Final Report: Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles

EPA Grant Number: R832415C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R832415
(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: Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
Investigators: Frampton, Mark W. , Zareba, Wojciech , Utell, Mark J. , Oakes, David , Phipps, Richard , Gelein, Robert
Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Human Health , Air

Objective:

The overall objective of our studies was to determine the pulmonary and cardiovascular effects of exposure to ultrafine and fine particulate matter (PM). The clinical studies in healthy humans and susceptible individuals in this research core focused on the effects of ambient ultrafine and fine particles on three major determinants of adverse cardiac events: 1) blood coagulation induced by effects on platelets and circulating microparticles; 2) cardiac output; and 3) cardiac rhythm and repolarization.
 
Our overall hypothesis was tested that inhalation of ambient PM causes small but measurable changes in coagulation and cardiovascular function that help explain the cardiovascular effects of PM exposure. We further hypothesize that the cardiovascular effects are determined by the ability of PM to generate reactive oxygen and nitrogen species, and are more pronounced in subjects with genetically determined reduced antioxidant defenses. Inhaled ultrafine particles increase the burden of reactive oxygen species to the endothelium. Mechanistic pathways include: Endothelial activation and vasoconstriction increase platelet adherence and release of thromboxane, activate and prolong the transit time of blood leukocytes, and deplete vascular nitric oxide (NO). Particles may also have direct effects on platelets and leukocytes. Vascular injury triggers release of procoagulant microparticles into the blood, and initiation of coagulation. In collaboration with the Vascular and Inflammation Facility Core, we measured the effects of inhaled ambient fine PM on platelet number, phenotype, and function, and quantitate intravascular microparticles derived from platelets and endothelial cells.

Summary/Accomplishments (Outputs/Outcomes):

Title: Ultrafine carbon particles: can they activate platelets in vitro?
Rationale: Exposure to ambient air particles is associated with increased risk for myocardial infarction. Our previous studies showed that ultrafine elemental carbon particles (UFCP) activated platelets in vitro. The hypothesis of this study was that prior exposure to UFCP would enhance the effects of platelet agonists. Methods: Blood was drawn atraumatically from 12 healthy adult non-smokers and diluted 1:10 in HEPES. Freshly generated UFCP (count median diameter 45 nm, geometric standard deviation 1.7) were added to blood at 0.2 and 20 µg/ml and incubated at 370C for 30 minutes with slow rotation. Platelet agonists (ADP, collagen, thrombin, TRAP), at concentrations below the level causing platelet aggregation, and immunostains were added and incubated for 20 minutes. Flow cytometry was used to determine platelet count, aggregates, activation, and platelet microparticles.
 
Results: UFCP at 20 µg/ml alone significantly increased platelet p-selectin expression (p = 0.04), but did not further enhance p-selectin expression in the presence of agonists. UFCP at 20 µg/ml also significantly reduced platelet count in the presence of all agonists except thrombin. There was no significant increase in platelet aggregates or microparticles. Conclusions: UFCP alone at 20 µg/ml activated platelet in vitro. Prior exposure to UFCP significantly reduced platelet count in response to platelet agonists in vitro. This may reflect the formation of platelet- leukocyte conjugates. Ten-fold lower UFC concentration did not induce these effects.
 
Title: Vascular effects of ultrafine particles in persons with type 2 diabetes.
Background: Diabetes confers an increased risk for cardiovascular effects of airborne particles. Objective: We hypothesized that inhalation of elemental carbon ultrafine particles (UFCP) would activate blood platelets and vascular endothelium in people with type 2 diabetes. Methods: In a randomized, double-blind, crossover trial, 19 subjects with type 2 diabetes inhaled filtered air or 50 μg/m3 elemental carbon UFP (count median diameter, 32 nm) by mouthpiece for 2 hr at rest. We repeatedly measured markers of vascular activation, coagulation, and systemic inflammation before and after exposure.
 
Results: Compared with air, particle exposure increased platelet expression of CD40 ligand (CD40L) and the number of platelet-leukocyte conjugates 3.5 hr after exposure. Soluble CD40L decreased with UFCP exposure. Plasma von Willebrand factor increased immediately after exposure. There were no effects of particles on plasma tissue factor, coagulation factors VII or IX, or D-dimer.
 
Conclusions: Inhalation of elemental carbon UFCP for 2-hr transiently activated platelets, and possibly the vascular endothelium, in people with type 2 diabetes.
 
Title: Effects of Outdoor Air Pollutants on Platelet Activation in People with Type 2 Diabetes.
Rationale: Exposure to air pollution is associated with increased morbidity and mortality from cardiovascular disease. We hypothesized that increases in exposure to ambient air pollution are associated with platelet activation and formation of circulating tissue factor-expressing microparticles. Methods: We studied 19 subjects with type 2 diabetes, without clinical evidence of cardiovascular disease, who had previously participated in a human clinical study of exposure to ultrafine particles (UFP). Blood was obtained for measurements of platelet activation following an overnight stay in the Clinical Research Center, prior to each of their two pre-exposure visits. Air pollution and meteorological data, including UFP counts, were analyzed for the 5 days prior to the subjects' arrival at the Clinical Research Center.
 
Results: Contrary to expectations, increases in UFP were associated with decreases in surface expression of platelet activation markers. The number of platelet-leukocyte conjugates decreased by -80 (95% confidence interval (CI) -123 to -37, p = 0.001) on the first lag day (20-44 h prior to the blood draw) and by -85 (CI -139 to -31, p = 0.005) on combined lag days 1 to 5, per interquartile range (IQR) increase in UFP particle number (2482). However, levels of soluble CD40L increased 104 (CI 3 to 205, p = 0.04) pg/ml per IQR increase in UFP on lag day 1, a finding consistent with prior platelet activation.
 
Conclusion: We speculate that, in people with diabetes, exposure to ambient UFP activates circulating platelets within hours of exposure, followed by an increase in soluble CD40L and a rebound reduction in circulating platelet surface markers.
 
Title: Cardiopulmonary effects of ambient ultrafine particles in healthy subjects.
Rationale: Exposure to ambient particulate matter is associated with increased cardiopulmonary mortality. In our previous studies in healthy subjects, inhalation of ultrafine elemental carbon particles during exercise reduced pulmonary capillary blood volume (Vc) and systemic vascular function, with no effect on lung function. We hypothesized that inhalation of concentrated ambient ultrafine particles (UFP) causes similar effects. Methods: 20 healthy non- smoking adults, aged 30-60 years, inhaled filtered air or concentrated ambient UFP at rest for 2 hours, in a randomized, double-blinded, cross-over study, with at least 3 weeks between exposures. The following were assessed on the day prior to, and 0.5, 3, 24, and 48 hours after exposure: vital signs, pulmonary function, Vc, and flow-mediated dilatation of the brachial artery (FMD).
 
Results: The mean particle number was 24.713.3 x 104 particles/cm3 (10.5 times higher than outside), with a mass concentration of 158.084.6 µg/m3. The particle count mean diameter was 94.147.90 nm, with a geometric standard deviation of 1.60. None of the subjects experienced exposure-related symptoms. The diastolic and mean blood pressure increased 0.5 hour after exposure to UFP vs air (mean blood pressure 97.62.2 vs 92.63.0 mmHg respectively, p=0.04), and returned to baseline 3 hours after exposure. 24 hours after UFP exposure, the FEV1 decreased 2.0% and the maximum mid-expiratory flow rate decreased 6.5%, significantly different from air exposure (p=0.01 and p=0.02, respectively). There were no significant changes in heart rate, Vc, or FMD.
 
Conclusions: Concentrated ambient UFP exposure transiently increased blood pressure and reduced pulmonary function 24 hours later in healthy subjects, with no effects on Vc or FMD. These findings suggest that exposure to ambient UFP at rest alters pulmonary and cardiovascular function.
 
Title: Ambient ultrafine particle exposure may alter circulating dendritic cell precursors in asthmatic subjects.
Background: Exposure to ambient ultrafine particulate matter (UFP) may worsen asthma, although the mechanisms involved are uncertain. We tested the hypothesis that UFP activates dendritic cells (DC), key sensors of the innate immune system. DC are heterogeneous and derive from circulating CD14+ myeloid precursors. Circulating myeloid DC (mDC) and plasmacytoid DC (pDC) can be detected using multi-color flow cytometry. Methods: We recruited 10 adult human asthmatic subjects (n=5 with the “null” polymorphism for glutathione-S-transferase M1) and exposed them for 2 hours at rest to concentrated ambient UFP (mean 2.96 x105 particles/cm3, count median diameter 90.9 nm) or filtered air using a double-blinded cross-over design. Phlebotomy, spirometry, and airway production of nitric oxide (NO) were performed at intervals up to 24 hours after exposure. The numbers and activation state of circulating CD14+ monocytes and DC subsets were analyzed using flow cytometry 3 hours after exposure. DC subsets were defined as lineage negative (lin-) cells not expressing CD3, CD16, CD19, CD32, or 7AAD, and separated into mDC1, mDC2 and pDC subsets with a panel of specific surface markers. CD14+ cells obtained 3 hours after exposure were differentiated into DC in vitro using standard techniques, and restimulated with CD40-ligand or lipopolysaccharide for 48 hours. We monitored cell activation using anti-CD40, CD80, CD86, and MHCII antibodies.
 
