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2007 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, 2006 through September 30, 2007
RFA: Particulate Matter Research Centers (2004)
Research Category: Particulate Matter

Description:

Objective:

In Project 2, we will examine the acute cardiovascular effects of coarse, fine and ultrafine concentrated ambient particles (CAPs) in healthy adults, using a newly constructed controlled particle exposure facility, in Toronto, ON, Canada. Based on recommendations of the Science Advisory Committee meeting in July 2006, a number of changes were made to improve the study. The proposed design, which included 50 subjects and 4 exposures per subject (filtered air and 3 size fractions of CAPs) would have taken 4 years to complete. In the revised design, we have decided not to include ultrafine CAPs, at least for the first series of studies. Twenty-four subjects will now each receive three 2-hr exposures selected from four possible treatments including: filtered (FA) air, 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. Once all 24 subjects have completed the three exposures, they will be asked if they wish to complete the 4th that they did not receive. If all subjects complete the optional 4th exposure there will be 48 coarse CAPs exposures, 24 fine CAPs and 24 FA. The 24 fine CAPs results @ 250 μg/m3 will be added to our CLEANAIR fine CAPs+O3 cardiovascular study (nearing completion) to look at dose-response relationships in common vascular endpoints. An interim analysis of this data has shown a positive association between the 2-hr exposure fine CAPs mass concentration, at levels ranging from 100 to 200 μg/m3 (gravimetric), and the change in diastolic BP during exposure, with greater BP increases at higher fine CAPs levels. Cardiovascular outcomes will be measured pre-, post- and 24 hrs post-exposure, and will include measures 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 using a Finometer finger cuff (pulse pressure) that includes calculated determinations of cardiac output, stroke volume and systemic vascular resistance. 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 (heart rate variability analyses). Filter samples will be collected during exposures for both mass and chemical composition (inorganic ions, trace elements, organic and elemental carbon) 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 and PM10, as well as pollen counts. 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:

Progress Summary:

Human Ethics Approval: The study protocol was submitted to St. Michael’s Hospital (SMH), Human Research Ethics Board (REB), Office of Research Administration in two stages: first for approval of study work in the 1st year, not involving human testing (approval date Dec 19, 2005); and second for the entire study, including human testing (approval date April 27, 2006). Annual renewal of approval from SMH REB was obtained. This includes amendments to the protocol. Application to the University of Toronto (U of T) REB was submitted, minor concerns of the board responded to and approval recently received. A point of clarification related the CAPs’ maximum and target levels was raised by Stacey Katz, our EPA project officer. In response, the target levels and maxima were clearly specified and a revised protocol sent to SMH REB for expedited review. Subsequent to this, human ethics approval and supporting documentation was forwarded to Harvard for human ethics approval from the U.S. EPA. This approval is pending. No human exposure testing for this project will be carried out until official approval from the U.S. EPA and the human CAPs exposure facility has been fully tested and characterized. It must be pointed out that a considerable amount of time and personnel costs have accumulated due to the ethics approval process required for two REBs in Toronto (SMH & U of T), as well as the EPA IRB. Each protocol amendment/revision has been consequent to discussions with the SAC and within the PM Center. We are now satisfied with the current design and anticipate no further delays.

New Harvard PM Concentrator Facility: Construction of our human CAPs (coarse, fine, ultrafine) exposure facility, funded through a Canadian infrastructure grant (CFI) and the Harvard/EPA Center, began the end of July, 2006. However, numerous meetings/discussions have been held to plan and finalize the facility, including GOEHU, University of Toronto, Harvard and the Southern Ontario Centre for Atmospheric Aerosol Research (SOCAAR). Both the coarse and fine concentrators have been installed and testing initiated. The ultrafine concentrator system has been delivered to the CAPs facility, and installation and operation training will be completed soon. The new CAPs exposure facility is nearing completion, including a 1.8 m3 plexiglass subject enclosure with facemask delivery of the CAPs. This larger enclosure will now afford us the potential opportunity of exercising subjects (to increase ventilation/inhaled dose) if deemed necessary in future studies. A full characterization of the facility will be carried out before human testing, including the facemask CAPs levels.

