2002 Progress Report: Clinical Studies of Ultrafine Particle Exposure in Susceptible Human SubjectsEPA Grant Number: R827354C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R827354
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Rochester PM Center
Center Director: Oberdörster, Günter
Title: Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects
Investigators: Frampton, Mark W. , Beckett, William , Cox, Christopher , Morrow, P. E. , Utell, Mark J. , Zareba, Wojciech
Current Investigators: Frampton, Mark W. , Utell, Mark J.
Institution: University of Rochester
Current Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2002 through May 31, 2003
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air
The objectives of this research project are to: (1) develop a facility for human studies of ultrafine particles; and (2) utilize controlled human exposures to examine, in healthy and potentially susceptible subjects, the deposition of inhaled ultrafine particles (UFP) of carbon, and the role of UFP in inducing respiratory and cardiovascular health effects.
We have developed a facility for experimental exposure of humans to ultrafine particles, which permits the quantitative determination of the exposure levels, respiratory intakes, and depositions of the aerosol. A detailed description of the facility has been presented at the 3rd Colloquium on Particle Matter and Human Health in Durham, North Carolina, in June 1999.
For our initial studies, exposures were conducted at rest with a relatively low concentration of pure carbon UFP (~10 µg/m3, ~2 x 106 particles/cm3, count median diameter 26.4 nm, geometric standard deviation 2.3). Twelve healthy nonsmoking subjects (6 male and 6 female) inhaled either filtered air or UFP by mouthpiece for 2 hours at rest, with a 10 minute break after the first hour. Exposures were double blinded, randomized, and separated by at least 2 weeks. The overall deposition fraction (DF) was 0.66 ± 0.12 (mean ± SD) by number, and 0.58 ± 0.14 by mass. The number of DFs during the first and second hours of exposure was similar within subjects, but the average overall DF varied from 0.43 to 0.79 among subjects. The DF decreased with increasing particle size in the UFP range, from 0.76 ± 0.11 at 7.5 nm to 0.47 ± 0.17 at 133.4 nm. We found no significant differences in respiratory symptoms, blood pressure, pulse-oximetry, spirometry, exhaled NO, blood markers of coagulation and endothelial activation, leukocyte activation, or sputum cell differential counts. There was no convincing evidence for significant effects on heart rate variability, repolarization, or arrhythmias. We concluded that exposure to 10 µg/m3 pure carbon UFP at rest does not cause significant respiratory or cardiac effects in healthy nonsmokers.
Ultrafine Particle Exposure-Response With Exercise
We initiated studies to examine concentration-response effects and to incorporate exercise. Subjects received each of three exposures (air, 10, and 25 µg/m3 UFP), which were separated by at least 2 weeks. A total of seven visits were required for each subject. Monitoring and measurements were identical to those for the resting exposures. The planned 12 subjects (6 male and 6 female) completed all phases of the study. Analysis of variance was performed by Dr. Cox from the Biostatistical Core. Preliminary analyses indicated that exercise further increased the relatively high resting deposition of UFP (number deposition fraction at rest: 0.63 ± 0.04; exercise: 0.76 ± 0.06; means ± SD).
There were no particle-related effects on symptoms, spirometry, airway nitric oxide production, or sputum cell differential counts. There was a significant exposure-gender interaction for an effect on oxygen saturation (p = 0.02), with oxygen saturation decreasing slightly in females at 21 hours after exposure (only with the highest concentration of UFP [25 µg/m3]).
There was evidence for a concentration-related effect of UFP exposure on the percentage of blood monocytes, with the response differing by gender. In addition, monocyte expression of CD54 (intercellular adhesion molecule-1 [ICAM-1]) decreased after exposure in a concentration-response pattern (p = 0.001), with the greatest effect occurring at 0 and 3.5 hours after exposure, and the differences resolved at 21 hours after exposure. Overall, the findings from UPDOSE appeared to provide evidence for modest effects of UFP exposure, with exercise, on blood monocyte number and leukocyte expression of surface markers. In general, surface marker expression decreased in association with UFP exposure, consistent with retention of higher expressing cells within the capillary bed.
