Final Report: Cardiovascular Toxicity of Concentrated Ambient Fine, Ultrafine and Coarse Particles in Controlled Human Exposures

EPA Grant Number: R832416C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R832416
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Harvard Particle Center
Center Director: Koutrakis, Petros
Title: Cardiovascular Toxicity of Concentrated Ambient Fine, Ultrafine and Coarse Particles in Controlled Human Exposures
Investigators: Silverman, Frances , Gold, Diane R. , Urch, Bruce
Institution: University of Toronto
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2011)
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air


Epidemiological studies have demonstrated strong and consistent associations between ambient PM exposure and increased short- and long-term morbidity and mortality from cardiopulmonary diseases. Current thinking suggests that the cardiopulmonary effects of PM inhalation are attributable to pulmonary/systemic inflammation, oxidative stress, endothelial activation/dysfunction, or autonomic dysfunction. The main objective of this project was to carry out controlled human exposures to fine, ultrafine and coarse concentrated ambient particles (CAPs) and to examine acute changes in cardiopulmonary outcomes to further our understanding of biological mechanisms of cardiopulmonary diseases attributed to PM. In the design phase of the project it was agreed to focus on coarse and fine CAPs and not to include ultrafine CAPs exposures. 

Controlled Human Exposures:  To facilitate these studies, a CAPs exposure facility for humans was established and characterized in Toronto, Ontario, Canada from 2006 to 2007. The facility was funded through a Canadian infrastructure grant to the Southern Ontario Centre for Atmospheric Aerosol Research, University of Toronto. Healthy non-smokers 19-60 years of age were recruited from the University of Toronto and Greater Toronto Area. Thirty-four subjects met inclusion criteria and were enrolled. Using a randomized block design, subjects had five 130 min exposures at rest with a minimum 2-week wash-out period between. Exposures included: one fine CAPs (PM2.5-0.1), two coarse CAPs (PM10-2.5), one HEPA filtered ambient air (HFAA) and one HEPA filtered medical air (HFMA). The target and maximum levels were as follows: fine CAPs (target 250 µg/m3; max 500 µg/m3); and coarse CAPs (target 200 µg/m3; max 400 µg/m3). A total of 141 exposures were completed.  Cardiovascular and respiratory health outcomes were measured before, during and after exposures.

Health Outcomes: Cardiovascular outcomes included: i) vascular dysfunction (brachial artery diameter and reactivity) by ultrasonography; ii) cardiac output by echocardiography; iii) blood pressure (BP) by automated arm cuff; iv) arterial pressure waveforms measures of large arterial compliance, central aortic BP and hemodynamics by SphygmoCor device; v) markers of systemic inflammation (CBCs, blood IL-6 and CRP); and vi) markers of oxidative stress in blood and urine. Respiratory outcomes included: i) pulmonary function by spirometry; ii) respiratory inflammation by induced sputum; and iii) respiratory and nasal symptomotology. In a subset of 11 subjects, DNA methylation was analyzed on candidate genes (TLR4, IL-12, IL-6, iNOS) and repeated elements (Alu, LINE-1) via bisulfite-pyrosequencing. Mortara Holter (ECG) monitors were worn by the subjects over 24 hours (started at ~8 am, prior to the pre-exposure ultrasound measures), as a measure of cardiac autonomic dysfunction (heart rate variability [HRV] analyses).

Exposure Characterization:  Exposures were characterized using continuous measures of particle mass (TEOM) and black carbon (PSAP) as well as integrated measurements (filter samples) of particle mass, sulfate, nitrate, ammonium, trace elements, organic and elemental carbon and biological material including airborne endotoxin and markers of fungi (β-(1,3)-D-glucan).  On-site daily measures included vehicle counts, meteorological data, TEOM PM2.5, gaseous air pollutants (NO, NO2, SO2, CO, O3), as well as pollen characterization (GRIPST-2000 pollen sampler) and fungal spores/pollen (Burkard sampler). Daily stationary central site monitoring data (gaseous and PM criteria pollutants) were obtained to statistically adjust for potential effects on baseline pre-exposure data.

