits: screening visit, training visit, and three sets of exposure visits consisting of the pre-exposure day, the exposure day, and the post-exposure day. After completing the pre-exposure day, subjects spent the night in a nearby hotel. On exposure days, the subjects were exposed for 3 hours in random order to 0 ppb O3 (clean air), 70 ppb O3, and 120 ppm O3. During the exposure period the subjects alternated between 15 minutes of moderate exercise and 15 minutes of rest. A suite of cardiovascular and pulmonary endpoints was measured on the day before, the day of, and up to 22 hours after each exposure.In MOSES Part 2 (MOSES 2), we used a longitudinal panel study design, cardiopulmonary biomarker data from MOSES 1, passive cumulative personal exposure samples (PES) of O3 and nitrogen dioxide (NO2) in the 72 hours before the pre-exposure visit, and hourly ambient air pollution and weather measurements in the 96 hours before the pre-exposure visit. We used mixed-effects linear regression and evaluated whether PES O3 and NO2 and these ambient pollutant concentrations in the 96 hours before the pre-exposure visit confounded the MOSES 1 controlled O3 exposure effects on the pre- to post-exposure biomarker changes (Aim 1), whether they modified these pre- to post-exposure biomarker responses to the controlled O3 exposures (Aim 2), whether they were associated with changes in biomarkers measured at the pre-exposure visit or morning of the exposure session (Aim 3), and whether they were associated with differences in the pre- to post-exposure biomarker changes independently of the controlled O3 exposures (Aim 4). RESULTS: Ambient pollutant concentrations at each site were low and were regularly below the National Ambient Air Quality Standard levels. In Aim 1, the controlled O3 exposure effects on the pre- to post-exposure biomarker differences were little changed when PES or ambient pollutant concentrations in the previous 96 hours were included in the model, suggesting these were not confounders of the controlled O3 exposure/biomarker difference associations. In Aim 2, effects of MOSES controlled O3 exposures on forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were modified by ambient NO2 and carbon monoxide (CO), and PES NO2, with reductions in FEV1 and FVC observed only when these concentrations were "Medium" or "High" in the 72 hours before the pre-exposure visit. There was no such effect modification of the effect of controlled O3 exposure on any other cardiopulmonary biomarker.As hypothesized for Aim 3, increased ambient O3 concentrations were associated with decreased pre-exposure heart rate variability (HRV). For example, high frequency (HF) HRV decreased in association with increased ambient O3 concentrations in the 96 hours before the pre-exposure visit (-0.460 ln[ms2]; 95% CI, -0.743 to -0.177 for each 10.35-ppb increase in O3; P = 0.002). However, in Aim 4 these increases in ambient O3 were also associated with increases in HF and low frequency (LF) HRV from pre- to post-exposure, likely reflecting a "recovery" of HRV during the MOSES O3 exposure sessions. Similar patterns across Aims 3 and 4 were observed for LF (the other primary HRV marker), and standard deviation of normal-to-normal sinus beat intervals (SDNN) and root mean square of successive differences in normal-to-normal sinus beat intervals (RMSSD) (secondary HRV markers).Similar Aim 3 and Aim 4 patterns were observed for FEV1 and FVC in association with increases in ambient PM with an aerodynamic diameter ≤ 2.5 µm (PM2.5), CO, and NO2 in the 96 hours before the pre-exposure visit. For Aim 3, small decreases in pre-exposure FEV1 were significantly associated with interquartile range (IQR) increases in PM2.5 concentrations in the 1 hour before the pre-exposure visit (-0.022 L; 95% CI, -0.037 to -0.006; P = 0.007), CO in the 3 hours before the pre-exposure visit (-0.046 L; 95% CI, -0.076 to -0.016; P = 0.003), and NO2 in the 72 hours before the pre-exposure visit (-0.030 L; 95% CI, -0.052 to -0.008; P = 0.007). However, FEV1 was not associated with ambient O3 or sulfur dioxide (SO2), or PES O3 or NO2 (Aim 3). For Aim 4, increased FEV1 across the exposure session (post-exposure minus pre-exposure) was marginally significantly associated with each 4.1-ppb increase in PES O3 concentration (0.010 L; 95% CI, 0.004 to 0.026; P = 0.010), as well as ambient PM2.5 and CO at all lag times. FVC showed similar associations, with patterns of decreased pre-exposure FVC associated with increased PM2.5, CO, and NO2 at most lag times, and increased FVC across the exposure session also associated with increased concentrations of the same pollutants, reflecting a similar recovery. However, increased pollutant concentrations were not associated with adverse changes in pre-exposure levels or pre- to post-exposure changes in biomarkers of cardiac repolarization, ST segment, vascular function, nitrotyrosine as a measure of oxidative stress, prothrombotic state, systemic inflammation, lung injury, or sputum polymorphonuclear leukocyte (PMN) percentage as a measure of airway inflammation. CONCLUSIONS: Our previous MOSES 1 findings of controlled O3 exposure effects on pulmonary function, but not on any cardiovascular biomarker, were not confounded by ambient or personal O3 or other pollutant exposures in the 96 and 72 hours before the pre-exposure visit. Further, these MOSES 1 O3 effects were generally not modified, blunted, or lessened by these same ambient and personal pollutant exposures. However, the reductions in markers of pulmonary function by the MOSES 1 controlled O3 exposure were modified by ambient NO2 and CO, and PES NO2, with reductions observed only when these pollutant concentrations were elevated in the few hours and days before the pre-exposure visit. Increased ambient O3 concentrations were associated with reduced HRV, with "recovery" during exposure visits. Increased ambient PM2.5, NO2, and CO were associated with reduced pulmonary function, independent of the MOSES-controlled O3 exposures. Increased pollutant concentrations were not associated with pre-exposure or pre- to post-exposure changes in other cardiopulmonary biomarkers. Future controlled exposure studies should consider the effect of ambient pollutants on pre-exposure biomarker levels and whether ambient pollutants modify any health response to a controlled pollutant exposure.

