Grantee Research Project Results

2010 Progress Report: Cardiovascular Toxicity of Concentrated Ambient Fine, Ultrafine and Coarse Particles in Controlled Human Exposures

Objective:

The main objective of this project is to carry out controlled human exposures to both fine and coarse ambient particulate matter (PM) size fractions and examine acute changes in cardiovascular and respiratory outcomes. We hope to better understand how specific particle characteristics (size, composition, sources) affect health responses. The exposures are to concentrated ambient particles (CAPs) using our controlled particle exposure facility in Toronto, Ontario, Canada. Each subject has five 130-min exposures, assigned in random order, with a minimum 2-week wash-out period between. Exposures include 1 fine CAPs (250 µg/m³), 2 coarse CAPs (200 µg/m³), 1 HEPA filtered ambient air (HFAA) and 1 HEPA filtered medical air (HFMA). Cardiovascular and respiratory health outcomes are measured before, during and after exposures. Cardiovascular outcomes include: i) vascular dysfunction (brachial artery diameter and reactivity) by ultrasonography; ii) cardiac output by echocardiography; iii) blood pressure (BP) by automate 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 and blood IL-6, CRP & endothelins); and vi) markers of oxidative stress in blood and urine. Respiratory outcomes include: i) pulmonary function by spirometry; ii) respiratory inflammation by induced sputum; iii) respiratory and nasal symptomotology; and iv) nasal inflammation by nasal smears, in a subset of allergic subjects. In addition, we will test for susceptibility genes (DNA methylation) of PM-induced oxidative stress in blood. During exposure, end-tidal CO₂ is measured every 60 min using nasal prongs and beat-to-beat arterial BP is measured continuously 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. Mortara Holter (ECG) monitors are worn by the subjects over 24 hours, as a measure of cardiac autonomic dysfunction (HRV analyses). Exposures are characterized using continuous measures of particle mass (TEOM) and black carbon (PSAP) as well as integrated measurements (filter samples) of particle mass, sulphate, 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 include vehicle counts, meteorological data, TEOM PM_2.5, 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) are obtained to statistically adjust for potential affects on baseline pre-exposure data.

The specific hypotheses addressed by this project are the following:

• Acute human exposures to CAPs of coarse and fine size fractions result in cardiovascular responses including increased BP, 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 will be greater with coarse CAPs than fine CAPs, compared to filtered air.
• Associations between CAPs and cardiovascular responses will differ by particle size fraction and PM composition.

Progress Summary:

Controlled Human Exposures: The first human exposures began late in November 2007.  As of July 31, 2010, a total of 126 exposures have been completed.  This includes 51 coarse CAPs and 26 fine CAPs, 28 filtered ambient air and 21 filtered medical air.  A total of 36 subjects have been enrolled (met inclusion criteria) of which 3 did not qualify for the exposure phase of the study. Eight subjects who were enrolled dropped out after completing 0-2 exposures.  Each exposure involves a 2-day protocol and we attempt to have two exposures each week, depending on subject availability and rescheduling due to upper respiratory tract infections (must be symptom free for 3 weeks), cancellations and holidays.  Sixteen subjects have completed all five exposures.  In addition, five subjects have completed four exposures and will do the fifth exposure.  Subjects continue to be recruited and exposures carried out.  The final goal is to have 25 subjects who have completed four to five exposures (four subjects did not complete the fifth exposure).  At the earliest, the 25 subjects will be completed by late September/early October 2010.

Data Analyses:

Final analyses will be carried once the 25 subjects have completed their exposure testing, results are compiled, data have been entered and quality control checks carried out.  Manuscripts then will be prepared.

Meetings began in early 2009 to initiate collaboration between Harvard and the Gage with respect to Behrooz Behbod, a PhD student under the supervision of Diane Gold, interested in the inflammatory exposure data as part of his doctoral thesis. In June 2009, weekly teleconferences were established and interim data sets/variable labels sent to Harvard. His PhD proposal was finalized recently and will include analyses of respiratory and systemic inflammatory markers.  Specifically, he will examine induced sputum leukocytes, CBCs and blood IL-6 and CRP.  He also will examine health effect associations with endotoxin and β-(1,3)-D-glucan comparing the coarse and fine PM size fractions.