Results: Exposure to concentrated UFP was well-tolerated, and did not alter the FEV1 or airway NO kinetics. Expression of CD40 was significantly reduced on CD14+ monocytes 3 hours after UFP exposure compared to filtered air (p=0.03). The numbers of lin- cells decreased significantly 3 hours after UFP but not filtered air (p=0.02), but this was not reflected by changes in the frequencies or activation of mDC1, mDC2, or pDC subsets, although expression of CD40 tended to be lower on mDC1. Inducible expression of CD40 also tended to be lower on DC differentiated from CD14+ precursors obtained 3 hours after UFP exposure compared to filtered air. The number of blood eosinophils increased 24 hours after UFP exposure relative to air (p=0.02). None of these results were significantly affected by GSTM1 genotype.
 
Conclusion: Exposure to UFP results in alterations in circulating eosinophils, monocytes, myeloid DC and probably DC precursors in asthmatic subjects. Future studies are needed to determine whether this reflects recruitment of DC subsets out of the circulation or direct activation in vivo. Myeloid DC may be a previously unsuspected target of inhaled UFP in human subjects.
 
 
Title: Gene expression profile in circulating mononuclear cells after exposure to ultrafine carbon particles.
Background: Exposure to particulate matter (PM) is associated with systemic health effects, but the cellular and molecular mechanisms are unclear. Objective: We hypothesized that, if circulating mononuclear cells play an important role in mediating systemic effects of PM, they would show gene expression changes following exposure. Methods: Peripheral blood samples were collected before (0 h) and at 24 h from healthy subjects exposed to filtered air (FA) and ultrafine carbon particles (UFCPs, 50 μg/m3) for 2 h in a previous study (n = 3 each). RNA from mononuclear cell fraction (>85% lymphocytes) was extracted, amplified and hybridized to Affymetrix HU133 plus 2 microarrays. Selected genes were confirmed in five additional subjects from the same study.
 
Results: We identified 1713 genes (UFCP 24 h vs. FA 0 and 24 h, P < 0.05, false discovery rate of 0.01). The top 10 upregulated genes (fold) were CDKN1C (1.86), ZNF12 (1.83), SRGAP2 (1.82), FYB (1.79), LSM14B (1.79), CD93 (1.76), NCSTN (1.70), DUSP6 (1.69), TACC1 (1.68), and H2AFY (1.68). Upregulation of CDKN1C and SRGAP2 was confirmed by real-time-PCR. We entered 1020 genes with a ratio >1.1 or <−1.1 into the Ingenuity Pathway Analysis and identified pathways related to inflammation, tissue growth and host defense against environmental insults, such as, insulin growth factor 1 signaling, insulin receptor signaling and NF-E2-related factor-2-mediated oxidative stress response pathway.
 
Conclusions: Two-hour exposures to UFCP produced gene expression changes in circulating mononuclear cells. These gene changes provide biologically plausible links to PM-induced systemic health effects, especially those in the cardiovascular system and glucose metabolism.
 
Title: ECG Parameters and exposure to carbon ultrafine particles in young healthy subjects.
Background: The mechanisms underlying the association between air pollution and cardiovascular morbidity and mortality are unknown. This study aimed to determine whether controlled exposure to elemental carbon ultrafine particles (UFCP) affects electrocardiogram (ECG) parameters describing heart rate variability; repolarization duration, morphology, and variability; and changes in the ST segment. Methods: Two separate controlled studies (12 subjects each) were performed using a crossover design, in which each subject was exposed to filtered air and carbon UFCP for 2 hours. The first protocol involved 2 exposures to air and 10 μg/m3 ( 2×106 particles/cm3, count median diameter 25 nm, geometric standard deviation  1.6), at rest. The second protocol included 3 exposures to air, 10, and 25 μg/m3 UFCP( 7×106 particles/cm3), with repeated exercise. Each subject underwent a continuous digital 12-lead ECG Holter recording to analyze the above ECG parameters. Repeated measures analysis of variance (ANOVA) was used to compare tested parameters between exposures.
 
Results: The observed responses to UFCP exposure were small and generally not significant, although there were trends indicating an increase in parasympathetic tone, which is most likely also responsible for trends toward ST elevation, blunted QTc, shortening, and increased variability of T-wave complexity after exposure to UFCP. Recovery from exercise showed a blunted response of the parasympathetic system after exposure to UFCP in comparison to air exposure.
 
Conclusion, transient exposure to 10-25 μg/m3 ultrafine carbon particles does not cause marked changes in ECG-derived parameters in young healthy subjects. However, trends are observed indicating that some subjects might be susceptible to air pollution, with a response involving autonomic modulation of the heart and repolarization of the ventricular myocardium.
 
Title: Cardiovascular Effects of Ultrafine Particles in Genetically Susceptible Subjects (CUSP).
Background: Our human clinical studies of UFP inhalation have shown evidence for acute effects on vascular function. We hypothesize that the acute vascular effects of UFP exposure are a consequence of reduced NO bioavailability. We have developed a new approach to test this hypothesis, involving the measurement of nitric oxide metabolites in both arterial and venous blood. Specifically, we hypothesized that exposure to ultrafine particles will deliver a burden of reactive oxygen species to the pulmonary vascular endothelium, altering the delivery of NO, specifically as nitrite, to the systemic vasculature. We expect that UFP exposure will alter the artery-to-vein nitrite gradient which is normally present.
 
This study was jointly funded by this center and by the NIH, to test this hypothesis in healthy subjects. For the EPA-funded part of this study, we added a new, separate set of analyses that was not part of the NIH application, but is highly relevant to the whole issue of PM effects on vascular function and NO bioavailability. This involves a set of analyses to detect subtle effects on the membrane of circulating red blood cells. Our hypothesis was that inhaled UFP or their products cause subtle alterations in the RBC membrane that shift the dynamics of RBC transport and storage of nitrite and other NO metabolites.
 
Results: Preliminary findings (11 subjects, 4 of which were GSTM1 null) presented at the ATS International Conference, May 2011 showed: Exposure to concentrated UFP did not cause symptoms or changes in heart rate or blood pressure. Arterial punctures were well tolerated, and no subject withdrew from the study because of discomfort. Of a total of 66 arterial puncture attempts, 88% were successful. The arterial-venous gradient for nitrite increased 3 hours after UFP relative to air exposure (air, 13.96 vs. UFP, 190.39 nm, p=0.06 by paired t-test), driven by an increase in arterial nitrite concentrations after UFP but not air exposure. There were no significant effects on the reactive hyperemia index.
 
Conclusion: Exposure to UFP non-significantly increased nitrite arterial-venous gradients 3 hours after exposure, without effects on vascular function.
 
Final data analyses are in progress for this study, and a manuscript is in preparation.
 
Title: Ultrafine Particles and Cardiac Responses: Evaluation in a Cardiac Rehabilitation Center
Investigators: Mark J. Utell (PI); Philip Hopke, David Rich, and William Beckett (Co-PIs) Co-Investigators: David Oakes; Wojciech Zareba; Mark Frampton; Yungang Wang; John Bisognano; Annette Peters, With technical support from Jan Bausch, David Chalupa, Karen O'Shea, and Laurie Kopin
 
OBJECTIVES OF THE RESEARCH
 
Airborne particulate matter is an environmental problem associated with adverse effects on human health and welfare, particularly in urban areas where populations live in close proximity to multiple pollution sources. Exposure to fine particulate matter in ambient air has been associated with increased mortality and morbidity, in some studies related to cardiovascular disease. Previous studies have estimated that each 50 µg/m3 increase in ambient PM10 (particles with an aerodynamic diameter less than 10 µm) concentration is associated with a 3% to 8% increase in relative risk of death (Schwartz and Dockery, 1992).
 