Multicenter Ozone Study in oldEr Subjects (MOSES): Part 1. Effects of Exposure to Low Concentrations of Ozone on Respiratory and Cardiovascular Outcomes.

INTRODUCTION: Exposure to air pollution is a well-established risk factor for cardiovascular morbidity and mortality. Most of the evidence supporting an association between air pollution and adverse cardiovascular effects involves exposure to particulate matter (PM). To date, little attention has been paid to acute cardiovascular responses to ozone, in part due to the notion that ozone causes primarily local effects on lung function, which are the basis for the current ozone National Ambient Air Quality Standards (NAAQS). There is evidence from a few epidemiological studies of adverse health effects of chronic exposure to ambient ozone, including increased risk of mortality from cardiovascular disease. However, in contrast to the well-established association between ambient ozone and various nonfatal adverse respiratory effects, the observational evidence for impacts of acute (previous few days) increases in ambient ozone levels on total cardiovascular mortality and morbidity is mixed.Ozone is a prototypic oxidant gas that reacts with constituents of the respiratory tract lining fluid to generate reactive oxygen species (ROS) that can overwhelm antioxidant defenses and cause local oxidative stress. Pathways by which ozone could cause cardiovascular dysfunction include alterations in autonomic balance, systemic inflammation, and oxidative stress. These initial responses could lead ultimately to arrhythmias, endothelial dysfunction, acute arterial vasoconstriction, and procoagulant activity. Individuals with impaired antioxidant defenses, such as those with the null variant of glutathione S-transferase mu 1 (GSTM1), may be at increased risk for acute health effects.The Multicenter Ozone Study in oldEr Subjects (MOSES) was a controlled human exposure study designed to evaluate whether short-term exposure of older, healthy individuals to ambient levels of ozone induces acute cardiovascular responses. The study was designed to test the a priori hypothesis that short-term exposure to ambient levels of ozone would induce acute cardiovascular responses through the following mechanisms: autonomic imbalance, systemic inflammation, and development of a prothrombotic vascular state. We also postulated a priori the confirmatory hypothesis that exposure to ozone would induce airway inflammation, lung injury, and lung function decrements. Finally, we postulated the secondary hypotheses that ozone-induced acute cardiovascular responses would be associated with: (a) increased systemic oxidative stress and lung effects, and (b) the GSTM1-null genotype. METHODS: The study was conducted at three clinical centers with a separate Data Coordinating and Analysis Center (DCAC) using a common protocol. All procedures were approved by the institutional review boards (IRBs) of the participating centers. Healthy volunteers 55 to 70 years of age were recruited. Consented participants who successfully completed the screening and training sessions were enrolled in the study. All three clinical centers adhered to common standard operating procedures (SOPs) and used common tracking and data forms. Each subject was scheduled to participate in a total of 11 visits: screening visit, training visit, and three sets of exposure visits, each consisting of the pre-exposure day, the exposure day, and the post-exposure day. The subjects spent the night in a nearby hotel the night of the pre-exposure day.On exposure days, the subjects were exposed for three hours in random order to 0 ppb ozone (clean air), 70 ppb ozone, and 120 ppm ozone, alternating 15 minutes of moderate exercise with 15 minutes of rest. A suite of cardiovascular and pulmonary endpoints was measured on the day before, the day of, and up to 22 hours after, each exposure. The endpoints included: (1) electrocardiographic changes (continuous Holter monitoring: heart rate variability [HRV], repolarization, and arrhythmia); (2) markers of inflammation and oxidative stress (C-reactive protein [CRP], interleukin-6 [IL-6], 8-isoprostane, nitrotyrosine, and P-selectin); (3) vascular function measures (blood pressure [BP], flow-mediated dilatation [FMD] of the brachial artery, and endothelin-1 [ET-1]; (4) venous blood markers of platelet activation, thrombosis, and microparticle-associated tissue factor activity (MP-TFA); (5) pulmonary function (spirometry); (6) markers of airway epithelial cell injury (increases in plasma club cell protein 16 [CC16] and sputum total protein); and (7) markers of lung inflammation in sputum (polymorphonuclear leukocytes [PMN], IL-6, interleukin-8 [IL-8], and tumor necrosis factor-alpha [TNF-α]). Sputum was collected only at 22 hours after exposure.The analyses of the continuous electrocardiographic monitoring, the brachial artery ultrasound (BAU) images, and the blood and sputum samples were carried out by core laboratories. The results of all analyses were submitted directly to the DCAC.The variables analyzed in the statistical models were represented as changes from pre-exposure to post-exposure (post-exposure minus pre-exposure). Mixed-effect linear models were used to evaluate the impact of exposure to ozone on the prespecified primary and secondary continuous outcomes. Site and time (when multiple measurements were taken) were controlled for in the models. Three separate interaction models were constructed for each outcome: ozone concentration by subject sex; ozone concentration by subject age; and ozone concentration by subject GSTM1 status (null or sufficient). Because of the issue of multiple comparisons, the statistical significance threshold was set a priori at P < 0.01. RESULTS: Subject recruitment started in June 2012, and the first subject was randomized on July 25, 2012. Subject recruitment ended on December 31, 2014, and testing of all subjects was completed by April 30, 2015. A total of 87 subjects completed all three exposures. The mean age was 59.9 ± 4.5 years, 60% of the subjects were female, 88% were white, and 57% were GSTM1 null. Mean baseline body mass index (BMI), BP, cholesterol (total and low-density lipoprotein), and lung function were all within the normal range.We found no significant effects of ozone exposure on any of the primary or secondary endpoints for autonomic function, repolarization, ST segment change, or arrhythmia. Ozone exposure also did not cause significant changes in the primary endpoints for systemic inflammation (CRP) and vascular function (systolic blood pressure [SBP] and FMD) or secondary endpoints for systemic inflammation and oxidative stress (IL-6, P-selectin, and 8-isoprostane). Ozone did cause changes in two secondary endpoints: a significant increase in plasma ET-1 (P = 0.008) and a marginally significant decrease in nitrotyrosine (P = 0.017). Lastly, ozone exposure did not affect the primary prothrombotic endpoints (MP-TFA and monocyte-platelet conjugate count) or any secondary markers of prothrombotic vascular status (platelet activation, circulating microparticles [MPs], von Willebrand factor [vWF], or fibrinogen.).Although our hypothesis focused on possible acute cardiovascular effects of exposure to low levels of ozone, we recognized that the initial effects of inhaled ozone involve the lower airways. Therefore, we looked for: (a) changes in lung function, which are known to occur during exposure to ozone and are maximal at the end of exposure; and (b) markers of airway injury and inflammation. We found an increase in forced vital capacity (FVC) and forced expiratory volume in 1 second (FEV1) after exposure to 0 ppb ozone, likely due to the effects of exercise. The FEV1 increased significantly 15 minutes after 0 ppb exposure (85 mL; 95% confidence interval [CI], 64 to 106; P < 0.001), and remained significantly increased from pre-exposure at 22 hours (45 mL; 95% CI, 26 to 64; P < 0.001). The increase in FVC followed a similar pattern. The increase in FEV1 and FVC were attenuated in a dose-response manner by exposure to 70 and 120 ppb ozone. We also observed a significant ozone-induced increase in the percentage of sputum PMN 22 hours after exposure at 120 ppb compared to 0 ppb exposure (P = 0.003). Plasma CC16 also increased significantly after exposure to 120 ppb (P < 0.001). Sputum IL-6, IL-8, and TNF-α concentrations were not significantly different after ozone exposure. We found no significant interactions with sex, age, or GSTM1 status regarding the effect of ozone on lung function, percentage of sputum PMN, or plasma CC16. CONCLUSIONS: In this multicenter clinical study of older healthy subjects, ozone exposure caused concentration-related reductions in lung function and presented evidence for airway inflammation and injury. However, there was no convincing evidence for effects on cardiovascular function. Blood levels of the potent vasoconstrictor, ET-1, increased with ozone exposure (with marginal statistical significance), but there were no effects on BP, FMD, or other markers of vascular function. Blood levels of nitrotyrosine decreased with ozone exposure, the opposite of our hypothesis. Our study does not support acute cardiovascular effects of low-level ozone exposure in healthy older subjects. Inclusion of only healthy older individuals is a major limitation, which may affect the generalizability of our findings. We cannot exclude the possibility of effects with higher ozone exposure concentrations or more prolonged exposure, or the possibility that subjects with underlying vascular disease, such as hypertension or diabetes, would show effects under these conditions.

Mutational spectra of a 100-base pair mitochondrial DNA target sequence in bronchial epithelial cells: a comparison of smoking and nonsmoking twins.