Electrocardiogram (ECG) recording analyses showed that the response of the parasympathetic system (measured by normalized units of high-frequency components) was blunted during recovery from exercise immediately after exposure to UFP in comparison to air exposure. This diminished vagal response was not observed 3.5 hour later. The analysis of QT interval duration and T-wave amplitude also showed a blunted response after UFP exposure in comparison to pure air exposure. The QT and heart rate-corrected (QTc) shortened during exercise more substantially during UFP exposure than during pure air exposure, and that the QT and QTc interval remained shortened for several hours after UFP exposure, but not after pure air exposure. These findings suggested that inhalation of UFP at both concentrations altered myocardial repolarization in healthy subjects.
UFP Exposure in Subjects with Asthma
Subjects with asthma may represent a group with increased susceptibility to the health effects of ultrafine particles because of the possibility of increased airways deposition of particles and because of underlying airway inflammation. We have completed a clinical exposure study (cofunded by the Health Effects Institute) of subjects with mild asthma. Sixteen subjects (8 male, 8 female) were exposed to air and to 10 µg/m3 carbon UFP for 2 hours with intermittent exercise. We measured effects on pulmonary function, symptoms, airway inflammation (exhaled NO and induced sputum), blood leukocyte activation, and cardiac electrophysiologic function. Results were analyzed in collaboration with the Biostatistical Core, as with the previous studies.
In the asthmatic subjects, we found that total respiratory fractional deposition by particle number was high at rest (0.77 ± 0.05) and increased during exercise (0.86 ± 0.04). Rest deposition was significantly increased compared with our previous study in healthy subjects at rest (0.63 ± 0.03). We conclude that ultrafine particle deposition is increased in mild asthmatic subjects compared with healthy subjects.
There were no significant changes in respiratory symptoms or pulmonary function in response to these exposures. Subjects did not report an increase in medication use following exposures. Blood studies revealed a particle-related decrease in blood eosinophils, basophils, and CD4+ T-lymphocytes. Blood monocytes showed a significant reduction in CD11b expression after exposure (p = 0.029). Expression of CD54 on polynuclear leukocyte (PMN) decreased in a time-related fashion, with the greatest difference from control at 45 hours after exposure and a significant time-exposure interaction (p = 0.031). Expression of CD62L on PMN showed a significant exposure-gender interaction, with an increase in expression of CD62L in males only. The most significant effect on leukocyte surface molecule expression in subjects with asthma appeared to be on eosinophils. As noted previously, there was a small exposure-related reduction in eosinophil percentage from the blood leukocyte differential count. In addition to this early reduction in eosinophil number, there was a delayed reduction in eosinophil expression of CD32 (time-exposure interaction, p = 0.015) and CD11b (main effect, p = 0.015).
In summary, data on leukocyte expression of adhesion molecules in subjects with asthma, similar to the findings for healthy subjects, suggest modest effects of UFP exposure with exercise. In general, the effect appears to be a reduction in expression or a blunting of the postexposure increase seen after air exposure. These findings are consistent with increased vascular retention of leukocyte subsets in the hours following UFP exposure, perhaps because of pulmonary vasoconstriction.
Effects of UFP Exposure on Endothelial Function
For the last six subjects studied in this project, we examined effects of UFP exposure on endothelial function by performing flow-mediated dilatation of the forearm circulation before exposure and 24 and 48 hours after exposure. After air exposure, flow-mediated dilation increased compared with preexposure baseline, an expected response to the exercise during the previous day’s exposure. However, this response was blunted following UFP exposure. At 48 hours, there appeared to be a rebound, with the change in total flow greater after UFP than after air.
Flow-mediated dilation is mediated by endothelial NO, so we measured the plasma NO products nitrite and nitrate before and after exposure in all 16 subjects in this study. We found a significant increase in blood nitrates 48 hours after exposure. This finding is consistent with a rebound increase in nitric oxide production, which likely mediates the rebound seen in flow-mediated dilation. These data support the hypothesis that exposure to very low concentrations of UFP alters systemic endothelial function. This is a novel finding, and we have now begun a study to determine if exposures in healthy subjects have similar effects on vascular function, and to determine the time course of the response.