Heart disease is a leading cause of mortality in the United States. Increasing numbers of heart disease patients are surviving coronary events, and thus living with coronary disease that places them potentially at high-risk for air pollution mediated cardiovascular events. The US EPA has estimated that approximately 60,000 excess deaths occur annually as a result of particulate air pollution, with the majority of these due to cardiovascular causes including myocardial infarction, sudden death, and congestive heart failure. Several reviews summarizing and evaluating health effects of particulate air pollution within epidemiological studies have been published (Committee of Environmental and Occupational Health, 1996; Pope et al., 2000). The most recent studies suggest that there is a higher risk for cardiovascular events for people who live near roads and traffic (e.g. within 150 meters of a major highway (Hoek et al., 2002; Grahame and Schlesinger, 2010). There is evidence from epidemiological studies on effects of particles on the cardiovascular system. Studies showed adverse effects of particulate air pollution on myocardial infarctions and ventricular fibrillation (Brook et al., 2004, 2010; Rich et al., 2005, 2006, 2008, 2010). Further, effects on autonomic function as seen by changes in heart rate and heart rate variability were observed (Brook et al., 2004, 2010). Other studies suggest an association between particulate air pollution and early blood markers of risk for sudden cardiac death such as plasma viscosity and C-reactive protein (CRP) (Peters et al., 1997, 2001). Overall the epidemiological findings suggest that individuals with cardiac disease are more susceptible to particulate air pollution than healthy individuals.
 
In the epidemiological studies, stronger effects were generally seen with smaller particle size fractions. If real, this may be due to different pollution sources and physiochemical properties of coarse, fine, and ultrafine, particles. In studies where both PM10 and PM2.5 (fine particles; mass of particles with an aerodynamic diameter less than 2.5 µm) were available to characterize the ambient concentrations of particle mass, there were indications that PM2.5 was more strongly associated with mortality than PM10 (Schwartz et al., 1996). Studies on subjects with respiratory diseases showed effects of ultrafine particle counts (UFP; number of particles <100nm in diameter) on peak expiratory flow, symptoms and medication use (Peters et al., 1997; Pekkanen et al., 1997). A study on daily mortality from Erfurt, Germany found comparable effects of fine and ultrafine particles in all size classes considered. However, effects of fine particles were more immediate while ultrafine particles showed rather delayed effects on mortality (Wichmann et al., 2000). A recent paper on changes in ST-segment depressions showed the strongest effects with both the particle number concentrations of particles in the size range of 0.1-1µm diameter and with UFP, both independent of fine particle mass (Pekkanen et al., 2002). Based on these findings not only the mass of particles but also the number concentrations, in particular in the fine and ultrafine range, seem to play a major role with respect to particle mediated acute cardiovascular health effects. Our study addresses the research gap in our understanding of what size fraction(s) of particulate air pollutions is/are responsible for the previously reported increases in cardiovascular morbidity and mortality associated with increased particulate matter air pollution concentration. Further, it tested whether ultrafine particles specifically contributed to adverse changes in these pathophysiologic biomarkers in this high-risk population of patients with recent coronary events.
 
Ultrafine particles are always present in ambient air, with background urban levels in the range of 40,000 to 50,000 particles/cm3, or estimated mass concentrations of 3-4 μg/m3 (Ogulei et al., 2007). Continuous monitoring by our group in Rochester, New York of UFP number and size between July 17, 2004, and July 25, 2005, revealed a maximum daily mean concentration of 25,500 particles/cm3. The mass concentrations were calculated by assuming spherical particles with a density of 1.5 g/cm3. The mean mass concentration of particles < 0.1 μm for this day was 2.45 μg/m3, the maximum mass concentration 6.25 μg/m3, and the average PM2.5 concentration 11.23 μg/m3 (Ogulei et al., 2007). In Germany, episodic increases in UFP have been documented up to 300,000 particles/cm3, or an estimated 50 μg/m3 of UFP as an hourly average (Tuch et al. 1997). Particle numbers inside a vehicle on a major highway reached 1x107 particles/cm3 or approximately 20 μg/m3 (Kittelson et al. 2001). In recent studies that exposed caged rats to UFP on a highway in New York State, the daily average number concentration in the control (filtered air) chamber was 0.01-12,000 particles/cm3. The incoming sampled air had a number concentration of 195,000-562,000 particles/cm3. No direct measurements of mass concentration were made, but it was estimated to be 37-106 μg/m3 (Elder et al., 2004). In a second truck on-road study performed by our group in New York State, the average concentrations of UFP were 1,600,000-4,300,000/cm3 in the plume (Elder et al., 2007).
 
Few studies have examined the role of UFP in cardiovascular disease (Timonen et al., 2006; Schneider et al., 2010; Ruckerl et al., 2007), and fewer still have evaluated the effects of the UFP fraction on highly susceptible groups recovering from an acute cardiovascular event. In Rochester NY, our research group has had extensive experience using controlled clinical studies to study health effects of UFP. In healthy subjects, we have found that inhalation of 10-50 µg/m3 for 2-hours results in: 1) high UFP deposition in the respiratory tract that further increases with exercise; 2) adverse changes in heart rate variability and cardiac repolarization; 3) adverse changes in circulating leukocytes (e.g. reduced blood monocytes); 4) effects consistent with adverse changes in blood vessel walls and how leukocytes move through the blood vessels; and 5) decrements in the pulmonary diffusing capacity (Daigle et al., 2003; Pietropaoli et al., 2004; Shah et al., 2008; Stewart et al., 2010). These clinical laboratory observations further support the link between air pollution exposure and adverse outcome for individuals with ischemic heart disease.
 
We suggest that ambient UFP are important with regard to cardiovascular health effects, for several reasons (Oberdörster et al., 2005):
 
1)      UFP are biologically more reactive on a volume basis than larger particles and elicit effects at lower concentrations.
2)      UFP at the same mass concentration in the air have a higher number concentration and surface area than larger particles. For example, on a mass basis, it takes roughly two million particles with diameters of 20 nanometers to equal the mass of a single 2.5 μm diameter particle (assuming spherical particles of the same material having unit density. Thus, without knowing the size of the particles present in an air sample, two samples having similar mass concentrations have significant differences in the number of particles present (i.e., it could reflect the presence of a few large particles or hundreds of thousands of smaller particles).
3)      Inhaled singlet UFP have a high deposition efficiency in the pulmonary region. For example: 20-nm particles have about 50% deposition efficiency.
4)      UFP have a high propensity to penetrate the epithelium and reach interstitial sites and the systemic circulation.
 
The objectives of the study were to assess the effects of ambient UFP exposure on biomarkers of pathophysiologic mechanisms thought to underlie previous reports of PM mediated cardiovascular morbidity, in a panel of patients with coronary artery disease. Few previous studies have examined this particularly high-risk group. Patients from an active cardiac rehabilitation program within the University of Rochester Medical Center were offered enrollment in our study as they entered the Cardiac Rehabilitation program. These were patients who have had a recent coronary event such as myocardial infarction or unstable angina leading to coronary stenting. As part of our study, patients participated in supervised, graded twice weekly exercise sessions for a total of 10 weeks with continuous cardiac monitoring. Concurrently, particle number and particle mass concentrations were measured continuously at a central measuring site in downtown Rochester. Previous studies have reported greater cardiovascular responses to accumulation mode particles (AMP; particles with diameters between 100 and 1000 nm) than UFP and/or particles with an aerodynamic diameter <2.5µm (PM2.5; Pekkanen et al., 2002). Other EPA Criteria Pollutants and outdoor temperature were also measured in downtown Rochester.
 
The project assessed the following specific hypotheses: (1) Elevated levels of ambient ultrafine and fine particles are associated with changes in autonomic nervous system function measured by heart rate variability parameters, and myocardial substrate and myocardial vulnerability measured by QRS duration, QT interval, ST segment changes and T wave abnormalities; (2) Elevated levels of ambient ultrafine and fine particles are associated with changes in biomarkers of enhanced cardiovascular risk, including systemic inflammation (C-reactive protein) and hypercoagulability (fibrinogen); and (3) Elevated levels of ambient ultrafine and fine particles are associated with slower and compromised rehabilitation.
 
 
METHODOLOGY
 
Study Design: Our findings revealed that the peak of ultrafine particles in the 10 - 50 nm size range is primarily related to traffic sources during the morning rush hour (7 to 9 AM). In contrast, the peak of ultrafine particles in the 50 to 100 nm size range were more correlated with the evening rush-hour (6 to 7 PM), while fine particles (100 to 500 nm) were marginally related to the traffic during both the morning and evening periods. In addition, we found that a second pattern of particle distribution follows the morning rush-hour peak, where large numbers of very small (approximately 10 nm) particles appeared just after noon. These particles grew rapidly through the early afternoon into particles in the 50 to 75 nm size range. This growth resulted from frequent nucleation events, which were most evident during the summer when photochemical processes were more intense. In addition to these nucleation events, where there were sufficient particles to observe subsequent growth, there were nucleation events without growth. There was a strong correlation between the occurrence of these nucleation events and the SO2 concentration measured at the same site. These events appeared to be the result of the plume from a local coal-fired power plant. The evening rush-hour traffic showed some additional peaks in the late afternoon (Wang et al., 2011). Importantly for the potential effects of cumulative exposures on cardiovascular events, we found large seasonal variation in UFP levels, as well as day-to-day variability due to meteorological conditions. Thus our panel study design using hourly measurements of ambient UFP levels is intended to detect variability in the occurrence of subtle cardiac effects in relation to the known hour-to-hour variability in UFP exposures.
 