Specific Hypotheses:

  • Acute human exposures to CAPs of coarse and fine size fractions result in cardiovascular responses including increased blood pressure, vascular narrowing of the brachial artery diameter (BAD), vascular/autonomic dysfunction (impaired flow-mediated dilatation [FMD]), inflammation (respiratory and systemic), and oxidative stress, compared to filtered air (control) exposures;
  • Respiratory inflammatory responses (induced sputum), pulmonary function (flow-volume curves) and nasal/respiratory symptom responses are greater for coarse CAPs than fine CAPs; and
  • Associations between CAPs and cardiovascular responses differ by particle size fraction and PM composition.

Summary/Accomplishments (Outputs/Outcomes):

Overview:  We recently completed the 141 exposures of 34 subjects. We are continuing testing of an additional 25 subjects through funding from Health Canada and support from Harvard University. These subjects will have three exposures including HFMA and two coarse CAPs. When completed in March 2012, in total we will have data for 100 coarse CAPs and 30 fine CAPs. In addition, we will include data from a previous study of 30 subjects (Brook, et al., 2009; Urch, 2010a) each receiving fine CAPs alone and fine CAPs+O3 at a lower CAPs level of 150 µg/m3. With this larger data set, we will have similar numbers of coarse (100 exposures) and fine (90 exposures) CAPs to compare cardiopulmonary responses. We also will examine dose-response associations between mass concentration and cardiopulmonary responses for each size fraction.
Statistical analyses were carried out at various stages throughout the exposures/data collection, and the results are summarized below. A number of significant collaborations were established as a result of the research project and our partnership with Harvard, leading to further studies, support of students/fellows and joint manuscripts. Below we present the findings to date:
Induced Sputum: Induced sputum is a non-invasive test used to collect cells from lung airways for examining acute lung inflammation. We previously have used this test to demonstrate adverse effects of controlled O3 exposures. Neutrophils, which are a gross marker of inflammation, are recruited from the blood into the lungs after an injury such as PM inhalation. We observed that sputum neutrophil counts were higher the morning after exposure for coarse CAPs compared to fine CAPs and both filtered air exposures. The results (Speck, et al., 2009) suggest a greater inflammatory response to coarse vs. fine CAPs, even though the mass concentration was lower for coarse CAPs (200 vs. 250 µg/m3). With the larger data set we will be able to examine differences in PM composition, which may explain the findings. Finally, we observed a stronger association between neutrophil levels and β-(1,3)-D-glucan concentration for coarse vs. fine CAPs suggesting that the biologic component of PM may be important, as described below.
Inflammatory Exposure Data: Currently, we are examining CBCs and blood IL-6/CRP as well as health effect associations with endotoxin and β-(1,3)-D-glucan for coarse and fine PM size fractions.
CAP and O3 Exposure and Blood IL-6: A retrospective analysis was carried out on blood IL-6, an acute phase protein and inflammatory marker, measured before and after controlled exposures to fine CAP and O3 (Urch, et al., 2010b). Blood IL-6 increased 3 hours after fine CAP exposure, but not as anticipated after CAP+O3 exposure. A possible explanation for the lack of IL-6 increase after CAP+O3 exposure was that some individuals switched to shallow breathing during CAP+O3 exposure, reducing their inhaled dose and resulting in a weakened IL-6 response. These findings highlight the importance of research examining co-pollutant mixtures vs. single pollutant exposures.
Ambient Air Pollution Effects on Blood IL-6 and Fibrinogen Levels: We were interested in assessing the effects of prior ambient exposures on baseline inflammatory measures from controlled exposure studies. This was a retrospective analysis using repeated-measures data in 45 non-smoking sub­jects (Thompson, et al., 2010). Hourly and daily moving averages were calculated for O3, NO2, SO2, and PM2.5. The association between pollutant exposure and baseline IL-6 and fibrinogen were assessed using up to 7-day moving averages. A significant association was observed for O3 as well as SO2 and IL-6 with similar trends for NO2 and PM2.5. These findings highlight the importance of considering the subject’s ambient pollution exposure before chamber exposures and suggest that the ambient pre-exposure data should be included in analyses of controlled exposure effects in order to control for potential baseline and carry-over effects during and after exposures.