Seventeen separate mitochondrial hot spot mutations in a 100-bp target sequence (mitochondrial bp 10,030-10,130) were detected and measured in bronchial epithelial cell samples isolated from smokers and nonsmokers. Among the individuals sampled were three pairs of monozygotic twins in which one twin had never smoked and had a nonsmoking spouse, and the other had a smoking history of >10 pack-years. Individual point mutations present at frequencies as low as 10(-6) were detected. Partially denaturing electrophoresis was used to separate mutant from nonmutant sequences on the basis of their melting temperatures, and the target sequence was subsequently amplified via high-fidelity PCR with Pfu DNA polymerase. Tests were performed to determine whether mismatch intermediates or DNA adducts present in the cellular DNA were converted to mutants during PCR. Hot spot mutations were clearly observed in bronchial epithelial cells, and the same hot spots were observed consistently in different samples. Significant numerical variability in the mutant fractions for individual mutants was observed among samples and are ascribed to unequal mitochondrial segregation in stem and transition cells. The mutational spectra in smokers' samples did not differ significantly from the mutational spectra in nonsmokers' samples for this 100 bp of mitochondrial DNA. No smoking-specific hot spots were detected. The overall mutant fractions in smokers' samples were not elevated compared to those of nonsmokers. As much variability was observed between two samples from the same individual's lung as between a sample from a smoker and a sample from a nonsmoker. These findings demonstrate that inhaled tobacco smoke does not induce prominent point mutations in this 100-bp target mitochondrial sequence in smokers' bronchial epithelial cells. Endogenous factors (e.g., DNA replication errors or DNA damage by endogenous reactive chemicals) are suggested to be more likely to represent the most important contributors to mitochondrial mutagenesis.

Systemic and cardiovascular effects of airway injury and inflammation: ultrafine particle exposure in humans.

The concentration of particles in the ambient air is associated with deaths from cardiovascular disease, and determining the biologic mechanisms involved has been identified as a high-priority research need. Hypotheses have focused on the possibility of direct cardiac effects, or indirect effects related to inflammatory responses, including increased blood viscosity or increased blood coagulability. Ultrafine particles (UFPs; those smaller than 100 nm) may be important in cardiovascular effects because of their very high deposition efficiency in the pulmonary region, and their high propensity to penetrate the epithelium and reach interstitial sites. We have initiated human clinical studies of the health effects of UFPs using a mouthpiece exposure system. Healthy, nonsmoking subjects 18-55 years of age are exposed at rest for 2 hr to 10 microg/m3 carbon UFPs and to filtered air as a control. Preliminary findings indicate a relatively high overall deposition fraction (0.66 +/- 0.12 by particle number) consistent with model predictions and an absence of particle-associated symptoms or changes in lung function. Planned studies examine responses in susceptible subject groups, and the effects of particles of varying composition. Human clinical studies using model particles will complement other approaches such as epidemiologic, animal exposure, and in vitro studies in determining the mechanisms for heath effects related to ambient particle exposure.

Toxicologic methods: controlled human exposures.

The assessment of risk from exposure to environmental air pollutants is complex, and involves the disciplines of epidemiology, animal toxicology, and human inhalation studies. Controlled, quantitative studies of exposed humans help determine health-related effects that result from breathing the atmosphere. The major unique feature of the clinical study is the ability to select, control, and quantify pollutant exposures of subjects of known clinical status, and determine their effects under ideal experimental conditions. The choice of outcomes to be assessed in human clinical studies can be guided by both scientific and practical considerations, but the diversity of human responses and responsiveness must be considered. Subjects considered to be among the most susceptible include those with asthma, chronic obstructive lung disease, and cardiovascular disease. New experimental approaches include exposures to concentrated ambient air particles, diesel engine exhaust, combustion products from smoking machines, and experimental model particles. Future investigations of the health effects of air pollution will benefit from collaborative efforts among the disciplines of epidemiology, animal toxicology, and human clinical studies.

Simultaneous measurement of nitric oxide production by conducting and alveolar airways of humans.

Human airways produce nitric oxide (NO), and exhaled NO increases as expiratory flow rates fall. We show that mixing during exhalation between the NO produced by the lower, alveolar airways (VL(NO)) and the upper conducting airways (VU(NO)) explains this phenomenon and permits measurement of VL(NO), VU(NO), and the NO diffusing capacity of the conducting airways (DU(NO)). After breath holding for 10-15 s the partial pressure of alveolar NO (PA) becomes constant, and during a subsequent exhalation at a constant expiratory flow rate the alveoli will deliver a stable amount of NO to the conducting airways. The conducting airways secrete NO into the lumen (VU(NO)), which mixes with PA during exhalation, resulting in the observed expiratory concentration of NO (PE). At fast exhalations, PA makes a large contribution to PE, and, at slow exhalations, NO from the conducting airways predominates. Simple equations describing this mixing, combined with measurements of PE at several different expiratory flow rates, permit calculation of PA, VU(NO), and DU(NO). VL(NO) is the product of PA and the alveolar airway diffusion capacity for NO. In seven normal subjects, PA = 1.6 +/- 0.7 x 10(-6) (SD) Torr, VL(NO) = 0.19 +/- 0.07 microl/min, VU(NO) = 0.08 +/- 0.05 microl/min, and DU(NO) = 0.4 +/- 0.4 ml. min(-1). Torr(-1). These quantitative measurements of VL(NO) and VU(NO) are suitable for exploring alterations in NO production at these sites by diseases and physiological stresses.

Determination of production of nitric oxide by lower airways of humans--theory.