The study design took advantage of the University of Rochester Medical Center's active cardiac rehabilitation program. The program enrolled approximately 10 new patients per week into its outpatient phases with 50 - 75 active participants at any given time. The program involves supervised, graded 2-3 exercise sessions per week, for a total of 10 weeks. For our study, each individual enrolled in the study could have participated in 20 individual (2 per week at maximum), study-related, exercise visits. Patients were in early post-myocardial infarction rehabilitation, post coronary bypass rehabilitation, or in rehabilitation for other conditions such as unstable angina, or cardiac valve replacement. Approximately 70% of participants completed the program. Most patients were enrolled within a few days of an acute coronary syndrome, which had been managed either medically or medically with invasive intervention (e.g., coronary artery bypass grafting or angioplasty with intracoronary stent placement). In general, this is a geriatric population with active cardiovascular disease who, by virtue of their enrollment in this intensive program, are a self-selected group of highly motivated patients. The study examined acute effects of UFP (within a few hours or days) on the electrocardiogram with parameters measured at rest and during exercise. Time domain heart rate variability parameters and frequency domain heart rate variability parameters were used to evaluate changes in the autonomic control of the heart while ST segment changes, QTc, QT variability, TpTe, and cardiac arrhythmias primarily reflected changes in myocardial vulnerability.
 
Study Population: Patients were offered enrollment in the study as they entered the Cardiac Rehabilitation program. A population of 76 subjects referred by their cardiologist to the University of Rochester Cardiac Rehabilitation Center (Center) after having a recent coronary event (MI or unstable angina) were included in the study. Individuals with cardiomyopathy in the absence of coronary disease, coronary bypass grafting within the last three months, type I diabetes, chronic atrial fibrillation, anemia, left bundle branch block, presence of a prosthetic heart valve or pacemaker, regular use of amiodarone, active smokers or living with an active smoker, or residing greater than 10 miles (Figure 1) from the particle monitoring site at the Center were excluded (see ECG Parameters below).
 
Study Protocol: Upon entering the rehabilitation program, subjects completed a standardized health database form, physical examination, and formal cardiac exercise testing; this information was collected prior to entering the study. This baseline information was entered into a standardized database. During each rehabilitation day, subjects completed a standardized questionnaire assessing changes in medications (including frequency of nitroglycerin use), and symptom scores. Before, during and after exercise, vital signs were monitored as per the rehabilitation protocol. Duration of exercise, achievement of workload goals, and workload parameters (e.g., peak heart rate and exercise time until peak heart rate) were also recorded. Concurrent ECG recording were used to monitor the occurrence of ischemic events as well as of signs of arrhythmia. In addition to the clinical outcomes, patients were asked to maintain a diary to record cardiac symptoms, compliance with prescribed medication, on demand medication, and information on individual behaviors.
 
Blood pressure measured at each visit, and an atraumatically drawn venous blood sample collected once weekly, were measured with the subject resting and seated, prior to exercise. Complete blood count, fibrinogen, and high sensitivity CRP analyses were measured in the Strong Memorial Hospital Clinical Laboratories. The study was approved by the Research Subjects Review Board of the University of Rochester, and informed written consent was obtained from all subjects.
 
Information on the subject's self-perceived exertion (RPE; on a scale of 6-"No Exertion" at all- to 20-"Maximum Exertion") (Borg, 1998) during the exercise component of each subject's visit to the Rehabilitation Center during the study was also collected.
 
ECG Parameters: Subjects attended at most 20 study-related exercise sessions during the 10- week program. At each visit, each subject was seated for 5 or more minutes (for blood pressure and ECG recording), did 2-5 minutes of "warm up" including gentle stretching, exercised for 30-45 minutes using one of various bicycles, treadmills, or rowing machines, and after a decreasing exercise "cool down" period, rested for 10 minutes (Figure 2). These 76 subjects and their 1489 subject-visits were used in all analyses.
 
At each visit, subjects underwent 3-lead (modified V2, V5, and AVF) Holter ECG recordings (Burdick Altair-Disc holter recorder; Cardiac Science, Bothell, WA), which were analyzed using the Vision Premier Burdick Holter System (Cardiac Science, Bothell, WA) and custom-made programs at the University of Rochester Medical Center. During both the pre- exercise supine period (Pre-exercise) and for the entire recording (Whole Session), we measured time domain HRV parameters including the mean NN interval time between successive normal to normal beats (MeanNN), the standard deviation of all normal to normal beat intervals (SDNN), and the square root of the mean of the sum of squared differences between adjacent NN intervals (rMSSD). Short-term, pre-exercise, supine recordings provided information regarding HRV parameters unaffected by sympathetic stimuli during exercise, whereas the whole session recording (including the exercise session) reflected the overall behavior of heart rate and autonomic responses to daily conditions including exercise. Across the whole session, we measured heart rate turbulence (HRT) and deceleration capacity. HRT, a measure of baroreflex sensitivity, is characterized by a brief acceleration of heart rate (turbulence onset) immediately following a spontaneous premature ventricular contraction, followed by a slower deceleration of heart rate (turbulence slope), before returning to normal sinus rhythm. In normal subjects, increased HRT is protective against adverse cardiac events. We focused on turbulence slope since this parameter was found to be more robust than turbulence onset in identifying subjects with increased risk of cardiac events (Cygankiewicz et al., 2008). Deceleration capacity (DC) is an additional measure of heart rate dynamics, reflecting the variability in heart rate during periods when the heart is slowing down, complementing information based on the other HRV and HRT parameters (Bauer et al., 2008). Repolarization duration was analyzed using the QT interval duration, which was measured manually in lead II, and corrected for heart rate using Bazett's formula (QTc). In addition, we also measured the Tpeak - Tend (TpTe), a measure of late repolarization duration, which might reflect heterogeneity in repolarization.
 
Particle and Weather Meteorological Measurements: We measured particle number distributions over the particle diameter range of 10 to 500 nm using a Wide-range Particle Spectrometer (Model 1000XP, MSP Corporation, Shoreview, MN)(Rodrigue et al., 2007; Liu et al., 2010) at the Cardiac Rehabilitation Center from June 2006 to November 2009 (Figure 1 and 3; Wang et al., 2010) every 3.5 minutes. This facility in Rochester is approximately 1500 m from an interstate highway beltway and on a diesel bus route. A technician from the University of Rochester Medical Center performed the required routine maintenance tasks including retrieving the data, monitoring the flow rates in the WPS, and refilling the butanol bottle in the CPC. The size distributions were then aggregated to provide particle number concentrations for 10-100 nm (ultrafine particles [UFP]) and 100-500 nm (accumulation mode particles [AMP]) diameters. These data were then utilized in the modeling described in the next section.
 
Particle size distributions were measured at the New York State Department of Environmental Conservation (NYS DEC) site in eastern Rochester using a Scanning Mobility Particle Sizer (SMPS), comprising a differential mobility analyzer (DMA) and a condensation particle counter (CPC). In the diameter range of 10 to 500 nm, ambient particles were classified by the DMA (TSI 3071) and counted with the CPC (TSI 3010) every 5 minutes. These measurements have been made continuously at the DEC site since May 2004 and were summarized by Wang et al. (2011). Continuous particle mass measurements were made with a 30°C Tapered Element Oscillating Microbalance (TEOM) with a Sample Equilibration System (SES) drier operated by NYS DEC. Carbon monoxide (CO), sulfur dioxide (SO2), and ozone (O3) were also measured by NYS DEC at this location and they provided these data. Hourly temperature, relative humidity, and barometric pressure were also measured at this NYS DEC site. The site-specific meteorology was supplemented by the National Weather Service measurements at the Rochester International Airport. Local meteorological data have been provided to us since February 2002. Table 2 provides correlation coefficients for each pair of daily pollutant concentrations and weather characteristics.
 
Statistical Methods: Data Analyses: A generalized linear model or analysis of variance is a common analytic methods used in panel studies with repeated measurements of outcomes and exposures for each subject (i.e. longitudinal data). These models allow an investigator to estimate the change in a continuous outcome per unit increase in a continuous exposure, test whether that estimated change is significantly greater than 0, and to control for additional covariates.
 