Inflammation and Oxidative Stress: Health Canada supported measures of inflammation and oxidative stress in blood (IL-6, CRP, endothlin-1, vascular endothelial growth factor [VEGF]) and in urine (8-hydroxy-2-deoxyguanosine [8-OHdG], thiobarbituric acid reactive substances [TBARS] and VEGF). Results of the urine biomarkers showed elevated levels of VEGF at 3 hours (p=0.078) and 24 hours (p=0.040) following fine CAPs exposures. TBARS also increased at 24 hours after (p=0.075) exposures. Exposure to coarse CAP showed a similar but non-significant pattern for urine VEGF and TBARS. VEGF is a vascular permeability-inducing agent and an important stimulator of angiogenesis. TBARS is a biomarker of lipid peroxidation caused by oxidative stress. The results were presented in a meeting abstract and as an extended abstract published by the Air & Waste Management Association as conference proceedings (Liu, et al., 2011).
Blood Pressure and Brachial Artery Changes: We previously have shown significant increases in diastolic BP (DBP) during fine CAPs+O3 vs. filtered air exposures, as well as decreases in flow-mediated dilation (FMD) after exposure (Brook, et al., 2009). Our results showed increases in systolic BP (SBP) during CAPs exposures, with responses greater for fine CAPs as compared to coarse CAPs and smaller increases for both HEPA-filtered medical air (HFMA) and HEPA-filtered ambient air (HFAA) exposures. However, the increases in SBP did not reach statistical significance vs. either filtered air (FA) response. DBP also increased during CAPs, but no more so than during FA. Brachial artery diameter and FMD responses were not different from those of FA. As with all our outcome measures, we plan to examine dose-response associations with PM composition to determine whether this affects the level and variability of response. These results were presented at a meeting and reported in abstracts (Urch, et al., 2010c; Urch, et al., 2011).
Epigenetic Changes: As a pilot study, we examined the epigenetic effects of coarse and fine CAPS, and their bioaerosol components LPS and glucan in a subgroup of 11 study participants. In preliminary analyses, we found that fine particles induced Alu and IL-12 hypomethylation, and that coarse particles, endotoxin and glucan induced TLR-4 hypomethylation. Moreover, mediation analyses showed that DNA methylation changes might be on the pathway to elevated systemic white blood cells (WBCs) and elevated SBP and DBP.
Filtered Air Exposures: Early in the study, we observed a pattern of similar changes in outcome measures (e.g., BP, blood neutrophils) for HEPA filtered ambient air (HFAA) and CAPs exposures. The HFAA changes were larger than the changes observed in previous studies. The facemask delivery system was the same for the new facility and the CAP facility used in the prior studies, ruling this out as a factor. The two main differences in the facilities were: 1) the inlet for the older facility was on the second floor compared to ground-level for the new facility; and 2) the dilution air for the older facility included a charcoal pre-filter, while the new facility uses only a HEPA filter. We hypothesized that in our study ambient VOCs may have contributed to the HFAA responses. In order to explore this possibility, we added a second filtered air exposure that contained no particles or ambient gases—HEPA-filtered medical air (HFMA), using humidified gas cylinder medical air as the inlet air to the exposure chamber. Subsequent results revealed an apparent difference in responses between the HFAA and HFMA, with the HFMA showing the smallest changes in most response variables. Thus, the medical air may be a more appropriate control, although the filtered ambient air response does suggest some effect of the ambient gases and volatiles that can pass through the HEPA filter. We have measured VOC exposure levels and gaseous pollutants and will explore potential associations.