Data sets are updated in batches, once sufficient data are collected, and will be used to prepare abstracts/posters for scientific meetings. Interim statistical analyses are carried out on each data set, but will be merged into one large dataset once the study is completed.  Statistical analyses are carried out using the SAS Mixed procedure with a random Subject Effect and exposure type as a four-level categorical Fixed Effect (coarse CAP, fine CAP, filtered ambient air and filtered medical air). Exposure means are presented as least square means estimates ± standard errors (SE). Comparisons between exposures (e.g., filtered medical air vs. fine CAP) are presented as differences of least squares means estimates ± SE. An initial analysis was carried out to test for significant differences between the first coarse CAP exposure (coarse-1) and the second course CAP (coarse-2) that each subject received. For a given outcome variable, if coarse-1 and coarse-2 were significantly different, they were treated as separate exposures in the analyses. Data that were not normally distributed were analyzed using the nonparametric Wilcoxon Signed Rank test. Due to batch compilation, the total number of subjects/exposures in each data set may differ.  A summary of the findings follows.

Blood pressure (BP) was measured in resting and seated subjects during exposure at the start (0-hr) and at 30-min intervals during the 130-min exposure (5 time points) using an automated BP arm cuff placed over the left arm brachial artery. Three measures were taken at each time point, 1 minute apart, and the average of the second and third measures averaged. Linear regression was used to fit a line over the five time points. Results are presented as the rate of change in BP over the exposure for 23 subjects and 99 exposures. Diastolic BP increased during exposures with similar responses for all four exposure types (figure 1). Only the coarse CAP response showed a significant increase (median change for all coarse CAPs: 2.49 mm Hg, p=0.08, n=20); however, this response was not significantly different from either the FA or medical air responses.

 

 

Figure 2. Sputum Neutrophils 20 hrs after FA, Coarse and Fine CAP.

 

 

Blood Analysis: Blood was taken before exposure (pre) and 2 and 20 hrs after completion of the exposure (post).  Blood neutrophils, a gross marker of systemic inflammation, also showed increases at 2-hrs post for all exposures including HFAA and HFMA (1 - 16%).  However, the coarse and fine CAP responses were not significantly different from the HFAA or HFMA responses. Neutrophil responses 20 hrs post presented as small non-significant decreases for coarse and fine CAPs. In future analyses, we also will look at additional CBC responses (e.g., eosinophil count, potential representing allergic responses, etc.).

 

Flow-mediated dilation (FMD), nitroglycerin-mediated dilatation (NMD) and brachial artery diameter (BAD) were measured 1 hr before exposure (pre), 1 hr after the end of the exposure (1-hr post) and 20 hrs after (20-hrs post). Intra-exposure changes (Δ=post - pre) in FMD, NMD and BAD were assessed for each exposure type. Statistical summaries from the mixed models are presented for 17 subjects and 81 exposures and shown in Figure 3.  At 1 hr after exposure, the overall coarse CAP response (+1.7 ±1.3%) was significantly different compared to the HFMA response (p=0.05), but a highly significant response was seen between the first coarse CAP response (+4.2±1.7%) and HFMA (p=0.004). No significant differences were seen for fine CAP.  There were no significant FMD changes 20-hrs post exposures.  NMD and BAD showed no significant changes at any time points for any exposures. 

 

 

Figure 3. Mean Changes in FMD Post - Pre-Exposures.

 

 

Pulmonary function changes were small with no significant adverse effects following CAPs exposures compared to FA.

 

Traffic Density: A video camera was set up to record traffic flow adjacent to the CAP inlet on College Street during the 130 min exposures. The videos are played back and a minute by minute summary obtained of eastbound and west bound traffic counts including diesel (mainly trucks) and non-diesel vehicles. These data will be linked with minute by minute data for gaseous ambient pollutants (NO, NO₂, SO₂, O₃, CO), DustTrak, PM₁₀ mass concentration and PSAP black carbon data measured by the CAP inlet to test for associations. Summary data of the traffic counts (% diesel, total counts, count variability, etc.) will be used as predictors of health outcomes in regression analyses.  

 

Finometer data have been sent to an expert (Gianfranco Parati) with customized software analyses, and we are awaiting analysis results. 

 

Echocardiography proved to be difficult to obtain in some patients and was time consuming. A preliminary analysis of the echo data showed no differences between FA and CAPs exposures. After consultation with Rob Brook it was agreed to drop the measure and replace it with a SphygmoCor device (large arterial compliance, central aortic BP and hemodynamics). The advantage of the SphygmoCor is that Rob Brook also will be using this measure in his recently funded EPA study of coarse CAPs, so it provides a common endpoint for inter-study comparisons. The Finometer test also gives an estimate of cardiac output, to replace the echocardiography measure.

 

Nasal Scrapings: Nasal scrapings and RNA gene expression (micro-array) have been included as an outcome measure for all newly recruited subjects with allergic nasal rhinitis (at least 1 positive skin prick test). 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. Unfortunately, only 1 subject has had allergic rhinitis.

 

Heart Rate Variability: Subjects wear a Mortara Holter ECG recorder during the entire exposure day and through to the next day (24-hr recording).  ECG digital data are sent to Harvard for analysis.  No results are available at this time. 