Within these models, the dependence of the observations from the same subject must be taken into account in order to estimate the model parameters and their variance correctly. In order to model an appropriate covariance structure, there are several different options. For example, an autoregressive structure is used if the correlation decreases with time. If the correlation is constant over time (i.e. outcome measurements within a subject are correlated but how far apart in time they are does not matter), then "compound symmetry" would be an appropriate structure. There are several other options that can be evaluated in the course of building a statistical model, but these are two common structures used in these types of studies. For continuous endpoints, diagnostic procedures have been developed that allow estimation of the correlation structure before fitting the final model. Proper modelling of the covariance structure increases the power of the final analysis (being able to find a significant association when one truly exists), without compromising the analysis of mean responses.
 
In our final models, repeated measures analysis of variance, with a compound symmetry covariance structure, were used to estimate the change in outcome (Pre-exercise resting period: MeanNN, SDNN, RMSSD, QTc, TpTe; Whole Session: MeanNN, SDNN, rMSSD, heart rate turbulence, deceleration capacity; Pre-exercise measurement: CRP, fibrinogen, white blood cell count, diastolic blood pressure, and systolic blood pressure) associated with each interquartile range increase in pollutant concentration (UFP, AMP, PM2.5). We evaluated several variables as potential confounders (i.e. variables correlated with exposure and predictive of the outcome that if not included in the model could result in biased/incorrect estimates of the change in an outcome associated with an exposure). We included visit number, days since study inception of study for each subject, calendar month, weekday, time of day, and mean temperature in the previous 24 hours in the model. Barometric pressure, relative humidity, sulfur dioxide, carbon monoxide, and ozone concentrations were not consistently associated with outcomes, and therefore were not confounders and not included in analyses. We log-transformed UFP and AMP concentrations to reduce skewness. Changes in each outcome associated with each of the three pollutant measures were estimated in separate analyses, using pollutants averaged over the 24 hours prior to the visit, as well as shorter (lag hours 0-5) and longer lag periods (lag hours 24-47, 48-71, 72-95, 96-119). The change in each outcome (and its 95% confidence interval) associated with each interquartile range (IQR) increase in pollutant concentration in the specified lag period are shown.
 
Next, to examine whether a change in an outcome (e.g. increased TpTe) associated with one pollutant (e.g. AMP lagged 24-47) was independent of a second pollutant (UFP or PM2.5) at the same lag time, the same model described above (same covariates and correlation structure as single pollutant model) including both pollutants at the lag time of interest (e.g. AMP and UFP counts lagged 24-47 hours) was built. The parameter estimates from the single and two pollutant models were then compared. For all analyses, SAS (version 9.1.3; SAS Institute Inc., Cary, NC) was used.
 
 
RESULTS
 
Particle Monitoring: The Cardiac Rehabilitation Program (Figure 1) is located approximately 2 miles from the DEC central monitoring site, and approximately 1500 yards from a portion of the city's outer loop interstate "beltway." Diesel-fueled buses provide public transportation in Rochester, with a route passing directly by the study site. The central monitoring site (Figure 4), in addition to monitoring PM2.5 and UFP, measured continuously CO, SO2 and ozone. For the cardiac rehabilitation study, descriptive statistics for each pollutant and weather conditions are shown in Table 1. AMP was moderately well correlated with both UFP (r=0.51) and PM2.5 (r = 0.62), but UFP and PM2.5 were not correlated (r = 0.11). UFP, AMP, and PM2.5 were less well correlated with temperature and relative humidity (r values ≤0.19). UFP, AMP, and PM2.5 were only minimally correlated with SO2, CO, and O3 (r values ≤0.35; Table 2). The interquartile ranges (IQR) for the 6 hour mean UFP count (2885 particles/cm3), AMP count (897 particles/cm3), and PM2.5 concentration (7.2 µg/m3) were used to scale all lag hour 0-5 effect estimates and confidence intervals. The IQRs for the 24 hour mean UFP count (2680 particles/cm3), AMP count (838 particles/cm3), and PM2.5 concentration (6.5 µg/m3) were used to scale all lag hour 0-23, 24-47, 48-71, 72-95, and 96-119 effect estimates and confidence intervals.
 
Clinical Study: The major task of the clinical study was the assessment of whether ambient UFP, AMP, and PM2.5 concentrations were associated with adverse alterations in sensitive electrophysiological measures during and prior to exercise, and in blood biomarkers prior to exercise.
 
Study Population. Of the 76 subjects, 83% (n=63) completed all 20 visits, with 8% (n=6) completing less than 10. Study subjects characteristics are shown in Table 3. Subjects were generally older, white, male, overweight, and were former smokers. Most had a history of MI, a prior stent, and a previous diagnosis of hypertension. Most were taking beta-blockers, ACE inhibitors, and statins (Table 3).
 
Outcomes. Mean (and standard deviation) HRV and repolarization and heart rate turbulence levels in both the pre-exercise resting period and across the whole session, as well as diastolic and systolic blood pressure, white blood cell count, CRP, and fibrinogen levels taken at the beginning of the first cardiac rehabilitation session are shown in Table 4.
 
Association between air pollution and outcomes: Pre-exercise holter outcomes. Changes in each outcome associated with IQR increases in UFP, AMP, and PM2.5 concentration in the previous five 24-hour lag periods, as well as in the previous 6 hours are presented in Table 5. There was no clear pattern of response to any pollutant for MeanNN or SDNN in the pre-exercise resting period, although we did observe a significant (p<0.05) 2.67 ms increase in SDNN associated with each IQR increase in PM2.5 72-95 hours before the clinic visit. However, IQR increases in AMP in both the previous 6 and 24 hours were associated with significant 3.65 and 4.33 ms decreases in rMSSD, respectively. Although not statistically significant, decreases in rMSSD were also associated with UFP and PM2.5 at the same lag times (Table 5). Although there was no pattern of QTc duration response to any pollutant, each IQR increase in AMP was associated with 0.78 ms and 1.05 ms increases in TpTe in the previous 24 hours and 24-47 hours, respectively, before the clinic session (Table 5; Figure 5).
 
Association between air pollution and outcomes. Whole Session Holter Outcomes. There was no clear pattern of response of MeanNN or deceleration capacity to any pollutant across the whole session (Table 6). However, each IQR increase in UFP 24-47 hours before the clinic session was associated with a 2.19 ms decrease in SDNN. rMSSD also decreased significantly with the same IQR increase in UFP within the previous 48 hours (lag hour 0-5, 0-23, and 24-47), with the largest change (-3.19 ms) observed with the UFP count in the 6 hours before the exercise session (Table 6; Figure 5). Each IQR increase in AMP 72-95 hours before the clinic session was also associated with a significant 0.67 ms/RR reduction in heart rate turbulence. There were similar, albeit non-significant, reductions in heart rate turbulence associated with increases in PM2.5 concentration during the same time period, but not UFP (Table 6; Figure 6).
 
Association between air pollution and outcomes. Pre-exercise blood pressure and blood outcomes. There were statistically significant 0.89 and 0.94 mmHg increases in SBP associated with IQR increases in UFP lagged 24-47 hours and PM2.5 lagged 0-5 hours, respectively, but little change in DBP (Table 7; Figure 7). However, each IQR increase in UFP lagged 96-119 hours was associated with significantly decreased DBP (Table 7). White blood cell counts did not respond to any lagged pollutant concentration, but IQR increases in UFP, AMP, and PM2.5 concentrations were associated with increases in CRP and fibrinogen at most lags, although not all were statistically significant (Table 7). Each IQR increase in PM2.5 was associated with a significant 0.069 mg/L increase in CRP lagged 72-95 hours, while each IQR increase in AMP lagged 24-47 hours was associated with a significant 0.120 g/L increase in fibrinogen. Similar significant increases in fibrinogen were associated with IQR increases in UFP and PM2.5 concentrations at the same lag time (Table 7; Figure 8).
 
Sensitivity analyses. Next we evaluated whether these effects were robust to inclusion of a second pollutant in the model by comparing pollutant specific effect estimates from the single and two pollutant models (Table 8). Our findings of increased TpTe associated with AMP lagged 24-47 hours, decreased HRT associated with increased AMP lagged 72-95 hours, and increased fibrinogen associated with increased AMP lagged 24-47 hours, all appeared independent of other pollutants at the same lag times, as there were only small changes in the AMP effect estimates when controlling for either UFP or PM2.5. In contrast, any change in these biomarkers associated with lagged UFP or PM2.5 was reduced or removed altogether when controlling for AMP. Similarly, the decreased rMSSD associated with UFP in the previous 5 hours and the increased SBP associated with increased PM2.5 lagged 0-5 hours both appeared independent of AMP, as the UFP/PM2.5 effect estimates were little changed from the single pollutant model to the two pollutant model including AMP (Table 8).
 