Biologic Components of PM: Innate immunity and inflammatory pathways have been shown to involve molecular structures called pathogen-associated molecular patterns, such as endotoxin and β-(1,3)-D-glucan that are important components of coarse PM. Few studies have considered that biologic components may contribute to respiratory and cardiovascular effects. Our study showed that ambient and exposure levels of endotoxin and β-(1,3)-D-glucan were highly correlated. Exposure levels of endotoxin and β-(1,3)-D-glucan were ~6-fold greater than respective ambient levels. Exposure levels of β-(1,3)-D-glucan were positively associated with lung and systemic neutrophil counts. Associations were stronger for individuals with atopy (positive skin tests) and for coarse CAP exposures. Associations with neutrophil counts were weaker but positive between exposure endotoxin levels and lung/systemic neutrophils.
Traffic Density: As a major source of air pollution, traffic-related emissions, commonly referred to as traffic-related air pollution (TRAP), are particularly important. Of note, diesel engine exhaust emissions produce nearly 100 times more PM compared to petroleum engines. In order to investigate the TRAP contribution to our exposures and to the cardiopulmonary responses, we set up a video camera to record traffic flow on the street adjacent to the CAP inlet. College Street is a four-lane street with two eastbound and two westbound lanes as well as streetcar tracks in the middle lanes. Although the streetcars are electric, they generate PM due to their brakes. Detailed data were obtained for traffic during exposures, including minute-by-minute eastbound and westbound counts, separately for cars, trucks (diesel) and streetcars. Total vehicle counts over the 130 minute-exposure period range from 2,300 to 2,700 vehicles of which 8-10% were diesel and ~2-3% were streetcars.
Heart Rate Vari­ability (HRV) Changes: A proposed mechanism of PM-induced cardiovascular effects is autonomic dysfunction, or an imbalance between the sympathetic and parasympathetic nervous systems. HRV has been used as a surrogate for autonomic tone in exposure stud­ies. In a retrospective analysis of controlled exposures to fine CAPs, O3, fine CAPs+O3 and filtered air, we assessed the impact of exposure on HRV (Fakhri, et al., 2009). We observed a dose-response association between CAPs mass concentration and HRV measures that differed for exposures with O3 versus without O3. The HRV data for the current coarse and fine CAPs exposures will be analyzed in 2012.
Cardiac Electrophysiology Changes: Cardiovascular mortality associated with air pollution exposure may be sudden or arrhythmic in nature. Changes in cardiac repolarization have been linked to an increased risk of arrhythmias. A retrospective analysis of 12-lead ECG recordings obtained from a previous CAP study was carried out (Sivagangabalan, et al., 2011). We examined the T-wave interval from the peak to the end (Tp-e), an estimate of repolarization time that relates to repolarization dispersion and arrhythmogenesis. Results showed a significant increase in the Tp-e interval from the start to the end of CAP+O3 exposure compared to filtered air. We also examined T-wave alternans (TWA), which are beat-to-beat fluctuation in the magnitude and shape of the T-wave. However, no significant exposure-induced changes were observed. A manuscript on the TWA results has been resubmitted to Environmental Health Perspectives and currently is under review (Kusha, et al., in review). A review paper on air pollution and arrhythmic risk was submitted to the Canadian Journal of Cardiology and is undergoing revision for resubmission (Watkins, et al., 2011). We plan to further study Tp-e and TWA in subjects with implanted defibrillators exposed to fine CAPs and O3, in single and co-pollutant mixtures.
Ultrafine CAPs: Health Canada also has funded studies using the ultrafine concentrator, initially for setup/characterization and then to carry out human exposures following the same protocol and outcome measures as the coarse and fine CAPs. Initial results have shown increases in blood pressure during ultrafine CAPs exposure as we previously reported for fine CAPs (Urch, 2010a; Fakhri, et al., 2009; Brook, et al., 2009). Ultrafine CAPs exposures studies on 20 subjects will be completed by March 2013.