Sub-award Agreement: A sub-award agreement between Harvard and SMH was signed July 11, 2006. Subsequent to this, an account was set up for ordering equipment and supplies. As well, a sub-contract was setup (June 2006) between SMH and the University of Michigan (Rob Brook), for analyses/consultation of ultrasound and echocardiography.

Training: Bruce Urch, the study coordinator and Research Assistant has been facilitating all aspects of the new CAPs facility construction with Harvard, Greg Evans (SOCAAR), the University of Toronto and on-site contactors. Steve Ferguson, from Harvard, ran through a 3-day CAPs training/familiarization session with Bruce and Mike Fila who will run the facility. We have been testing the new Finometer BP finger cuff for stability, outcome measures and use during the 2-hr exposure. Mary Speck, the Laboratory Technologist at GOEHU currently doing ultrasound measurements was trained in Michigan to do the echo cardiac measures by an echo technologist under the supervision of Rob Brook and Julie Kovach. Subsequently, Mary has been practicing the echo techniques in order to perfect echo testing using our Terason system, sending echo images of GOEHU staff to Michigan for review. In discussion with Rob Brook, we decided that he would train us to do the software analyses of the ultrasound and echo images in-house. Strict guidelines will be established to ensure quality data. Julie Kovach, the cardiologist working with Rob Brook has recently left and is being replaced by Ted Kolias, a cardiologist specializing in echocardiography.

Collaborations: 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.

New Collaborations: We are in the process of establishing a collaboration with Health Canada on coarse PM human heath effects. They would like to collaborate with Harvard and us on our PM Center study by adding a few measures of inflammation & oxidative stress that they would measure in the urine and blood. Specifically in urine, measures 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 researcher, Jeremy Scott, is the PI of a study examining: 1) the effect of coarse CAPs exposure in humans with seasonal allergic rhinitis (in and out of season and ± pollution exposure; 2) whether specific gene-environment interactions are responsible for the development and/or exacerbation of symptoms; 3) the development of a system to expose nasal/airway epithelial cells to real-world pollutants; and 4) the contribution of specific gene pathways responsible for the pollutant-induced allergic rhinitis in a murine model. Filter samples will be collected during exposures of people, cells and animals for biological material including airborne endotoxin (Limulus Amebocyte Lysate test method) and markers of fungi (β1→3 D-glucans; Glucatell kit). Samples will also be provided to James Scott for characterization of the microbial community in the air.

AllerGen researcher, James Scott, is also the PI of a study that is part of the Canadian Healthy Infant Longitudinal Development study (CHILD- a Canada-wide multidisciplinary, longitudinal, population-based birth-cohort study). The specific objective is to: adapt DGGE and DNA macroarray technologies for the characterization of airborne and dust-borne fungal contaminants; (2) investigate the value of these data in the context of studying indoor and outdoor exposures and health outcome measures. In addition, endotoxin analyses samples will be made available from the CAPs exposures of the Toronto PM Center study. James Scott has analyzed endotoxin samples collected during the Toronto CLEANAIR fine CAPs+O3 study starting from April 2006. Results from 31 samples analyzed show up to a 20-fold range in endotoxin levels for the CAPs.

University of Michigan collaborator Rob Brook has written a proposal to: 1) examine vascular dysfunctions following 2-hr coarse CAPs exposures; 2) compare responses to CAPs from both urban and rural sources; 3) compare responses in two races (blacks and whites); 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. Additional vascular measures will be added to the Toronto study including Endo-PAT (microvascular resistance, artery endothelial function) and SphygmoCor (large arterial compliance, central aortic BP and hemodynamics) devices. Biological constituents will be collected on appropriate filters, with the characterization coordinated by Diane Gold at HSPH for measures of airborne endotoxin (LAL kit) and markers of fungi (β1→3 D-glucans; Glucatell kit).

Evaluation of Autonomic Responses to CAPs in Controlled Exposure Studies: Holter ECG recordings are available for our previous Toronto exposure studies, and have been sent to Boston for heart rate variability (HRV) analyses at no additional cost to the EPA Center. This will inform the new study by enabling the investigators to assess the interrelation between pollution exposures, HRV autonomic responses, vascular, and inflammatory responses.