Air pollution and perceived exertion. The distribution of RPE across these n=1,489 subject visits are shown in Table 9. Using the same modeling approach as described for the main analysis but, without the other covariates in the model, we estimated the change in this self-perceived exertion score associated with ambient concentrations of UFP, AMP, and PM2.5 in the previous 6 hours (lag hours 0-5) and five previous 24 hours periods (lag hours 0-23, 24-47, 48-71, 72-95, 96-119). As shown in Table 10, there was no clear association between air pollution and this exertion score. The largest effect was a non-significant 0.05 decrease in RPE (95% CI = -0.12, 0.02) associated with each IQR increase in UFP lagged 48-71 hours.
 
 
DISCUSSION
 
In a panel of post-infarction patients participating in a cardiac rehabilitation exercise program, we found significant adverse changes in SDNN, rMSSD, late repolarization duration (TpTe), heart rate turbulence, systolic blood pressure, CRP, and fibrinogen associated with increased UFP, AMP, and PM2.5 concentrations in the previous few hours and days. Associations were more common and parameter estimates generally larger for lagged AMP and UFP concentrations than for PM2.5 concentrations. These effects were independent of PM2.5 concentrations, long-term time trends, season, week day, hour of the day, temperature, and duration of participation in the rehabilitation program. These acute, asymptomatic, and subclinical decreases in parasympathetic modulation, prolongation of late repolarization duration, and acute increases in blood pressure and coagulation/inflammation are factors that may predispose post-infarction patients to increased risk of arrhythmic events, cardiac ischemia, and sudden death (Rich et al. 2012).
 
We generally found larger and more frequent associations with ambient UFP and AMP concentrations than with ambient PM2.5. UFP are an important component of combustion-related or secondary aerosol-related air pollution, and have been linked to adverse vascular, inflammatory, and autonomic effects (USEPA 2009; Brook et al. 2010). Current mass-based regulatory monitoring only measures ambient PM2.5 and PM10, and not UFP. UFP may be particularly important with regard to CV effects because their high specific surface area (Oberdörster et al. 1995), enhanced oxidant capacity (Li et al. 2003), and propensity to enter cells (Stearns et al. 1994), which may facilitate delivery of reactive chemical species to the pulmonary and systemic vasculature. The subset of AMP measured in this study (100 to 500 nm) represents the size range covering the peak of the surface area distribution of the ambient aerosol. Thus, material condensed onto the particle surface can be effectively transported into the lungs. Even with the relatively low deposition probability of these particles, those that do deposit could provide a substantial dose to the lung. The effects we observed are consistent with a greater cardiovascular response to ambient UFP and AMP than with PM2.5 mass concentrations, and are consistent with human controlled exposure studies suggesting vascular and thrombotic effects of UFP inhalation (Stewart et al. 2010; Shah et al. 2008).
 
Across the whole exercise session, rMSSD and SDNN decreased with increased levels of UFP, reflecting a decrease in parasympathetic modulation of the heart. Associations were observed as short as 6 hours before the exercise session, suggesting an immediate HRV response to UFP. Although previous studies are not entirely consistent, most have reported decreased HRV associated with increased PM in the previous few hours and days, with the strongest associations with the high frequency component of HRV (Brook et al. 2010). Since rMSSD is highly correlated with this high frequency component of HRV (USEPA 2009), our findings are consistent with this.
 
We also assessed whether increased UFP, AMP, or PM2.5 concentrations were associated with adverse changes in HRT. We found a decreased HRT slope (i.e. impaired baroreflex sensitivity) associated with increased AMP concentration, which may lead to an increased risk of acute CV events including sudden cardiac death (Bauer et al. 2008; Cygankiewicz et al. 2008).
 
Regarding repolarization, QTc evaluated at baseline was not affected by particulate air pollution at any lag. However, we did observe increased TpTe levels (considered as a measure of repolarization heterogeneity in the myocardium) associated with increased AMP in the previous 24 hours. Repolarization responses to ambient particulate air pollutant concentrations have been reported previously in humans (Zareba et al. 2009; USEPA 2009; Brook et al. 2010). Taken together, these findings suggest that cardiac rhythm is affected by ambient particulate air pollution, and specifically AMP.
 
Associations between increased SBP and ambient particulate air pollution have not been consistently observed (Brook et al. 2010; USEPA 2009). However, our finding of increased SBP associated with UFP and PM2.5 is consistent with that reported by Zanobetti and Schwartz (2004) in a similar rehabilitation patient panel. However, the effect size (1.37 mmHg SBP associated with each 10.5 µg/m3 increase in PM2.5 concentration; scaled to the same IQR as in Zanobetti and Schwartz, 2004) and lag time of response we observed (previous 6 hours) were smaller and earlier than reported in that study (2.8 mmHg SBP associated with each 10.5 µg/m3 increase in mean PM2.5 concentration over the previous 5 days).
 
CRP and fibrinogen are "acute phase" proteins known to increase in the hours and days following an inflammatory stimulus. Fibrinogen, in the presence of thrombin, forms fibrin, a key component of the blood clot. Both CRP and fibrinogen are associated with increased cardiovascular risk, and have been associated with short-term increases in ambient particulate air pollution, especially in subjects with underlying cardiovascular risk factors (Brook et al. 2010). Some controlled human exposure studies, including those of UFP, have not shown particle effects on CRP or fibrinogen (Pietropaoli et al. 2006; Samet et al. 2009; USEPA 2009). In patients with clinical coronary artery disease, however, we found significant increases in fibrinogen lagged 24-47 hours for all three particulate air pollutant size fractions, providing evidence for particulate air pollution mediated increases in systemic inflammation and coagulation in these susceptible subjects.
 
The results presented in Tables 5, 6, and 7 illustrate differences in terms of the pollutants and lags found to be statistically significant. We believe that ultrafine particle (UFP; 10 to 100 nm), accumulation mode particle (AMP; 100-500nm), and fine particle concentrations (PM2.5) may represent a spectrum of particulate matter associated with different responses depending on their ability to penetrate and their ability to trigger responses. For example, AMP was predominantly associated with rMSSD at rest with a weaker association with UFP. However, when rMSSD was assessed during the entire session including repeated exercise (associated with more pronounced inhalation and exercise breathing), increased UFP concentrations were associated with the most significant response, with AMP and PM2.5 exerting less effect. These differences could be explained by different exposures at rest and during exercise. At the same time, variables like fibrinogen were associated with all three sizes of particles suggesting that inflammation may occur with any form or size of air pollution particulate matter.
 
Although our study had several strengths including a large sample size due to repeated rehabilitation sessions for each subject, and ambient monitoring of size fractioned particle counts at a location immediately adjacent to the rehabilitation site, there were a few limitations. We used data from stations measuring PM2.5 and particle counts (UFP and AMP) and assigned concentrations from those sites to study subjects regardless of how close they lived to the monitoring site resulting in some exposure error. However, since this exposure error is likely non-differential with respect to the level of these HRV and repolarization markers, this likely just resulted in a bias towards the null and underestimates of effect.
 
On the other hand, differences in the spatial variability of UFP, AMP, and PM2.5, and differences in the particle infiltration capacity (from outside to inside of a building) of these different sized particles, may result in differing magnitudes of exposure misclassification bias. This could potentially explain why we observed larger effects associated with AMP than UFP, for several outcomes. First, there is generally greater spatial variability in UFP than AMP (Wang et al., 2011). Second, since most people spend a majority of their time indoors (Klepeis et al. 2001), exposure to ambient particulate air pollution mostly occurs indoors. Therefore, particle size fractions with greater penetration indoors would also have less exposure misclassification bias in epidemiology studies (i.e. AMP; Hussein et al, 2005) than smaller particles that do not penetrate as efficiently (i.e. UFP). Therefore, our finding of more consistent and larger effects associated with AMP than with UFP may, in part, reflect differences in the magnitude of exposure misclassification bias for AMP and UFP, and not a greater physiologic response to AMP than UFP.
 
