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2008 Progress 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.
Institution: University of Toronto
EPA Project Officer: Stacey Katz/Gail Robarge,
Project Period: October 1, 2005 through September 30, 2010
Project Period Covered by this Report: October 1, 2007 through September 30,2008
RFA: Particulate Matter Research Centers (2004)
Research Category: Particulate Matter

Description:

Objective:

Our original design included controlled human exposures of 50 subjects each with 4 exposures including filtered air (FA), fine CAPs, coarse CAPs and ultrafine CAPs.  Based on recommendations from our Science Advisory Committee (SAC) (July 2006, meeting) a number of changes were made to the study design.  According to the revised design, each of the 25 subjects will receive four 2-hr exposures including: FA, fine CAPs (target 250 µg/m3; max 500 µg/m3), coarse CAPs (target 200 µg/m3; max 400 µg/m3), and a 2nd coarse CAPs exposure at the same levels.  Exposures are randomized and at least 2-weeks apart to allow for washout. We will examine and compare the acute cardiovascular and respiratory effects of coarse and fine CAPs in healthy adults, using our newly constructed controlled particle exposure facility, in Toronto, ON, Canada.  The revised design does not include ultrafine CAPs, at least for the first series of studies (25 subjects).  When completed, the 25 fine CAPs results @ 250 mg/m3 will be added to our EPA-STAR fine CAPs+O3 cardiovascular study (33 subjects, completed November 2007) with a mean CAPs mass of 140 µg/m3.  This will allow us to look at dose-response relationships using common vascular and inflammatory endpoints.  Cardiovascular outcomes will be measured pre-, post- and 24 hrs post-exposure, and will include measurements of: i) vascular dysfunction (brachial artery diameter and reactivity) measured by ultrasonography; ii) cardiac output by echocardiography; iii) blood pressure by automated arm cuff and; iv)  markers of systemic inflammation (CBCs and blood IL-6, CRP & endothelins). In addition, we will test for susceptibility genes of PM-induced oxidative stress using blood samples.  During exposure, we will continuously measure beat-to-beat arterial BP for 10-min periods every 30 min using a Finometer finger cuff (pulse pressure) that includes calculated determinations of cardiac output, stroke volume and systemic vascular resistance.  Simultaneously, we will measure end-tidal CO2 using nasal prongs, so as to directly compare the two outcome measures.  Mortara Holter (ECG) monitors (the same used by other PM Centers doing human studies) will be worn by the subjects from the start of the exposure day for 24-hrs, as a measure of cardiac autonomic dysfunction (HRV analyses).  Filter samples will be collected during exposures for both mass and chemical composition (inorganic ions, trace elements, OC/EC) and biological material including airborne endotoxin (Limulus Amebocyte Lysate test method) and markers of fungi (beta-glucans; Glucatell kit).  On-site daily measures will include meteorological data, TEOM PM2.5, 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) will also be obtained to statistically adjust for potential affects on baseline pre-exposure data.
 
The specific hypotheses to be addressed by this project are the following:

Approach:

We propose to expose 50 healthy adults to fine, ultrafine and coarse CAPs and particle-free (filtered) air. Each participant will receive 4 exposures in random order, separated by at least two weeks. Cardiovascular outcomes will be measured both pre-, post- and 24 hrs post-exposure and will include measures of: brachial artery diameter, flow- and nitroglycerin-mediated dilatation by ultrasonography; stroke volume (SV) and cardiac output (CO) by echocardiography; blood pressure (BP); and venous blood CBCs, IL-6, CRP and endothelins. Also, during exposures, continuous measurements of: beat-to-beat arterial BP by Finometer monitor, including calculated determinations of SV, CO and systemic vascular resistance; and HRV using 24 hr Holter monitoring will be performed. PM exposures will be characterized for particle mass, number, diameter, size and composition (inorganic ions, trace metals, organic and elemental carbon and black carbon). Gaseous co-pollutants (carbon monoxide, CO2, NO, NO2, SO2, O3), temperature and humidity will be monitored continuously during the exposure experiments. In addition, on the days before and after exposures, 24-hr measurements will be conducted for each participant using a multi-pollutant personal sampler. For each of the observed biological effects, repeated measures ANOVA models will be employed to assess differences among treatments. These models will contain a random effect for subject and a categorical variable for the four exposure treatments (fine, ultrafine, coarse, CAPs and particle-free air). To assess exposure-response relationships between biological outcomes and CAPs mass or individual components concentrations, single pollutant analyses will be conducted in which a separate linear mixed regression model will be used for each exposure parameter. These models will use biologic response as the dependent variable, subject as a random effect, and either particle mass, number, diameter or component as the exposure metric in the model. Hierarchical linear models will be developed to account for the multiple levels of data, including measurements taken at different time points within an exposure, for a subject.