Exercise and inflammatory lung disorders such as asthma and acute lung injury increase exhaled nitric oxide (NO). This finding is interpreted as a rise in production of NO by the lungs (VNO) but fails to take into account the diffusing capacity for NO (DNO) that carries NO into the pulmonary capillary blood. We have derived equations to measure VNO from the following rates, which determine NO tension in the lungs (PL) at any moment from 1) production (VNO); 2) diffusion, where DNO(PL) = rate of removal by lung capillary blood; and 3) ventilation, where V A(PL)/(PB - 47) = the rate of NO removal by alveolar ventilation (V A) and PB is barometric pressure. During open-circuit breathing when PL is not in equilibrium, d/dt PL[V(L)/ (PB - 47)] (where V(L) is volume of NO in the lower airways) = VNO - DNO(PL) - V A(PL)/(PB - 47). When PL reaches a steady state so that d/dt = 0 and V A is eliminated by rebreathing or breath holding, then PL = VNO/DNO. PL can be interpreted as NO production per unit of DNO. This equation predicts that diseases that diminish DNO but do not alter VNO will increase expired NO levels. These equations permit precise measurements of VNO that can be applied to determining factors controlling NO production by the lungs.

Rate of nitric oxide production by lower alveolar airways of human lungs.

This report describes methods for measuring nitric oxide production by the lungs' lower alveolar airways (VNO), defined as those alveoli and bronchioles well perfused by the pulmonary circulation. Breath holding or vigorous rebreathing for 15-20 s minimizes removal of NO from the lower airways and results in a constant partial pressure of NO in the lower airways (PL). Then the amount of NO diffusing into the perfusing blood will be the pulmonary diffusing capacity for NO (DNO) multiplied by PL and by mass balance equals VNO, or VNO = DNO(PL). To measure PL, 10 normal subjects breath held for 20 s followed by exhalation at a constant flow rate of 0.83 +/- 0.14 (SD) l/s or rebreathed at 59 +/- 15 l/min for 20 s while NO was continuously measured at the mouth. DNO was estimated to equal five times the single-breath carbon monoxide diffusing capacity. By using breath holding, PL equaled 2.9 +/- 0.8 mmHg x 10(-6) and VNO equaled 0.39 +/- 0.12 microl/min. During rebreathing PL equaled 2.3 +/- 0.6 mmHg x 10(-6) and VNO equaled 0.29 +/- 0.11 microl/min. Measurements of NO at the mouth during rapid, constant exhalation after breath holding for 20 s or during rebreathing provide reproducible methods for measuring VNO in humans.

Chemiluminescent measurements of nitric oxide pulmonary diffusing capacity and alveolar production in humans.

Measurements of nitric oxide (NO) pulmonary diffusing capacity (DL(NO)) multiplied by alveolar NO partial pressure (PA(NO)) provide values for alveolar NO production (VA(NO)). We evaluated applying a rapidly responding chemiluminescent NO analyzer to measure DL(NO) during a single, constant exhalation (Dex(NO)) or by rebreathing (Drb(NO)). With the use of an initial inspiration of 5-10 parts/million of NO with a correction for the measured NO back pressure, Dex(NO) in nine healthy subjects equaled 125 +/- 29 (SD) ml x min(-1) x mmHg(-1) and Drb(NO) equaled 122 +/- 26 ml x min(-1) x mmHg(-1). These values were 4.7 +/- 0.6 and 4.6 +/- 0.6 times greater, respectively, than the subject's single-breath carbon monoxide diffusing capacity (Dsb(CO)). Coefficients of variation were similar to previously reported breath-holding, single-breath measurements of Dsb(CO). PA(NO) measured in seven of the subjects equaled 1.8 +/- 0.7 mmHg x 10(-6) and resulted in VA(NO) of 0.21 +/- 0.06 microl/min using Dex(NO) and 0.20 +/- 0.6 microl/min with Drb(NO). Dex(NO) remained constant at end-expiratory oxygen tensions varied from 42 to 682 Torr. Decreases in lung volume resulted in falls of Dex(NO) and Drb(NO) similar to the reported effect of volume changes on Dsb(CO). These data show that rapidly responding chemiluminescent NO analyzers provide reproducible measurements of DL(NO) using single exhalations or rebreathing suitable for measuring VA(NO).

Acute respiratory exposure of human volunteers to octamethylcyclotetrasiloxane (D4): absence of immunological effects.

Humans are exposed to silicones in a number of commercial and consumer products. Some of these silicones, including octamethylcyclotetrasiloxane (D4), are volatile. Therefore, there is a potential for respiratory exposure. A pharmacokinetic analysis of respiratory exposure to D4 is presented in the accompanying paper (M. J. Utell et al., 1998, Toxicol. Sci. 44, 206-213). Possible immune effects of respiratory exposure to D4 are investigated in this paper. Normal volunteers were exposed to 10 ppm D4 or air for 1 h via a mouthpiece using a double-blind, crossover study design. Assays were chosen to screen for immunotoxicity or a systemic inflammatory response. Assessment of immunotoxicity included enumeration of peripheral lymphocyte subsets and functional assays using peripheral blood mononuclear cells. Because in humans there is no direct test for adjuvant effect of respiratory exposure, we analyzed proinflammatory cytokines and acute-phase reactants in peripheral blood, markers for a systemic inflammatory response, as surrogate markers for adjuvancy. These tests were repeated when the volunteers were reexposed to D4 approximately 3 months after this initial exposure. Blood was obtained prior to exposure, immediately postexposure, and 6 and 24 h postexposure. In these short-term, controlled human exposures, no immunotoxic or proinflammatory effects of respiratory exposure to D4 were found.