Brook RD, Urch B, Dvonch JT, Bard RL, Speck M, Keeler G, Morishita M, Marsik FJ, Kamil AS, Kaciroti N, Harkema J, Corey P, Silverman F, Gold D, Wellenius G, Mittleman MA, Rajagopalan S, Brook JR. Insights into the mechanisms and mediators of the effects of air pollution exposure on blood pressure and vascular function in healthy humans.  Hypertension 2009;54:659-667.

Fakhri AA, Ilic LM, Wellenius GA, Urch B, Silverman F, Gold DR, Mittleman MA. Autonomic effects of controlled fine particulate exposure in young healthy adults: effect modification by ozone.  Environ Health Perspect 2009;117(8):1287-1292.

Sivagangabalan  G, Spears D, Masse S, Urch B, Brook RD, Silverman S, Gold DR, Lukic KZ, Speck M, Kusha M, Farid T, Poku K, Shi E, Floras J,  Nanthakumar K. The Effect of air pollution on spatial dispersion of myocardial repolarization in healthy human volunteers.  J Am Coll Cardiol 2011;57(2):198-206.

Thompson AMS, Zanobetti A, Silverman F, Schwartz J, Coull B, Urch B, Speck M, Brook JR, Manno M, Gold DR. Baseline repeated-measures from controlled human exposure studies: associations between ambient air pollution exposure and systemic inflammatory biomarkers IL-6 and fibrinogen.  Environ Health Perspect 2010;118(1):120-124.

Urch RB. Controlled Human Exposures to Concentrated Ambient Fine Particles and Ozone: Individual and Combined Effects on Cardiorespiratory Outcomes.  PhD Thesis.  University of Toronto, Department of Medicine.  Archived in University of Toronto T-Space, 2010a.

Urch B, Speck M, Corey P, Wasserstein D, Manno M, Lukic KZ, Brook JR, Liu L, Coull B, Schwartz J, Gold DR, Silverman F. Concentrated ambient fine particles and not ozone induce a systemic interleukin-6 response in humans.  Inhal Toxicol 2010b;22(3):210-218.

Kusha M, Masse S, Farid T, Sivagangabalan G, Urch B, Silverman F, Brook RD, Gold DR, Lukic KZ, Mangat I, Speck M, Nair K, Poku K, Mittleman M, Wellenius GA, Nanthakumar K.  The effect of air pollution on temporal dispersion of repolarization under basal conditions in volunteers with no pre-existing cardiovascular disease.  Re-submitted to Environmental Health Perspectives (September 2011).  Resubmitted October 14, 2011 (under review).

Watkins A, Spears D, Urch B, Kusha M, Quadros K, Danilewitz M, Farid T, Chauhan V, Nanthakumar K.  Air pollution and arrhythmic risk: The smog is yet to clear.  Canadian Journal of Cardiology (September 2011).  Currently being revised for resubmission.

Liu L, Poon R, Szyszkowicz M, Urch B, Speck M, Silverman F, Brook JR, Gold D.  2011. Controlled human exposure to fine and coarse ambient particles and effects on systemic biomarkers.  Extended abstract.  Air & Waste Management Association Conference Proceedings, June 21-24, 2011, Orlando, FL (conference proceedings, on-line).

Speck M, Urch RB, Fritscher L, Corey P, Brook JR, Gold DR, Silverman F. 2009. Exposure to coarse but not fine concentrated ambient particles increases airway leukocytes in humans.  Am J Respir Crit Care Med 2009;179:A3153 (poster presentation).

Urch B, Speck M, Corey P, Brook JR, Brook RD, Gold DR, Behbod B, Silverman F. 2010b.  Blood pressure changes during and after controlled exposures to Toronto concentrated ambient coarse and fine particles in healthy subjects.  Poster presented at the NIEHS-EPA Symposium on Air Pollution and Cardiovascular Disease. Seattle, WA, June 2010.

Urch BSpeck M, Scott J, Ewaze J, Brook JR, Gold DR, Behbod B, Silverman F.  2010c. Associations between inhaled endotoxin/β-(1,3)-D-glucan levels and airway/systemic neutrophils after controlled exposures to coarse and fine particles: effect of atopy on responses. Poster presented at the American Association for Aerosol Research's Air Pollution and Health: Bridging the Gap from Sources to Health Outcomes. San Diego, CA, March 2010.