Progress Summary:

Human Ethics Approval:  Since the last report, we submitted amendments to the protocol including: 1) addition of sputum induction – to examine acute lung inflammation; 2) addition of nasal scrapings and a nasal symptoms questionnaire – to examine nasal irritation/inflammation; and 3) addition of blood tests to measure DNA Methylation (based on the work of Andrea Baccarelli).  Amendments 1 & 2 were in response to the SAC comments that course CAPs may cause more respiratory changes and thus we should include tests of respiratory function.  The three amendments were approved by the St. Michael’s Hospital (SMH) Human Research Ethics Board (REB) in March, 2008 and the tests subsequently added in all subjects. Of note, the SMH REB is the primary Board, as SMH handles our grant money, but ethics approval has also been obtained from the University of Toronto, Health Canada and the US EPA.
 
New CAPs Human Exposure Facility:  Construction of our CAPs (coarse, fine, ultrafine) exposure facility, funded through a Canadian infrastructure grant (CFI) and the Harvard/EPA Center, began the end of July, 2006 and the coarse and fine CAPs were ready for initial testing and calibrations in October 2007.  The first human exposures began late in November 2007.  The ultrafine concentrator testing (by ProFlow) was delayed due to electrical installation delays, but is scheduled for final testing July 10-11, 2008.  The new CAPs exposure facility, including newly designed plexiglass subject enclosure with facemask delivery of the CAPs has been running well with no problems in attaining target CAPs levels.  As of July 2, 2008 we have recruited 15 subjects and carried out 33 human exposures: 17 coarse and 7 fine CAPs and 9 FA.
 
Preliminary Results: To date, four subjects have completed all four exposures (FA, fine CAP and 2 coarse CAPs).  Findings are summarized as follows:  Blood pressure (BP) was measured during the exposure at 30-min intervals, using an automated BP cuff.  Three BP measures were taken at each time point.  As we have observed in our previous CAP studies, more consistent findings were observed when the average of the 2nd and 3rd BP measures was used. Linear regression analysis was then used to determine the slope of the line fitted over the 5 times points (0, 0.5, 1, 1.5 & 2-hrs).  The mean ± SE change in diastolic BP over 2-hrs for 16 coarse CAPs exposures increased by 2.93±1.12 mmHg (9 subjects).  In our previous EPA-STAR fine CAPs±O3 study, the observed mean change for the 2-hr fine CAPs alone (no O3) exposure was similar, 2.86±0.86 mmHg (29 subjects).  This compared to a smaller increase in the EPA-STAR CAPs±O3 study (1.28±1.27 mmHg, n=27).  An even larger change in diastolic BP was observed when BP measured just before and immediately after coarse CAPs exposures were compared (a mean increase of 4.38±1.21 mmHg or 7.0%).  This is an interesting finding because we have previously reported that diastolic BP measured just before and after fine CAPs+O3 was not significantly different. Systolic BP for the 16 coarse CAPs exposures also increased, 3.46±1.27 mmHg compared to 2.29±1.12 mmHg (n=29) for the EPA-STAR fine CAPs.  However, to date, we only have data for six fine CAPs exposures and seven for FA exposures, too few to report given the variability of this measure.  DNA Methylation.  Blood samples of two subjects were analyzed as a pilot study.  Results showed decreased global methylation with exposures to coarse and fine CAPs and a suggestion of resolution with iNOS 24-hrs after CAPs exposures.  Pulmonary function.  Flow-volume curves were performed before, just after and 24-hrs after exposures.  As we have reported in previous controlled exposure studies, flow and volume parameters increased slightly from pre to post exposure with FA.  For example, in the seven subjects who have completed filtered air tests, FVC and FEV1 increased 1.14±0.68 and 3.03±1.39%, respectively.  Changes were small for both coarse and fine CAPs exposures.  For example, after coarse CAPs (n=14), FVC decreased 0.07±0.71% and FEV1 increased 1.53±0.79%. After fine CAPs (n=6), FVC increased 0.27±0.57% and FEV1 increased 2.70±1.49%.  Traffic Density.  In May 2008, we set up a video camera, to record traffic flow adjacent to the CAP inlet on College Street.  Detailed data was obtained for traffic during the exposures, including minute-by-minute eastbound and westbound counts, separately for cars and trucks.  Data to date includes eight exposures, with counts ranging from 5-42 vehicles per min, of which 5-9% were trucks (diesel). Total vehicle counts over the exposure period, now extended to 2 hours and 10 minutes, ranged from 2,341 to 2,550 vehicles.  We do expect to see more variation in traffic counts as we obtain more data over the entire year.  The traffic counts will be used as predictors of health outcomes in regression analyses and to examine potential relationships between traffic flow and changes in PM10 mass concentrations at the concentrator inlet.  
 