Ultrafine Particle Concentrations in a Hospital.

Ultrafine particles (UFP) may contribute to the morbidity and mortality associated with exposure to ambient particles, but few data are available on ultrafine particle numbers in indoor air, where susceptible subjects spend most of their time. We measured particle number, UFP size distribution, and total suspended particulate (JSP) mass in three locations: (I) a medical floor in a large tertiary care hospital, (2) outdoor air above a construction site outside the hospital, and (3) an environmental exposure chamber with purification of intake air. Mass and number concentrations were recorded continuously in each location over 70-110 h. Mean ± SD particle (p) numbers were 3.63 ± 1.l5 } 10(3) p/cm(3) in the hospital, 3.05 ± 6.65 } 10(4) p/cm(3) outside, and 5.86 ± 2.11 } 10(2) p/cm(3) in the environmental chamber. In the hospital, particle number and mass declined during the evening hours when the unit was less active, with the particle number as low as 1.15 } 10(3) p/cm(3). Particle numbers peaked (2.78 } 10(4) p/cm(3)) in the morning hours when activity on the unit was the most intense. "Spikes" in fine particle number were often not accompanied by increases in TSP mass. In the hospital, a distinct population of ultrafine particles (median diameter approximately 23 nm) was observed during the lunch hour, suggesting a change in particle source during this time. Outdoor fine particle numbers above the construction site were highly variable, reaching peaks of greater than 1.7 } 10(6) p/cm(3). These data suggest that, in the indoor environment, particle numbers and size distribution vary with intensity and type of local activity, and significant peaks in particle number are not detected with daily averages. Monitoring of particle mass may be an inaccurate measure of exposure to ultrafine particles indoors.

Aldehydes (nonanal and hexanal) in rat and human bronchoalveolar lavage fluid after ozone exposure.

We hypothesized that exposure of healthy humans to ozone at concentrations found in ambient air causes both ozonation and peroxidation of lipids in lung epithelial lining fluid. Smokers (12) and nonsmokers (15) were exposed once to air and twice to 0.22 ppm ozone for four hours with exercise in an environmental chamber; each exposure was separated by at least three weeks. Bronchoalveolar lavage (BAL) was performed immediately after one ozone exposure and 18 hours after the other ozone exposure. Lavage fluid was analyzed for two aldehyde products of ozonation and lipid peroxidation, nonanal and hexanal, as well as for total protein, albumin, and immunoglobulin M (IgM) as markers of changes in epithelial permeability. Ozone exposure resulted in a significant early increase in nonanal (p < 0.0001), with no statistically significant relationship between increases in nonanal and lung function changes, airway inflammation, or changes in epithelial permeability. Increases in hexanal levels were not statistically significant (p = 0.16). Both nonanal and hexanal levels returned to baseline by 18 hours after exposure. These studies confirm that exposure to ozone with exercise at concentrations relevant to urban outdoor air results in ozonation of lipids in the airway epithelial lining fluid of humans.

Oxidant and acid aerosol exposure in healthy subjects and subjects with asthma. Part II: Effects of sequential sulfuric acid and ozone exposures on the pulmonary function of healthy subjects and subjects with asthma.

These studies were undertaken to evaluate pulmonary responses of humans sequentially exposed to acidic aerosols and ozone at levels that could reasonably be encountered in actual environmental exposures. Subjects first were exposed to sulfuric acid (H2SO4) aerosol to sensitize the airways to ozone. The exposure protocols were designed to provide more quantitative information about the threshold levels of ozone that produce adverse biological effects and to provide exposure-response data on ozone. Two groups of 30 nonsmoking volunteers of both sexes, between the ages of 18 and 45 years, were recruited. The healthy study population comprised 16 men and 14 women with an average age of 28 years and no airway hyperreactivity. The second group comprised 10 men and 20 women comparable in age to the control group, but with allergic asthma and positive skin tests. The study examined an exposure-response relationship using three levels of ozone ranging from below the current standard to one and one-half times the ambient air quality standard (0.08, 0.12, and 0.18 ppm* [parts per million]) with preexposure 24 hours earlier to H2SO4 (100 micrograms/m3) or sodium chloride (NaCl) (control) aerosol in a 45-m3 environmental chamber. The study used an incomplete block design in which each subject was exposed to four of the six paired experimental atmospheres. Both the selection of paired exposures and the order in which they were presented were randomized. The exposure protocol required nine days: Day 1, training and baseline preexposure measurements; Day 2, the first of the three-hour particle (H2SO4 or NaCl) exposures; Day 3 (24 hours after Day 2), ozone exposure at 0.08, 0.12, or 0.18 ppm for three hours; Day 4 (two to four weeks later), exposure to the same ozone concentration as on Day 4. After at least another two weeks, Days 6, 7, 8, and 9 repeated Days 2, 3, 4, and 5 using a second ozone concentration. All three-hour exposures included several predetermined periods of exercise and pulmonary function measurements. To examine for delayed effects, pulmonary function tests were measured two and four hours after exposure on the ozone days. Data were analyzed over the time course of exposure and by exposure level of ozone at each time point to reveal dose-response relationships more closely. The main findings of the study are as follows. No significant symptomatic or physiologic effects of exposure to either aerosol or ozone on lung function were found for the healthy group.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of nitrogen dioxide toxicity in humans.