 

Exposure Characterization:  Teflon filters (47 mm) are sent to Environment Canada (EC) for pre- and post-exposure conditioning and weighing. The median integrated mass concentrations for 43 coarse CAPs exposures was 199 µg/m³ (interquartile range: 188-216 µg/m³) and for 22 fine CAPs was 246 µg/m³(interquartile range: 218-269 µg/m³).  The median mass for the first coarse CAP exposure was very similar to that of the second coarse exposure (200 vs 196 µg/m³), thus does not account for any of the observed differences in health outcomes that were observed between the two course CAP exposures (e.g., FMD). After gravimetric measurements are completed, filters are analyzed at EC for sulphate, nitrate and ammonium 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 also will be taken for selected organics, including a motor vehicle tracer (engine lubricant), by GC-MS. Teflon filters (37 mm) are collected and sent to the DRI lab for trace element analyses by XRF. A batch of 45 coarse and fine CAP filters has been analyzed for trace elements. The seven main elements for the coarse CAP were calcium (median, 22 μg/m³), silicon (11 μ/m³), iron (10 ng/m³), aluminum (4 μg/m³), chlorine (2-4 μg/m³), sulfur and potassium (2 μg/m³) with little difference in concentrations between the two coarse CAPs exposures.  For fine CAP the seven main elements were sulfur (median 11 μg/m³), calcium 6 μg/m³), iron (6 μg/m³), sodium (2 μg/m³), aluminum (1.7 μg/m³), silicon 1.6 μg/m³) and potassium 1.1 μg/m³). Aluminum, calcium and iron levels were significantly higher for the coarse CAP vs. fine CAP (p<0.0001); a previous source apportionment study (Lee, et al., 2003) at the same site showed that a “vehicle/road dust” profile explained the majority of the variation of the latter three metals as well as manganese, magnesium, barium, chromium and organic/elemental carbon.  Sulfur levels (fossil fuel combustion) were significantly higher for fine CAP vs. coarse CAP (p<0.0001).  Figure 4 shows metal results for aluminum, calcium, iron and sulfur.

 

 

Figure 4. Selected Metal Concentrations for Coarse and Fine CAP Exposures.

 

 

Biologic components are collected on 25 mm polycarbonate filters for endotoxin and β-(1,3)-D-glucan, both exposure and outdoor samples. Data from 43 exposures (30 coarse and 13 fine CAPs) showed temporal variations in ambient endotoxin and glucan levels, with lower values during the winter months. Both ambient and concentrated endotoxin and glucan were highly correlated with each other (r=0.72 and 0.73, respectively, p<0.0001). Exposure glucan levels were more strongly associated with blood neutrophils in atopic subjects (>1 positive skin test) vs. non atopics (Figure 5, r=0.68 vs 0.20).  Positive associations also were observed between exposure glucan levels and induced sputum neutrophils (r=0.55 for coarse CAP and r=0.90 for subjects with atopy). Continuous data also are collected for PM_2.5 mass (TEOM, DustTrak), black carbon (PSAP), temperature, RH% and criteria gases (Table 1).

 

Figure 5. Association of β-(1,3)-D-Glucan and Change in Blood Neutrophils after CAP Exposure: Effect of Atopy on Response.

 

 

Table 1.  PM Characterization – Sampling from CAP Airstream

Mass Concentration	Method
TEOM	real-time
Gravimetric	47 mm Teflon
DustTrak	real-time
Composition
Inorganic Ions	Environment Canada (EC) - IC, 47 mm Teflon filters
Carbon	EC - TOT, OC/EC, 25 mm Quartz filters
Black Carbon	PSAP - continuous
Metals	with Harvard samples at DRI - XRF, 37 mm Teflon filters
Selected Organics	e.g. PAH, motor vehicle tracers, EC - GC-MS, 25 mm Quartz filters
VOCs	Multi-bed thermal desorption tubes
Sources
Motor vehicle tracers	see selected organics
Back trajectory analyses	Identify the path(s) of air masses and potential sources in the days prior to exposures
Biologic Components
Endotoxin	Gage - J Scott, LAL assay, 25 mm polycarbonate filter
b-(1,3)- D-glucan	Gage - J Scott, Glucatell assay, 25 mm polycarbonate filter
Pollen/spores	Burkhard sampler, Gripst rotation impaction sampler - ambient levels
Ambient Gases
NO, NO2, SO2, CO,O3, CO2	Continuous gas analyzers

 

 

Meetings: There have been regular scientific communications/meetings between the study investigators (GOEHU, Michigan & Harvard). A weekly 1-hr teleconference between Toronto and Harvard was initiated in June 2009, to discuss project-related issues. These meetings have proven to be both fruitful and beneficial to the development/progress of this project, thus they will continue throughout to its completion.  Posters were presented at the American Association of Aerosol Research meeting on Air Pollution and Health: Bridging the Gap from Sources to Health Outcomes in San Diego, CA, March 2010, and the NIEHS-EPA Symposium on Air Pollution and Cardiovascular Disease in Seattle, WA, June 2010. Data from the human exposures study was presented by Diane Gold and Petros Koutrakis at the PM Centers Directors Meeting at University of California-Davis, CA in January 2010.  Four posters and late-breaking FMD data were presented at the annual Harvard Science Advisory Committee Meeting, in Boston, MA, in June 2010.