In summary, our study indicates that asthmatics have increased airway deposition of ultrafine particles, and that exposure to even low mass concentrations of ultrafine particles alters circulating leukocyte subsets. These data are most consistent with an alteration in leukocyte retention in the pulmonary circulation. In addition, our preliminary results suggest there also are effects on systemic endothelial function. As a result of these findings, we have undertaken discussions with other Center Research Cores to develop additional strategies to investigate the vascular effects of exposure to UFP.
Progress Report, Year 4
In our progress report for Years 1-3 of the project, we described findings indicating that exposure to ultrafine carbon particles at low mass concentrations (10 and 25 µg/m3), in both healthy subjects and subjects with asthma, may cause subclinical effects on pulmonary ventilation/perfusion matching, circulating leukocytes, and cardiac repolarization, particularly in females. If these findings are confirmed, they will represent the most convincing support for the ultrafine hypothesis to date, and would suggest that females experience increased susceptibility to UFP exposure relative to males. We have designed a study to confirm and extend these observations in a larger group of healthy men and women, using a higher, yet still environmentally relevant, concentration of UFP. Our hypothesis is that inhalation of UFP alters pulmonary vascular function, circulating leukocyte activation, and cardiac repolarization. We speculate that these alterations reflect mechanisms involved in the observed increase in cardiovascular morbidity and mortality associated with particulate air pollution. Furthermore, we postulate that these effects are influenced in part by gender.
Objective #1: Perform a human clinical inhalation study of exposure to filtered air and to carbon UFP, 50 µg/m3 for 2 hours, with intermittent exercise.
Objective #2: Measure UFP effects on cardiac electrical activity, circulating leukocyte activation, and blood oxygenation.
We now have initiated studies at the higher concentration of 50 mg/m3, using an exposure protocol identical to our previous studies. Sixteen subjects will be studied, 8 men and 8 women. The study design incorporates features to avoid bias and reduce the impact to intersubject variability. Each subject undergoes both air and particle exposure in a randomized, double-blinded, cross-over design, so that each subject serves as his/her own control. Exposures are for 2 hours, with intermittent exercise, to filtered air and to 50 µg/m3 carbon UFP (~1 x 107 particles/cm3). Exposures are separated by at least 3 weeks to avoid the possibility of carry-over effects. Subject assessments and measurements are performed before and at intervals after exposure: immediately, 3.5, 24, and 48 hours after exposure. Digital 12-lead high-resolution ECG recordings are obtained at each measurement time point before and after exposure, and 24-hour ambulatory cardiac monitoring is initiated at the start of exposure. Systemic endothelial function is assessed using flow-mediated dilatation of the forearm, before and at intervals after exposure. Pulmonary vascular function is assessed using measurement of the diffusing capacity for carbon monoxide (DLCO). In addition, subjects undergo continuous digital pulse oximetry monitoring throughout the postexposure period, including overnight at home. Other assessments at each time point include respiratory symptoms, blood pressure, heart rate, and phlebotomy. Blood samples are immediately sent to the clinical laboratory for complete blood count, leukocyte differentials, and platelet count. Fresh whole blood is anticoagulated and prepared for immunofluorescence staining with monoclonal antibodies to a variety of cell surface markers and adhesion molecules. These are analyzed using multiparameter flow cytometry. Samples of blood plasma and serum are stored at -80° C for subsequent analysis of soluble cytokines and adhesion molecules, and for markers of endothelial activation.
At the time of completion of this report, all 16 subjects have been enrolled in the study, and 12 subjects have completed both exposure to air and ultrafine particles. We have performed a preliminary analysis of the first 12 subjects completing this exposure protocol. As in our previous studies, there were no symptoms associated with exposure, and subjects were unable to identify which exposure day involved inhaling particles. There were no significant changes in the forced expiratory volume in 1 second, or in the forced vital capacity in association with exposure. However, small reductions were seen in midexpiratory flow rates, suggesting that particle inhalation may have induced very mild construction of the small airways of the lung.
There was a highly statistically significant decline in DLCO 21 hours after exposure to UFP when compared with air exposure at the same time point (p = 0.0001). DLCO returned to normal, but remained somewhat reduced 45 hours after exposure. This finding, although preliminary, supports our hypothesis that inhalation of carbon UFP may affect the pulmonary circulation. The DLCO is influenced by pulmonary capillary blood volume, and pulmonary vasoconstriction with reductions in pulmonary capillary blood volume result in reductions in DLCO. This finding provides further support for the concept that low mass concentrations of ultrafine particles may have vascular effects, even in healthy subjects.