Health outcomes with no results to report at this time:  Flow-mediated dilation (FMD).  In our recently completed EPA-STAR study, we observed a significant decrease in FMD 24-hrs following fine CAPs exposure, compared to an increase with FA.  We anticipate that we will see a larger decrease with fine CAP exposures at the higher target mass concentration level of 250 µg/m3 (mean level of 140 µg/m3 for the EPA-STAR fine CAPs study).  Finometer.  In discussions with Dr. Rob Brook, and after some trial testing, we decided to use 10-min continuous recordings every 30-min for the Finometer, as it was very uncomfortable to keep the finger cuff on longer.  We have thus extended the exposure length by 10 min, to accommodate the Finometer reading at the end of the exposure.  Finometer data has recently been sent to an expert in this area (Gianfranco Parati) with customized software analyses, and we are awaiting analyses.  Echocardiography.  We have added echocardiography to this study as a non-invasive determination of cardiac output (CO) – a primary determinant of systemic BP.  Briefly, cardiac images of the aortic annulus (AA) are obtained for determination of the (diameter) cross sectional area.  Next, the aortic velocity waveform at the AA is obtained by Doppler, and the velocity time integral measured (Terason software).  The product of the two measures, stroke volume, is multiplied by heart rate to obtain CO.  Nasal scrapings.  Nasal scrapings and RNA gene expression (micro-array) will shortly be included as an outcome measure for all newly recruited subjects.  This test is a component of another study (AllerGen NCE, Dr. Jeremy Scott) in subjects with allergic rhinitis, recruited from the PM Center study subjects.   CBCs, cytokines and endothelins/NO.  Data are batch analyzed for cytokines and endothelins/NO.  We have previously observed increased white cell counts and IL-6 following exposure to fine CAPs.  Heart Rate Variability.  Subjects wear a Mortara holter during the entire exposure day and keep it on overnight, returning the next day, thus giving a 24-hr recording.  ECG digital data are stored on flash cards and sent to Harvard every 2-3 exposures for data recovery and analyses.  Exposure characterization.  We have been working closely with Jeff Brook at Environment Canada (EC) and Harvard to coordinate our filter sample collections. Teflon filters (47 mm) have been sent to EC for pre- and post-exposure conditioning and weighing. Mean ± SD mass concentrations for 12 coarse CAPs exposures were 199.3±26.5 µg/m3 (target 200 µg/m3) and four fine CAPs 276.5±16.8 µg/m3 (target 250 µg/m3). After gravimetric measurements are completed, filters are batch analyzed at EC for inorganic ions by IC. Quartz filters (25 mm) are pre-fired to remove organics and after exposure sent back for OC/EC analyses by TOT using a 1.45 cm2 punch. A punch will also be taken for selected organics, including a motor vehicle tracer (engine lubricant), by GC-MS.  Teflon filters (37 mm) are collected for trace element analyses and will be sent to the DRI lab for XRF as a batch analyses (with Harvard samples).  Biologic components are collected on 25 mm polycarbonate filters for endotoxin and β-glucans, both exposure and outdoor samples. Continuous data are also collected for PM mass (TEOM, DustTrak), black carbon (PSAP), temperature, RH% and criteria gases.
 
Collaborations/meetings:  There have been regular scientific communications/meetings between the study investigators (GOEHU, Michigan & Harvard), mostly by teleconference but also in-person, which have proven to be both fruitful & beneficial to the development/progress of this project, thus will continue throughout to its completion.  In April, 2008, Dr. Petros Koutrakis came to Toronto to give a presentation and to see our new CAP facility and discuss our project.  During the American Thoracic Society (ATS) Conference in Toronto May 18-21, 2008, we had an open house at our new CAPs facility and investigators involved with PM research were present to view our facility and discuss our recent findings.  At the ATS conference we set up several meetings with our Harvard collaborators (Drs. Joel Schwartz, Brent Coull and, Diane Gold) to discuss our project progress/results, new collaborations, statistical issues and new manuscripts. 
 