 
Major Findings:
 
The major objective of the panel study in cardiac rehabilitation patients was to determine if elevated levels of ambient ultrafine and fine particles were associated with adverse changes in autonomic nervous system function measured by heart rate variability parameters as well as in myocardial substrate and myocardial vulnerability measured by QRS duration, QT interval, ST segment changes and T wave abnormalities.
  • Significant adverse changes were observed in most outcomes associated with increased UFP, AMP, and PM2.5 within the previous 5 days using repeated measures analysis of variance.
  • The largest changes were decreased rMSSD (square root of the mean of the sum of the squared differences between adjacent NN intervals) associated with UFP in the previous 6 hours.
  • Increased TpTe (Time from peak to end of T-wave) was associated with AMP lagged 72-95 hours.
  • Decreased heart rate turbulence was associated with AMP lagged 72-95 hours.
  • Increased systolic blood pressure was associated with PM2.5 in the previous 6 hours.
  • Increased fibrinogen was associated with all three particulate air pollutant size fractions lagged 24-47 hours.
  • There was little evidence of an air pollution effect on subjects’ self-perceived exertion during the exercise sessions.
  • In these cardiac rehabilitation patients, particles were associated with asymptomatic and subclinical decreases in parasympathetic modulation, prolongation of late repolarization duration, increased blood pressure, and systemic inflammation, factors predisposing to increased risk of arrhythmic events and sudden death in post-infarction patients.
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
FIGURE LEGENDS
 
Figure 1. Locations of monitoring stations and clinic
 
Figure 2. Patients participating in exercise session at Rochester Cardiac Rehabilitation Center
 
Figure 3. Rochester Cardiac Rehabilitation Center
 
Figure 4. NY DEC - Rochester Monitoring station
 
Figure 5. Change in QT duration (ms) and TpTe (ms) measured at the beginning of the exercise session (and 95% confidence interval) associated with each interquartile range increase in ultrafine particle count (UFP; 10-100nm), accumulation mode particle count (AMP 100-500nm), and PM2.5 concentration, by lag hours.
 
Figure 6. Change in SDNN (ms), rMSSD (ms), and Heart Rate Turbulence (unitless) measured across the whole exercise session (and 95% confidence interval) associated with each interquartile range increase in ultrafine particle count (UFP; 10-100nm), accumulation mode particle count (AMP 100-500nm), and PM2.5 concentration, by lag hours.
 
Figure 7. Change in Diastolic and Systolic Blood Pressure (mmHb) measured at the beginning of the exercise session (and 95% confidence interval) associated with each interquartile range increase in ultrafine particle count (UFP; 10-100nm), accumulation mode particle count (AMP 100-500nm), and PM2.5 concentration, by lag hours.
 
Figure 8. Change in White Blood Cell Count (x109/L), C-reactive protein (mg/L, and fibrinogen (g/L) measured at the beginning of the exercise session (and 95% confidence interval) associated with each interquartile range increase in ultrafine particle count (UFP; 10-100nm), accumulation mode particle count (AMP 100-500nm), and PM2.5 concentration, by lag hours.
 
For all Figures:
H = 6 hour mean (lag hours 0-5)
0 = lag 0 (lag hours 0-23)
1 = lag 1 (lag hours 24-47)
2 = lag 2 (lag hours 48-71)
3 = lag 3 (lag hours 72-95)
4 = lag 4 (lag hours 96-119)
 
Figure 1.
 
 
 
Figure 3.
 
Figure 4
 
 
Figure 5
 
 
Figure 6
 
 
Figure 7
 
 
Figure 8.
 
 

Conclusions:

Conclusions of the clinical exposure studies:
 
  • UF carbon particles activate platelets in vitro at high concentrations
  • Inhalation of UF carbon particles activate platelets in type 2 diabetics
  • Ambient UFP exposure activate circulating platelets in type 2 diabetics
  • Concentrated ambient UFP transiently increase blood pressure and reduce pulmonary function 24 hrs. after exposure in healthy people
  • Concentrated ambient UFP alters circulating eosinophils, monocytes, myeloid dendritic cells and their precursors in asthmatic subjects
  • Exposure of healthy subjects to UF carbon particles induced gene expression changes in circulating mononuclear cells
  • Exposure of healthy young subjects to UF carbon particles did not induce significant changes in ECG-derived parameters; trends in some subjects indicating autonomic modulation of the heart and repolarization of ventricular myocardium.
  • Exposure to concentrated UFP non-significantly increased nitrite arterial-venous gradients without effects on vascular function.
 
 
 

References:

Bauer A, Malik M, Schmidt G, Berthel P, Bonnemeier H, Cygankiewicz I, et al: 2008. Heart rate turbulence: standards of measurement, physiological interpretation, and clinical use. JACC 52:1353-1365.
 
Borg G: 1998. Borg's Perceived Exertion and Pain Scales: Champaign, IL, Human Kinetics. Brook R, Rajagopalan S, Pope AC, Brook J, Bhatnagar A, Diez Roux A, Holguin F, Hong Y, Luepker R, Mittleman M: 2010. American Heart Association Council on Epidemiology and Prevention, Council on the Kidney in Cardiovascular Disease, and Council on Nutrition, Physical Activity and Metabolism: Particulate matter air pollution and cardiovascular disease: An update to the scientific statement from the American Heart Association. Circulation, 121(21): p. 2331 2378.
 
Brook RD, Franklin B, Cascio W, Hong Y, Howard G, Lipsett M, Luepker R, Mittleman M, Samet J, Smith Jr, SC: 2004. Air pollution and cardiovascular disease: a statement for healthcare professionals from the Expert Panel on Population and Prevention Science of the American Heart Association. Circulation, 109(21): p. 2655.
 
Committee of the Environmental and Occupational Health Assembly of the ATS: 1996. Health effects of outdoor air pollution. Am J Respir Crit Care Med 1996; 153(1): 3-50.
 
Cygankiewicz I, Zareba W, Vazquez R, Vallverdu M, Gonzalez-Juanatey JR, Valdes M: 2008. Heart rate turbulence predicts all-cause mortality and sudden death in congestive heart failure patients. Heart Rhythm 5:1095-1102.
 
Daigle CC, Chalupa DC, Gibb FR, Morrow PE, Oberdörster G, Utell M, et al: 2003. Ultrafine particle deposition in humans during rest and exercise. Inhal Toxicol. 15(6):539-552.
 
Elder A, Couderec JP, Gelein R, Eberly RS, Cox C, Xia X, Zareba W, Hopke P, Watts W, Kittelson D, Frampton M, Utell M, Oberdorster G: 2007. Effects of on-road highway aerosol exposures on autonomic responses in aged, spontaneously hypertensive rats. Inhalation Toxicol. 19: 1-12.
 
Elder A, Gelein R, Finkelstein J, Phipps R, Frampton M, Utell M, Topham D, D. Kittelson, Watts W, Hopke P, Jeong C-H, Kim E, Liu W, Zhao W, Zhou L, Vincent R, Kumarathasan P, Oberdörster G: 2004. On-Road Exposure to Highway Aerosols. 2. Exposures of Aged, Compromised Rats. Inhal Toxicol 16(Suppl.1): 41-53.
 
Grahame TJ, and Schlesinger RB: 2010. Cardiovascular health and particulate vehicular emissions: a critical evaluation of the evidence. Air Quality, Atmosphere and Health 3(1):3-27.
 
Hoek G, Brunekreef B, Goldbohm S, Fischer P, et al: 2002. Association between mortality and indicators of traffic-related air pollution in the Netherlands: a cohort study. Lancet 360: 1203-
1209.
 
Hopke PK, Utell MJ: 2005. Ambient air quality monitoring of ultrafine particles in Rochester, New York. NYSERDA Final Report 05-04.
 
Hussein T, Hameri K, Heikkinen MSA, et al. 2005. Indoor and outdoor particle size characterization at a family house in Espoo, Finland. Atmosphere and the Environment 39:3697-3709.
 
Kittelson DB, Watts WF, Johnson JP, Remerowski ML, Ische EE, Oberdörster G, Gelein RM, Elder A, Hopke PK: 2004. On-Road Exposure to Highway Aerosols. 1. Aerosol and Gas Measurements. Inhal Toxicol; 16 (Suppl. 1): 31-39.
 
Klepeis NE, Nelson WC, Ott WR, Robinson JP, Tsang AM, Switzer P, et al: 2001. The National Human Activity Pattern Survey (NHAPS): a resource for assessing exposure to environmental pollutants, Journal of Exposure Analysis and Environmental Epidemiology. 11: 231-252.
 
Li N, Sioutas C, Cho A, Schmitz D, Misra C, Sempf J, et al: 2003. Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environmental Health Perspectives 111:455-460.
 
Liu, B.Y.H., Romay, F.J., Dick, W.D., Woo, K.-S., Chiruta, M. 2010. “A Wide- Range Particle Spectrometer for Aerosol Measurement from 0.010 μm to 10 μm” Aerosol and Air Quality Research 10: 125-139.
 
Oberdorster G, Gelein RM, Ferin J, Weiss B: 1995. Association of particulate air pollution and acute mortality: involvement of ultrafine particles? Inhal Toxicol 7:111-124.
 