Summary of Human Clinical Studies of UFP Exposure
We have developed and validated an exposure system for human studies of inhalation of ultrafine particles. We have demonstrated that the total respiratory deposition of ultrafine particles is relatively high, consistent with prediction models. However, UFP deposition increased further with exercise and in the presence of mild asthma. Inhalation of ultrafine carbon particles at concentrations up to 25 µg/m3 caused no symptoms, changes in lung function, or evidence for airway inflammation. Our hypothesis that inhalation of ultrafine carbon particles would cause pulmonary inflammation and an acute phase response was not confirmed. However, we observed subtle changes in blood leukocyte subsets and adhesion molecule expression that suggest there may be effects on endothelial function. Preliminary findings from our study of exposure to 50 µg/m3 appear to confirm these observations. We also found evidence for effects on heart rate variability, and on cardiac repolarization in healthy subjects. The finding that these very low mass concentrations of particles have vascular effects would have important implications for future particulate matter regulatory strategies. Further studies are needed to confirm our observations, and to determine effects in people with underlying vascular dysfunction.
We expect to complete the current exposure protocol, healthy subjects breathing 50 µg/m3 UFP with exercise, in the next few weeks. The results will then be analyzed with the help of the Biostatistics Core. We then plan to initiate studies in healthy elderly subjects and aged-matched subjects with Type II diabetes mellitus. Patients with diabetes have been identified in recent epidemiological studies as being particularly susceptible to the effects of particulate matter exposure. We hypothesize that patients with diabetes, who are known to have underlying endothelial dysfunction, will show enhanced vascular responses to particle inhalation. Our findings will be interpreted in conjunction with ongoing and planned studies in the other research cores, examining effects of UFP on vascular endothelium.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
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Supplemental Keywords:urban air pollution, atmosphere, air, health, atmospheric sciences, biochemistry, environmental chemistry, health risk assessment, risk assessments, air toxics, tropospheric ozone, PM2.5, particulates, ultrafine particles, particulate matter, particle exposure, particle size, aerosol, ambient air, ambient air monitoring, ambient air quality, atmospheric, cardiopulmonary, cardiopulmonary responses, cardiovascular disease, cardiovascular vulnerability, coronary artery disease, fine particles, human exposure, human health, human health effects, environmental health effects, inhalation toxicology, lung, lung inflammation, morbidity, mortality, pathophysiological mechanisms, pulmonary, pulmonary disease, stratospheric ozone, sensitive populations, susceptible populations,, RFA, Health, Scientific Discipline, Air, particulate matter, air toxics, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, Atmospheric Sciences, Molecular Biology/Genetics, ambient air quality, cytokine production, particle size, particulates, sensitive populations, health effects, risk assessment, cardiopulmonary responses, fine particles, human health effects, morbidity, ambient air monitoring, lung, cardiovascular vulnerability, pulmonary disease, susceptible populations, animal model, carbon particles, environmental health effects, particle exposure, ambient monitoring, human exposure, particulate exposure, lung inflamation, pulmonary, coronary artery disease, inhalation toxicology, urban air pollution, mortality, urban environment, aerosol, cardiopulmonary, human health, aerosols, cardiovascular disease, ultrafine particles, pathophysiological mechanisms
Progress and Final Reports:Original Abstract
Main Center Abstract and Reports:R827354 Rochester PM Center
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827354C001 Characterization of the Chemical Composition of Atmospheric Ultrafine Particles
R827354C002 Inflammatory Responses and Cardiovascular Risk Factors in Susceptible Populations
R827354C003 Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects
R827354C004 Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
R827354C005 Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
R827354C006 Development of an Electrodynamic Quadrupole Aerosol Concentrator
R827354C007 Kinetics of Clearance and Relocation of Insoluble Ultrafine Iridium Particles From the Rat Lung Epithelium to Extrapulmonary Organs and Tissues (Pilot Project)
R827354C008 Ultrafine Oil Aerosol Generation for Inhalation Studies