B, Speck M, Corey P, Brook JR, Brook RD, Gold DR, Silverman F.  Changes in blood pressure and vascular responses with controlled exposures to fine and coarse concentrated ambient particles (CAPs).  Am J Respir Crit Care Med 2011;183:A5615.

Journal Articles on this Report : 3 Displayed | Download in RIS Format

Other subproject views: All 8 publications 5 publications in selected types All 5 journal articles
Other center views: All 200 publications 194 publications in selected types All 194 journal articles
Type Citation Sub Project Document Sources
Journal Article Sivagangabalan G, Spears D, Masse S, Urch B, Brook RD, Silverman F, Gold DR, Lukic KZ, Speck M, Kusha M, Farid T, Poku K, Shi E, Floras J, Nanthakumar K. The effect of air pollution on spatial dispersion of myocardial repolarization in healthy human volunteers. Journal of the American College of Cardiology 2011;57(2):198-206. R832416 (Final)
R832416C002 (2010)
R832416C002 (Final)
R834798 (2012)
R834798 (2014)
R834798 (Final)
R834798C002 (2014)
R834798C002 (Final)
R834798C004 (2012)
R834798C004 (2014)
R834798C004 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
  • Abstract: ScienceDirect-Abstract
  • Other: ScienceDirect-Full Text PDF
  • Journal Article Thompson AM, Zanobetti A, Silverman F, Schwartz J, Coull B, Urch B, Speck M, Brook JR, Manno M, Gold DR. Baseline repeated measures from controlled human exposure studies: associations between ambient air pollution exposure and the systemic inflammatory biomarkers IL-6 and fibrinogen. Environmental Health Perspectives 2010;118(1):120-124. R832416 (2009)
    R832416 (Final)
    R832416C002 (2009)
    R832416C002 (2010)
    R832416C002 (Final)
    CR830837 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: EHP-Full Text HTML
  • Other: EHP-Full Text PDF
  • Journal Article Urch B, Speck M, Corey P, Wasserstein D, Manno M, Lukic KZ, Brook JR, Liu L, Coull B, Schwartz J, Gold DR, Silverman F. Concentrated ambient fine particles and not ozone induce a systemic interleukin-6 response in humans. Inhalation Toxicology 2010;22(3):210-218. R832416 (2009)
    R832416 (Final)
    R832416C002 (2009)
    R832416C002 (2010)
    R832416C002 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Abstract: Taylor & Francis-Abstract
  • Supplemental Keywords:

    concentrated air particles, acute cardiovascular effects, coarse particles, fine particles, vascular dysfunction, inflammation, oxidative stress, RFA, Health, Scientific Discipline, Air, particulate matter, Environmental Chemistry, Health Risk Assessment, Risk Assessments, ambient air quality, atmospheric particulate matter, chemical characteristics, human health effects, cardiovascular vulnerability, automobile exhaust, airborne particulate matter, chemical composition, biological mechanisms, biological mechanism , traffic related particulate matter, human exposure, mobile sources, ambient particle health effects, autonomic dysfunction, human health risk

    Progress and Final Reports:

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

  • Main Center Abstract and Reports:

    R832416    Harvard Particle Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832416C001 Cardiovascular Responses in the Normative Aging Study: Exploring the Pathways of Particle Toxicity
    R832416C002 Cardiovascular Toxicity of Concentrated Ambient Fine, Ultrafine and Coarse Particles in Controlled Human Exposures
    R832416C003 Assessing Toxicity of Local and Transported Particles Using Animal Models Exposed to CAPs
    R832416C004 Cardiovascular Effects of Mobile Source Exposures: Effects of Particles and Gaseous Co-pollutants
    R832416C005 Toxicological Evaluation of Realistic Emission Source Aerosol (TERESA): Investigation of Vehicular Emissions