These studies were undertaken to evaluate short-term respiratory effects and identify markers of nitrogen dioxide toxicity during exposures designed to approximate realistic conditions. With the development of bronchoalveolar lavage as a clinical investigative technique, the evaluation focused on the assessment of effects induced at the alveolar level. The exposure protocols were designed to assess the duration of nitrogen dioxide-induced effects and determine exposure-response relationships. Groups of normal, nonsmoking volunteers of both sexes between the ages of 18 and 40 years, without airway hyperreactivity, constituted the study population. The exposure protocols required a total of three to five days for each subject, depending on the timing of bronchoalveolar lavage. Subjects were exposed to nitrogen dioxide or air for three hours in a double-blind, randomized fashion in a 45-m3 environmental chamber, with intermittent exercise sufficient to quadruple minute ventilation. Pulmonary function was measured during and after exposure, and airway reactivity to carbachol was assessed before and after exposure. Lavaged cells were examined for their capacity to inactivate influenza virus and secrete IL-1 in vitro. Cell-free lavage fluid was analyzed for total protein, albumin, alpha 2-macroglobulin, arylsulfatase, and alpha 1-protease inhibitor. The studies were undertaken in three phases, each of approximately one year's duration. In Phase 1, 15 subjects were exposed to a background concentration of 0.05 parts per million2 (ppm) nitrogen dioxide and to three 15-minute peaks of 2.0 ppm, and underwent bronchoalveolar lavage 3.5 hours after nitrogen dioxide exposure. During Phase 2, 8 subjects were exposed to continuous 0.60 ppm nitrogen dioxide and underwent bronchoalveolar lavage 18 hours later. Finally, in Phase 3, 15 subjects were exposed to continuous 1.5 ppm nitrogen dioxide and underwent bronchoalveolar lavage 3.5 hours after exposure. No significant symptomatic or pulmonary function changes could be detected in response to any of the nitrogen dioxide exposures. However, a small but significant increase in airway reactivity was observed in normal subjects after exposure to 1.5 ppm nitrogen dioxide. Following the highest dose of carbachol (10 mg/mL), the forced expiratory volume in one second decreased 7.5 +/- 1.1 percent after nitrogen dioxide exposure compared to 4.8 +/- 1.1 percent after exposure to air (p less than 0.05). No symptoms were induced in any of the groups by the carbachol exposures. Analyses of cells recovered by bronchoalveolar lavage during all three phases revealed no differences in total cell recovery, cell viability, or differential cell counts.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of ozone on normal and potentially sensitive human subjects. Part II: Airway inflammation and responsiveness to ozone in nonsmokers and smokers.

Exposure to ozone at levels near the National Ambient Air Quality Standard causes respiratory symptoms, changes in lung function, and airway inflammation. Although ozone-induced changes in lung function have been well characterized in healthy individuals, the relationship between airway inflammation and changes in pulmonary function have not been prospectively examined. The purpose of this study was to determine whether individuals who differ in, lung function responsiveness to ozone also differ in susceptibility to airway inflammation and injury. A secondary goal was to determine whether ozone exposure induces airway inflammation in smokers, a population known to have airway inflammation and an increased burden of toxic oxygen species. Healthy nonsmokers (n = 56) and smokers (n = 34) were exposed to 0.22 parts per million (ppm)* ozone for 4 hours, with intermittent exercise, for the purpose of selecting ozone "responders" (decrement in forced expiratory volume in 1 second [FEV1] > 15%) and "nonresponders" (decrement in FEV1 < 5%). Selected subjects then were exposed twice to ozone (0.22 ppm for 4 hours with exercise) and once to air (with the same exposure protocol), each pair of exposures separated by at least 3 weeks, in a randomized, double-blind fashion. Nasal lavage (NL) and bronchoalveolar lavage (BAL) were performed immediately after one ozone exposure and 18 hours after the other, and either immediately or 18 hours after the air exposure. Indicators of airway effects in lavage fluid included changes in inflammatory cells, proinflammatory cytokines, protein markers of epithelial injury and repair, and generation of toxic oxygen species. In the classification exposure, fewer smokers than nonsmokers were responsive to ozone (11.8% vs. 28.6%, respectively); an insufficient number of smoker-responders were identified to study as a separate group. In the BAL study, all groups developed a similar degree of airway inflammation, consisting of increases in interleukins 6 and 8 (maximal immediately after exposure), and increases in polymorphonuclear leukocytes (PMNs), lymphocytes, and mast cells (maximal 18 hours after exposure). The increase in PMNs was inversely correlated with age (p = 0.013), but gender, nonspecific airway responsiveness, and allergy history were not predictive of inflammation. Alveolar macrophage production of toxic oxygen species decreased after ozone exposure in nonsmokers; however, not in smokers. Findings from nasal lavage did not mirror lower airway inflammatory responses in these studies. We conclude that, in response to ozone exposure, smokers experienced smaller decrements in lung function and fewer symptoms than nonsmokers; however, the intensity of the airway inflammatory response was independent of smoking status or airway responsiveness to ozone. Furthermore, the burden of toxic oxygen species following ozone exposure was greater for smokers than for nonsmokers. Subjects were young, healthy, and able to sustain exercise; the results may not be representative of nonsmokers or smokers in general. Nevertheless, the findings indicate that measuring symptoms and spirometric changes is not sufficient to assess the potential risks associated with ozone exposure.