Health Canada: Oxidative Stress Collaboration: We have established collaboration with Health Canada on oxidative stress measures in response to coarse CAPs exposure, as part of our EPA PM Center project.  Blood and urine samples are provided to Health Canada, initially for 9 subjects (study completed) with ongoing plans to add 16 more subjects in 2008-2010.  Specifically, measures in urine will include: isoprostane-8 & thiobarbituric acid reactive substances (TBARS) for oxidative stress, D-Glucaric acid for liver response & enzyme stimulation, and VEGF an angiogenesis factor. Measures in blood will include: TNF-α for inflammation, oxidative potential, Isoprostane-8, TBARS and conjugated diene for oxidative stress.
 
AllerGen NCE Collaborations: We have collected filter samples during all FA (control) coarse and fine CAP exposures for determination of biological material including airborne endotoxin and markers of fungi.  Samples will be batch analyzed at a later date.  In addition, we have recently started to collect filter samples from pre-exposure calibration runs of coarse and fine CAPs for the specific objective of adapting DGGE and DNA macro-array technologies for the characterization of airborne and dust-borne fungal contaminants and to investigate the value of these data in the context of studying indoor/outdoor exposures and health outcome measures.  The latter information will inform the Canadian Healthy Infant Longitudinal Development study (a multi-disciplinary, longitudinal, birth-cohort study).
 
EPA-STAR Coarse CAP Study Collaboration: University of Michigan collaborator Dr. Rob Brook has been funded to: 1) examine vascular dysfunction following 2-hr coarse CAPs exposures; 2) compare responses to CAPs from both urban and rural sources; 3) compare racial differences in responses; and 4) elucidate the CAPs constituents/sources responsible for the cardiovascular responses. This study will be a comprehensive integration of a series of supplemental human experiments with our PM Center study of coarse PM in Toronto.  Through funding from the grant an additional vascular measure will be added to the Toronto study; a SphygmoCor, large arterial compliance, central aortic BP and hemodynamics device.  Common measures will be compared between the two sites, including: ultrasound baseline diameter and reactivity measures; Finometer continuous BP measures; BP measures during exposure; SphygmoCor measures; and biological constituents collected on filters, with the characterization coordinated by Dr. Diane Gold at Harvard for measures of airborne endotoxin and markers of fungi.
 
Evaluation of Autonomic Responses to CAPs in Controlled Exposure Studies:  Holter recordings from our previous Toronto exposure studies have been sent to Harvard and analyzed for HRV.  Statistical analyses are ongoing and initial results show an association between increasing diastolic BP and decreasing HRV during exposures.  The results will inform the current study by enabling the investigators to assess the interrelation between pollution exposures, HRV autonomic responses, vascular, and inflammatory responses.
 
Evaluation of Ambient Exposures on Baseline Systemic Inflammation:  Dr. Aaron Thompson from the Toronto group just completed a work term at Harvard as part of his training in Occupational Medicine.  In collaboration with Harvard and the Toronto group, he evaluated the contribution of prior ambient pollution exposure on pre-exposure (baseline) systemic inflammatory measures.
 
Acute Blood Pressure Changes in Outside Workers:  As a direct result of the blood pressure responses observed in our CAPs studies, an MSc student is carrying out a pilot project in outside workers (e.g., University of Toronto grounds keepers).  Starting July 11, 2008, over the summer and fall he will measure blood pressure at the start and end of a work day and also obtain both personal and area PM measures in 20-30 outside workers to test for associations between BP changes and PM levels.

Expected Results:

We expect to find physiologic responses consistent with vascular narrowing (increased BP, decreased brachial artery diameter) in response to all three CAPs size fractions, as compared to particle-free air. Also, we expect that the cardiovascular responses may vary by CAP treatment (fine, ultrafine, and coarse).

Future Activities:

Human Exposures. Exposure testing will continue until 25 subjects are completed.  Interim analyses will be carried out to guide future work.

Supplemental Keywords:

Air pollution, concentrated air particles, acute cardiovascular effects, coarse particles, fine particles, vascular dysfunction
Relevant Websites:

http://www.hsph.harvard.edu/epacenter/

Progress and Final Reports:
2006 Progress Report
2007 Progress Report
Original Abstract


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

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.

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