Evaluation of Baseline Inflammatory Responses to CAPs in Controlled Exposure Studies: Aaron Thompson from the Toronto group will be at HSPH in the Occupational Medicine program for the coming year, and as part of his training will, with the help of Jeff Brook and the rest of the Toronto group, be evaluating the contribution of the prior 24-72 hr ambient pollution to the baseline and post-exposure inflammatory responses to CAPs +/- O3.

Summary of Other Findings used to inform the Current Project: Our EPA CLEANAIR fine CAP+O3 study, with Rob Brook in Michigan, is nearing completion (healthy non-asthmatics, 18-50 yrs). To date, 28 of 38 subjects have completed the Toronto part of the study. Each subject receives 4 exposures, including: 1) FA; 2) fine CAPs (target 150-200 μg/m3); O3 (120 ppb); & fine CAPs+O3. An interim analysis has shown a small but significant increase in diastolic BP during the 2-hr exposures, more so for the combined CAP+O3 (3.6 mm Hg increase over 2 hrs; p=0.0003), followed by CAPs alone (2.5 mmHg/2hrs; p=0.055) and O3 alone (1.8 mmHg/2hrs; p=0.09) with the smallest and non-significant changes for FA (1.6 mmHg/2hrs; p=0.2). A similar exposure trend of about the same size effect was shown for systolic BP. In addition, the magnitude of the BP increase was positively associated with the level of CAPs exposure mass concentration. Blood neutrophils also showed a similar pattern of response to BP, with the greatest pre to post exposure increase shown for CAPs+O3 (15%; p=0.001), followed by CAPs alone (9%; p=0.04) and O3 alone (8%; p=0.07), with the smallest and non-significant changes for FA (4%; p=0.3). Data is not as complete for the brachial artery measures, but there appears to be a trend for flow-mediated dilation to decrease post relative to pre-exposure, for CAPs (-1.9%) & CAPs+O3 (-2.0%), compared to no change for O3 and an increase of 2.6% for FA.

Rob Brook’s part of the CLEANAIR fine CAPs+O3 study is completed and he will be sending the data to GOEHU for statistical analyses/consultation. This was a randomized cross-over study with 50 subjects exposed to CAPs+O3, pre-treated with 1) placebo (control) vs 2) Bosentan (an anti-endothelin) and 3) vitamin C, an anti-oxidant.

James Scott has analyzed endotoxin samples from the Toronto CLEANAIR fine CAPs+O3 study for April - December 2006. Results show up to a 20-fold range in endotoxin concentration for CAPs/CAPs+O3 exposures. This data will be used in regression analyses to test for associations with CAPs exposure-induced changes in BP, brachial diameter and reactivity, HRV and inflammatory measures.

Future Activities:

The CAPs facility will be completed and exposure characterization carried out. The Mortara Holters were ordered in July 2007 and we will do some ECG measures on staff and send the recordings to Harvard for QA/QC. We will complete training with the echo/ultrasound and Finometer and all other aspects of exposure testing including SOPs. After ethics approval from EPA, we will begin recruiting subjects, after which exposure testing will begin. An interim analysis will be carried out after the 24 subjects have completed the three exposures, to guide subsequent work.

At present, there are no direct publications. However, in the meantime, we have been analyzing data and the following manuscripts that will inform the current project, are in preparation. Diane Gold from Harvard will work closely with GOEHU in the preparation of the three manuscripts listed below:

  1. “Concentrated Ambient Fine Particles induce an IL-6 inflammatory response in asthmatics and non-asthmatics.”
  2. Blood endothelins and nitric oxide metabolites (systemic vascular peptides) in response to controlled fine CAPs +/- O3 in young healthy asthmatics and non-asthmatics and in children.
  3. End-tidal CO2 (capnography) as sensitive marker of ventilation/perfusion changes in response to fine CAPs +/- O3 in young healthy individuals.

Supplemental Keywords:

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

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

Progress and Final Reports:
2006 Progress Report
Original Abstract
2008 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

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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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