 

Stat Core and Exposure Core: The Harvard Stat and Exposure Core groups have informed the work of the Toronto human exposure studies, both in the initial design and ongoing statistical and exposure analyses. The Harvard-Toronto collaboration has been very beneficial and productive, informing the papers that have been written with data coming from previous work funded by EPA, and also data analyses and papers forthcoming from this current EPA Center.

 

Health Canada Oxidative Stress Collaboration: Our collaboration with Health Canada on oxidative stress markers will include a total of 50 subjects. This includes 25 subjects from the PM Center studies and an addition 25 subjects. The latter 25 subjects will have only three exposures including two coarse CAPs and one HEPA filtered medical air.  Specifically, measures in urine include: 8-hydroxy-2'-deoxyguanosine (8-OHdG) for oxidative DNA damage and thiobarbituric acid reactive substances (TBARS) for oxidative stress. Measures in blood include: VEGF an angiogenesis factor, ET-1, IL-6 and CRP for inflammation; and TBARS for oxidative stress.

 

Health Canada Ultrafine Collaboration: We have new funding to set up and carry out pilot studies of concentrated ambient ultrafine particles (~ 8 human exposures) examining similar cardiovascular outcomes. Additional funding is provided to extend the VOC measures/analyses and to carry out analyses of the PM composition data from the previous CAP+O₃ study. Exposures are due to begin in September. 

 

EPA-STAR Coarse CAP Study Collaboration: University of Michigan (Ann Arbor) collaborator Rob Brook will be ready to start human exposures for his new coarse CAPs facility in September/October 2010. Common coarse CAPs outcome measures will be compared between our Toronto site and Ann Arbor, including arm cuff BP, CBC and SphygmoCor as well as biological constituents collected on filters (endotoxin, β-(1,3)-D-glucan, with the characterization coordinated by Diane Gold at Harvard for measures of airborne endotoxin and markers of fungi.

Future Activities:

We will continue the exposure testing to complete the 25 subjects.  Data reduction and QA/QC will be carried out and data sets updated and merged. Final statistical analyses then will be carried out. Some interesting findings will be further explored.

• First, we observed greater adverse responses to the first vs. second coarse CAPs exposures in FMD and some trends in other health outcomes, which were not explained by total mass concentration or elemental composition differences. If this were an adaptive response similar to that of O₃, one would expect that the differences would be smaller the greater the time between the two coarse CAPs exposures. We will examine this and other possible explanations.
• Second, there was an apparent difference in responses between the filtered ambient air and filtered medical air, with the medical air showing the smallest changes. 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.
• Third, we observed some interesting findings with the biologic data - endotoxin and β-(1,3)-D-glucan. This is an important component of the coarse CAPs exposures and few studies have considered that biologic components may contribute to respiratory and cardiovascular effects. We showed associations with both respiratory and systemic outcomes.
• Fourth, our exposure site has a significant contribution from traffic sources in close proximity to the CAP inlet; it thus provides the opportunity to explore specific associations using traffic markers of exposure. Specifically, we have obtained traffic counts, continuous black carbon measures and gaseous pollutants and will obtain motor vehicle tracers from filter samples as well as back trajectory analyses to determine sources during exposures. The fine CAPs exposures at higher concentrations than used in previous studies (250 μg/m³ vs. 150-200 μg/m³) does not appear to show more adverse responses, based on total mass concentration, but further analyses will be carried out to test for associations with individual constituents, as these may have varied across studies.

Once the exposure testing is completed, we will have data for more than 50 coarse CAPs exposures and with the continuation of the study (Health Canada) another 50 coarse CAPs. This will provide sufficient power to explore health effect associations in the largest coarse CAPs data set to date. Overall, the changes observed were small, but do provide important insights into the mechanisms of response to inhaled particles, including both respiratory and cardiovascular pathways. Furthermore, potential differences in response were shown between the coarse and fine PM size fractions. The final analyses will be the most comprehensive and it is hoped that they will uncover the most significant findings.  

Supplemental Keywords:

Progress and Final Reports:

Main Center Abstract and Reports:

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