Effects of ozone on normal and potentially sensitive human subjects. Part III: Mediators of inflammation in bronchoalveolar lavage fluid from nonsmokers, smokers, and asthmatic subjects exposed to ozone: a collaborative study.

To provide bases of comparison between the studies described in Parts I and II of this Research Report, concentrations of interleukin 6 (IL-6)*, interleukin 8 (IL-8), and alpha 2-macroglobulin (a2M) were measured in airway lavage fluids obtained in the Balmes study (Part I) and compared with the same measurements in the Frampton study (Part II). For healthy subjects in the Balmes study, IL-6 and a2M, but not IL-8, increased in association with ozone exposure. Statistical analyses suggested that effects of ozone on IL-8 levels observed in the first exposure and bronchoscopy may have carried over to the second exposure and bronchoscopy, which may have obscured an effect of ozone on IL-8 after the second exposure. For asthmatic subjects in the Balmes study, IL-6 and IL-8 increased in both bronchial and alveolar lavage fluid, but not in proximal airway lavage fluid. The mean interval between exposures was longer for asthmatic subjects than for healthy subjects, and no carryover effects were seen. When the Balmes and Frampton data were analyzed together, subject groups in the two studies (nonsmokers, smokers, and subjects without and with asthma) did not differ significantly in the response of cytokines to ozone exposure. The finding of possible carryover effects in one group suggests that subtle effects of ozone exposure, or bronchoscopy including proximal airway lavage and biopsy, or both, may persist for three weeks in some subjects.

Acute health effects of ambient air pollution: the ultrafine particle hypothesis.

A strong and consistent association has been observed between adjusted mortality rates and ambient particle concentration. The strongest associations are seen for respiratory and cardiac deaths, particularly among the elderly. Particulate air pollution is also associated with asthma exacerbations, increased respiratory symptoms, decreased lung function, increased medication use, and increased hospital admissions. The U.S. Environmental Protection Agency (EPA) has recently promulgated a new national ambient air quality standard for fine particles, and yet the mechanisms for health effects at such low particle mass concentrations remain unclear. Hypotheses to identify the responsible particles have focused on particle acidity, particle content of transition metals, bioaerosols, and ultrafine particles. Because ultrafine particles are efficiently deposited in the respiratory tract and may be important in initiating airway inflammation, we have initiated clinical studies with ultrafine carbon particles in healthy subjects. These studies examine the role of ultrafines in: (1) the induction of airway inflammation; (2) expression of leukocyte and endothelial adhesion molecules in blood; (3) the alteration of blood coagulability; and (4) alteration in cardiac electrical activity. These events could lead to exacerbation of underlying cardiorespiratory disease. For example, airway inflammation may activate endothelium and circulating leukocytes, and induce a systemic acute phase response with transient hypercoagulability; this could explain the epidemiologic linkages between pollutant exposures and cardiovascular events. These approaches should be useful in identifying mechanisms for pollutant-induced respiratory and systemic effects, and in providing data for determining appropriate air quality standards.

Human performance during exposure to toluene.

PURPOSE: The purpose of this research was to examine the effects of inhalation of toluene on respiratory function and neuropsychological performance of humans. METHODS: We exposed six healthy adults to 100 ppm toluene or air (control) for 6 h, in a double-blind, randomized fashion, with exposures separated by at least 14 d and including 30 min of exercise at a level that quadrupled minute ventilation. Blood and exhaled air toluene levels were measured before, during, immediately, and 1 and 2 h post-exposure. Lung function was measured before and immediately after exposure. Three repetitions of two computerized neuropsychological tests were performed, including a brief standard neuropsychological battery (ANAM) and a 1-h complex performance test (SYNWORK). Statistical analysis of the psychological data was conducted as a repeated measures ANOVA. FINDINGS: Following exercise, the mean blood and exhaled air toluene levels averaged 1.5 micrograms and 28 ppm, respectively. Lung function was unchanged post-exposure. On the SYNWORK test, the Composite score obtained over time during toluene exposure was lower than that during room air (F = 29.20, p = 0.005), with the score from the final hour reduced by 10%. On standard neuropsychological tests, latency but not accuracy proved the sensitive measure for five of the seven subtests presented. CONCLUSIONS: Performance of complex tests and response time to simple brief tests can be disrupted by toluene inhalation at 100 ppm. Differences in performance between air and toluene conditions were greatest after exercise, indicating that physical activity may enhance the response to volatile organic solvents.