Oberdörster G, Oberdörster E, Oberdörster J: 2005. Nanotoxicology: An emerging discipline from studies of ultrafine particles. Environ Health Perspect. 113:823–839.
 
Ogulei D, Hopke PK, Chalupa DC, Utell MJ: 2007. Modeling sources contributions to ultrafine particle number concentrations measured in Rochester, NY. Aerosol Science and Technol, 41:179-201.
 
Pekkanen J, Peters A, Hoek G, Tiittanen P, et al: 2002. Particulate air pollution and risk of ST- segment depression during repeated submaximal exercise tests among subjects with coronary heart disease: the Exposure and Risk Assessment for Fine and Ultrafine Particles in Ambient Air (ULTRA) study. [see comments.]. Circulation 106(8):933-938.
 
Pekkanen J, Timonen KL, Ruuskanen J, Reponen A, Mirme A: 1997. Effects of ultrafine and fine particles in an urban air on peak expiratory flow among children with asthmatic symptoms. Environ Res. 74:24-33.
 
Peters A, Döring A, Wichmann HE, Koenig W: 1997. Increased plasma viscosity during air pollution episode: A link to mortality? Lancet, 349:1582-1587.
 
Peters A, Fröhlich M, Döring A, Immervoll T, Wichmann HE, et al: 2001. Particulate air pollution is associated with an acute phase response in men. Eur Heart J. 22(14): 1198-1204.
 
Peters A, Wichmann HE, Tuch T: 1997. Respiratory effects are associated with the number of ultra-fine particles. Am J Respir Crit Care Med. 155:1376-1383.
 
Pietropaoli AP, Frampton MW, Oberdörster G, Cox C, Huang L-S, Marder V: 2004. Blood markers of coagulation and inflammation in healthy human subjects exposed to carbon ultrafine particles. In: Effects of Air Contaminants on the Respiratory Tract - Interpretations from Molecular to Meta Analysis, (Heinrich U, Ed). Stuttgart, Germany:INIS Monographs, Fraunhofer IRB Verlag, 181-194.
 
Pope CA, III: 2000. Epidemiology of fine particulate air pollution and human health: biologic mechanisms and who's at risk? Environ Health Perspect. 108 Suppl 4:713-723.
 
Rich DQ, Freudenberger RS, Ohman-Strickland P, Cho Y, Kipen HM: 2008. Right heart pressure increases following acute increases in ambient particulate concentration. Environmental Health Perspectives, 116(9):1167-1171.
 
Rich DQ, Kim MH, Turner JR, Mittleman MA, Schwartz J, Catalano PJ, Dockery DW: 2006. Association of ventricular arrhythmias detected by implantable cardioverter defibrillator and ambient air pollutants in Saint Louis, Missouri. Occupational and Environmental Medicine 63(9):591-596.
 
Rich DQ, Kipen HM, Zhang J, Kamat L, Wilson AC, Kostis JB, et al: 2010. Triggering of transmural infarctions, but not nontransmural infarctions, by ambient fine particles. Environ Health Perspect. 118:1229-1234.
 
Rich DQ, Schwartz J, Mittleman MA, Luttmann-Gibson H, Link M, Catalano PJ, Speizer FS, Dockery DW: 2005. Association of ambient air pollution and ICD-detected ventricular arrhythmias in Boston, MA. American Journal of Epidemiology, 161:1123-1132.
 
Rich DQ, Zareba W, Beckett W, Hopke PK, Oakes D, Frampton MW, Bisognano J, Chalupa D, Bausch J, O’Shea K, Wang Y, Utell MJ: 2012. Are Ambient Ultrafine, Accumulation Mode, and Fine Particles Associated With Adverse Cardiac Responses in Patients Undergoing Cardiac Rehabilitation? Environ Health Perspect. 120:1162-1169.
 
Rodrigue, J., Ranjan, M., Hopke, P.K., Dhaniyala, S. 2007. Performance Comparison of Scanning Electrical Mobility Spectrometers. Aerosol Sci. Technol. 41: 360–368.
 
Ruckerl R, Phipps RP, Schneider A, Frampton M, Cyrys J, Oberdorster G: 2007. Ultrafine particles and platelet activation in patients with coronary heart disease – results from a prospective panel study. Part Fibre Toxicol 4:1.
 
Samet JM, Rappold A, Graff D, Cascio WE, Berntsen JH, Huang YC, et al: 2009. Concentrated ambient ultrafine particle exposure induces cardiac changes in young healthy volunteers. Am J Respir Crit Care Med 179(11): 1034-1042.
 
Schneider A, Hampel R, Ibald-Mulli A, Zareba W, Schmidt G, Schneider R: 2010. Changes in deceleration capacity of heart rate and heart rate variability induced by ambient air pollution in individuals with coronary artery disease. Part Fibre Toxicol 7:29.
 
Schwartz J, Dockery DW, Neas LM: 1996. Is daily mortality associated specifically with fine particles? J Air Waste Manag Assoc. 46(10): 927-939.
 
Schwartz J, Dockery DW: 1992. Increased Mortality in Philadelphia Associated with Daily Air Pollution Concentrations. Am Rev Respir Dis 145: 600-604.
 
Shah AP, Pietropaoli AP, Frasier LM, Speers DM, Chalupa DC, Delehanty JM: 2008. Effect of inhaled carbon ultrafine particles on reactive hyperemia in healthy human subjects. Environ Health Perspect 116: 375-380.
 
Stearns RC, Murthy GGK, Skornik W, Hatch V, Katler M, Godleski JJ: 1994. Detection of ultrafine copper oxide particles in the lungs of hamsters by electron spectroscopic imaging. ICEM 13-Paris. International Congress of Electron Microscopy. July 17-22:763-764.
 
Stewart JC, Chalupa DC, Devlin RB, Frasier LM, Huang L-S, Little EL, et al: 2010. Vascular effects of ultrafine particles in persons with type 2 diabetes. Environ Health Perspect 118: 1692-1698.
 
Timonen KL, Vanninen E, De Hartog J, Ibald-Mulli A, Brunekreef B, Gold DR: 2006. Effects of ultrafine and fine particulate and gaseous air pollution on cardiac autonomic control in subjects with coronary artery disease: the ULTRA study. J Expo Sci Environ Epidemiol 16:332-341.
 
Tuch TH, Brand P, Wichmann H-E, Heyder J: 1997. Variation of Particle Numebr and Mass Concentration in Various Size Ranges of Ambient Aerosols in Eastern Germany. Atmos Environ. 31: 4193-4197.
 
United States Environmental Protection Agency (US EPA): December 2009 Integrated Science Assessment for Particulate Matter. National Center for Environmental Assessment, Office of Research and Development. Research Triangle Park, NC.
 
Wang Y, Hopke PK, Chalupa DC, Utell MJ: 2010. Long term characterization of indoor and outdoor ultrafine particles at a commercial building. Environ. Sci. Technol. 44: 5775-5780.
 
Wang Y, Hopke PK, Chalupa DC, Utell MJ: 2011. Long-term study of urban ultrafine particles and other pollutants. Atmospheric Environ. in press.
 
Wichmann HE, Spix C, Tuch T, Woelke G, Peters A, Heinrich J et al: November 2000. Daily mortality and fine and ultrafine particles in Erfurt, Germany. Part I: Role of particle number and particle mass. Health Effects Institute Research Report 2000; 98.
 
Zanobetti A, Canner MJ, Stone PH, Schwartz J, Sher D, Eagan-Bengston E, et al: 2004.Ambient pollution and blood pressure in cardiac rehabilitation patients. Circulation110:2184-2189.
 
Zareba W, Couderc JP, Oberdorster G, Chalupa D, Cox C, Huang LS, et al: 2009. ECG parameters and exposure to carbon ultrafine particles in young healthy subjects. Inhal Toxicol 21:223-233.
 

Journal Articles:

No journal articles submitted with this report: View all 57 publications for this subproject

Supplemental Keywords:

RFA, Health, PHYSICAL ASPECTS, Scientific Discipline, Air, particulate matter, Health Risk Assessment, Risk Assessments, Physical Processes, atmospheric particulate matter, atmospheric particles, long term exposure, acute cardiovascular effects, airway disease, exposure, human exposure, ambient particle health effects, atmospheric aerosol particles, ultrafine particulate matter, PM, aersol particles, cardiovascular disease

Progress and Final Reports:

Original Abstract
  • 2006 Progress Report
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011 Progress Report

  • Main Center Abstract and Reports:

    R832415    Rochester PM Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832415C001 Characterization and Source Apportionment
    R832415C002 Epidemiological Studies on Extra Pulmonary Effects of Fresh and Aged Urban Aerosols from Different Sources
    R832415C003 Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
    R832415C004 Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
    R832415C005 Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition