2012 Progress Report: Cardiometabolic, Autonomic, and Airway Toxicity of Acute Exposures to PM2.5 from Multipollutant Atmospheres in the Great Lakes RegionEPA Grant Number: R834797C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R834797
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Great Lakes Air Center for Integrative Environmental Research
Center Director: Harkema, Jack
Title: Cardiometabolic, Autonomic, and Airway Toxicity of Acute Exposures to PM2.5 from Multipollutant Atmospheres in the Great Lakes Region
Investigators: Harkema, Jack , Fink, Greg , Wagner, James
Institution: Michigan State University
EPA Project Officer: Ilacqua, Vito
Project Period: December 1, 2010 through November 30, 2015 (Extended to December 31, 2016)
Project Period Covered by this Report: December 1, 2011 through November 30,2012
RFA: Clean Air Research Centers (2009) RFA Text | Recipients Lists
Research Category: Health Effects , Air
Our objectives in Project 2 arise out of GLACIER’s overarching hypothesis that the major air pollutants, fine particulate matter (PM2.5) and ozone (O3), are (1) capable of eliciting multiple important adverse cardiometabolic health effects that are dependent on (2) the local multipollutant milieu, (3) an individual’s pre-existing cardiovascular (CV) and metabolic condition (susceptibility factors), and (4) the interactive toxicity of PM2.5 and O3 co-exposure. Goals of Project 2 are to determine the cardiovascular (CV), autonomic nervous system (ANS), and airway toxicity in rats acutely exposed to concentrated ambient PM2.5 (CAP) from distinct multipollutant atmospheres commonly found in the Great Lakes Region of the United States. Our studies are extensions of our previous findings that CAP-induced alterations in heart rate variability are dependent on specific PM2.5 emission sources in distinct locations in the Great Lakes Region. We will use a mobile air research facility (AirCARE 1) that is fully equipped with inhalation toxicology and atmospheric monitoring labs to conduct toxicology studies of rats exposed to CAP derived from real-world PM2.5 in three distinct locations dominated by industrial/urban, transported/ regional, or near-roadway/residential emission sources. Blood pressure, heart rate, heart rate variability and direct measurements of autonomic nerve activity will be continuously monitored during CAP and/or O3 exposures in lean or obese rats with and without diet-induced facets of the cardiometabolic syndrome (CMS; hypertension, insulin resistance, endothelial dysfunction), respectively. Acute functional responses will be measured by radiotelemetry and will be correlated with specific PM constituents and their emission sources determined for the same highly resolved 30-minute timeframes, thereby making associations of exposure and health effects especially robust. Studies will feature novel real-time sympathetic nerve recordings during PM2.5 and/or O3 inhalation exposure. In addition, our project will highlight the unique integrative capabilities of our research team to link specific health cardiovascular effects in a sensitive obese population with PM content by a combined technological expertise that is unavailable elsewhere. Our GLACIER project will extend and complement the research of lean and obese human subjects (Project 1), conducted at the same exposure sites, by making invasive and prolonged measurements that could not be practically or ethically done in humans (e.g., repeated CAP exposures, continuous recordings of CV and autonomic nerve function, and microscopic examination of multiple organs for exposure-related pathology). Our acute animal studies will also overlap and integrate scientifically with the animal toxicology study of long-term multipollutant exposures in Project 3.
There have been no changes in the study investigators or personnel in year 2 of this Project. Our objectives in Project 2 have also remained the same in year 2. Specifically, our studies have been designed to address GLACIER’s overarching hypothesis that the major criteria air pollutants, fine particulate matter (PM2.5) and ozone (O3), are (1) capable of eliciting multiple important adverse cardiometabolic health effects that are dependent on (2) the local multipollutant milieu, (3) an individual’s pre-existing cardiovascular (CV) and metabolic condition (susceptibility factors), and (4) the interactive toxicity of PM2.5 and O3 co-exposure. Research in year 2 addressed all four of these hypotheses, and focused on initial field exposure studies during summer months, and was followed by mechanism-driven experiments during the winter months. These studies were extensions of our previous findings that CAPinduced alterations in heart rate variability are dependent on specific PM2.5 emission sources in distinct locations in the Great Lakes Region. Our project leveraged the unique integrative capabilities of our research team to link specific health cardiovascular effects in a sensitive obese population with PM content. Our results in Years 1 and 2 were designed to extend and complement the initial research of lean and obese human subjects (Project 1), by making invasive and prolonged measurements in laboratory rats that could not be practically or ethically done in humans (e.g., repeated CAP exposures, semi-continuous cardiovascular recordings, and microscopic examination of multiple organs for exposure-related pathology). Our acute animal studies were also designed to contrast and complement the initial animal toxicology studies of long-term multipollutant exposures in Project 3.
In year 2, several research milestones were achieved that led directly to a better understanding of the health effects of multipollutant exposures in the face of experimentally induced facets of the cardiometabolic syndrome (CMS). Specifically, we (1) established a rodent model of CMS using a high fructose diet in Sprague-Dawley rats, (2) conducted a large field study of acute inhalations of rodents to O3 and concentrated PM2.5 aerosols in urban Dearborn MI, (3) demonstrated exaggerated exposure induced drops in heart rate and blood pressure in rats with CMS using high resolution telemetry techniques, (4) documented novel changes in hepatic lipid storage and altered expression of genes related to lipid mobilization, and (5) revealed the role of transient receptor potential (TRP) channels in O3- induced acute cardiovascular and airway alterations. Our results from these initial studies are being extended in two ongoing experiments that address the potential mechanisms underlying metabolic and cardiovascular dysregulation that are induced or enhanced by multipollutant exposure. In addition, we will be starting a intraCLARC collaborative animal toxicology study with Harvard University in the Fall of 2012 employing our rat model of diet-induced CMS and their inhalation exposure system to traffic emissions from the Boston Tunnel. A brief summary of these Project 2 accomplishments in the last year and future studies in Year 3 are briefly described below.
- Established a Rodent Model of CardioMetabolic Syndrome.
Sprague-Dawley rats fed a diet with 60% fructose-derived calories induced multiple facets of the human CMS. These rats were insulinresistant, hypertensive and had hypertriglyceridemia, which are three major factors of the CMS (Figure 1A, B, C). In addition, high fructose (HFr) fed rats developed hepatic steatosis and elevated heart rate. As such, HFr-fed rat model demonstrated cardiovascular and metabolic abnormalities that are consistent with human CMS, and provided a reproducible rodent model to further elucidate the adverse health effects of multipollutant exposures in a potentially susceptible population suffering from CMS.
- Successful Completion of Field Exposure Studies.
From July 25-August 26, 2011 we conducted a series of acute inhalation exposures of rats, on normal or HFr-diets, to O3 and/or concentrated PM2.5 (CAP), for 9 days, 8 h/day, in our mobile air research laboratory (AirCARE I) parked in an urban/industrial setting in Dearborn, MI. Two 9-day exposure regimens were conducted in order to acquire data from all experimental groups. Inhalation exposures were coordinated with Drs. Dvonch and Morishtia in the Exposure Characterization Core at the University of Michigan. They characterized the physicochemical composition of both ambient and concentrated PM2.5 at this exposure site. Initiated by Drs. Harkema, Wagner and Fink (MSU-Project 2), the details of the study design and data and tissue collection protocols were finalized after consultation with Drs. Rajagopalan (Project 3) and Brook (Project 1), so as to ensure cohesion of endpoints in acute and chronic rodent and acute human studies. Acute cardiovascular responses (e.g., blood pressure, heart rate, heart rate variability) were measured every 5 minutes by radiotelemetry during and after daily exposures. Twenty-four hours after the last exposure, tissue samples were taken at necropsy from a wide-range of targeted organs, along with blood and bronchoalveolar lavage fluid samples, and analyzed for biochemical, molecular, and morphometric alterations related to the CMS.
- Demonstration of Enhanced Cardiovascular Depression in High Fructose (HFr)-Fed Rats Caused by Multipollutant Exposure.
Inhalation of either concentrated PM2.5 or O3 alone caused significant drops in mean arterial pressure (10-15 mmHg) in HFr-fed rats that were maintained for most days of the exposures (Figure 2A, B). These decreases persisted during post-exposure periods on the weekends (7:30 a.m.-3:30 p.m.) and during evening hours of weekdays (12:00-5:00 a.m.) when rats were removed from the inhalation chambers. In comparison, vascular responses of rats fed a normal diet were minimal and less frequent, with episodic elevations in blood pressure on certain days. Interestingly, coexposure to both pollutants caused marked decreases in blood pressure in HFr-fed rats on the first two days of exposure, yet this vascular response did not persist with repeated exposures, as was observed with single pollutant exposures (Figure 2C).
Both PM2.5 and O3 exposure alone caused drops in heart rate that were consistently greater and persistent in HFr-fed rats (Figure 3A, B). Decreases of up to 25 beats per minute (bpm) in normal chow-fed rats and up to 60 bpm in HFr-fed rats were observed. During coexposures to both O3 and PM2.5, drops in heart rate were remarkable during the first two days (decreases of 40-70 bpm), but were less dramatic with repeated exposures (0-20 bpm decreases; Figure 3C). Thus, decreases in both blood pressure and heart rate were greater and more sustained with single pollutant exposures, whereas cardiovascular responses during coexposures appeared to attenuate more quickly. The reason for this difference in the persistence of these cardiovascular responses to repeated single or multiple pollutant exposures is unknown. We speculate that the rapid adaptation of copollutant-induced blood pressure and heart rate responses in HFr-fed rats may have been the result of a greater adaptive defense mechanism(s) due to a greater total pollutant dose with the co-pollutant exposures. The biological mechanisms underlying these physiologic responses to exposure will be the focus of future studies.
- Multipollutant Exposures Induce Novel Changes in Hepatic Lipid Storage and Altered Expression of Genes Related to Lipid Mobilization.
In pollutant-exposed HFr-fed rats, we detected enhanced hepatic gene expression of stearoyl-coenzyme A desaturase-1 (SCD1) the primary ratelimiting step in the synthesis of unsaturated fatty acids (Figure 4A), as well as the low density lipoprotein receptor (Figure 4B). Downregulation of gluconeogenesis was also exaggerated in these rats, as evidenced by decreases in PCK1, a critical enzyme used in the metabolic pathway of gluconeogenesis (Figure 4C). Storage patterns of hepatic lipids were also altered after exposures, with more diffuse and smaller lipid droplets microscopically evident in hepatic tissue sections in HFr-fed rats exposed to O3, PM2.5 or O3±PM2.5, as compared to larger and more frequent lipid droplets in the liver sections of HFr-fed rats exposed to filtered air.
- Blockade of Transient Receptor Potential (TRP) Channels Alters Cardiovascular Responses Induced by Ozone Exposure.
Building on studies by EPA researchers, Drs. Farraj and Hazari, who have previously reported the dependence of transient receptor potential (TRP) channels for dieselinduced arrhythmia, we used TRP channel antagonists to explore the biological mechanisms underlying ozone-induced decreases in blood pressure and heart rate in HFr-fed rats. Rats were treated with Ruthenium Red, a cation channel blocker that blocks TRPV channels, and HC030031, a specific TRPA1 antagonist, immediately before a single, 5h exposure to ozone (Figure 5). While treatment with Ruthenium Red caused a mild decrease in blood pressure and increased heart rate in filtered air-exposed HFr-fed rats, the ozone-induced decreases in blood pressure were inhibited with this TRP channel antagonist. In comparison, treatment with the selective TRPA1 antagonist had no effects on this cardiovascular response to O3. TRPA1 treatment did, however, result in an enhancement of ozone induced airway epithelial toxicity in terminal bronchioles and alveolar ducts, suggesting attenuation of upper airway irritant-induced respiratory apnea and resulting in a greater toxic dose of O3 to the distal pulmonary airways.
- Sensitivity of Nasal Airway Epithelium to the Toxicity of Multipollutant Exposure.
Because the airway epithelium that lines the proximal aspects of the nose is known to be sensitive to O3-induced toxicity, we investigated inflammatory responses to multipollutant exposure on rats fed normal versus HFr diets in this sensitive region of the upper respiratory tract. Both O3 and O3+PM2.5 coexposure caused neutrophilic rhinitis and nasal epithelial cell proliferation (hyperplasia) in response to toxic injury (Figure 6A, B). In HFr-fed rats, but not normal diet-fed rats, PM2.5 exposure resulted in neutrophilic inflammation similar to that caused by O3 or O3+PM2.5 exposures. In addition, nasal epithelial hyperplasia caused by O3+PM2.5 coexposure was significantly greater in HFr-fed versus normal diet-fed rats.
- Cardiotelemetry Data: Analysis of heart rate and blood pressures responses in rats acutely exposed to O3 (single 8h exposure; 0.5 ppm) and treated with TRPV4 and TRPA1 channel antagonists is ongoing. In addition, data analysis of heart rate variability is being calculated for all cardiotelemetry studies and ECG waveform analyses are being planned.
- Associations of Exposure/Health Outcomes: With the help of the Biostatistics and Data Management Core, we will use data of cardiovascular responses (e.g., heart rate, mean arterial pressure, heart rate variability) and PM2.5 metrics (e.g., trace elements, gases, sources) collected over the same 30-minute timeframes to provide a high resolution correlation of specific PM2.5 components to specific health effects.
- Brain Neurochemistry: With the help of collaborators and neuroscientists Drs. P.S. and Sheba MohanKumar at MSU, exposure- and diet-related responses in specific brain regions that control autonomic functions are being examined. For example, neurochemical and molecular analyses in the paraventricular nucleus of the hypothalamus are being conducted for neurotransmitters and inflammatory cytokines after inhalation exposures in both normal and high fructose-fed rats.
New Studies in Project 2:
- 1. Effect of Metformin on Ozone-Induced Cardiometabolic Responses in HFr Rats.
Metformin is the most commonly prescribed drug for the treatment of type 2 diabetes, and the only anti-diabetic therapy that has been demonstrated to attenuate the cardiovascular complications of diabetes and metabolic syndrome. It also helps to reduce LDL cholesterol and triglyceride levels in the blood, and improves insulin sensitivity. Our new study is designed to determine if Metformin will protect the animals from air pollutant- and/or diet-induced changes in metabolic and cardiovascular functions. Initially, we will intervene with Metformin in normal and HFr rats two weeks prior to repeated exposure to O3 (8h/day, 4 days) to determine the therapeutic effects on diet- and exposure-dependent cardiovascular, respiratory and metabolic alterations.
- Effects of Subchronic Ozone Exposure on the Development of Cardiometabolic Sydrome in C57Bl/6 mice.
We will test the effects of chronic ozone inhalation exposure (4h/day, 4d/wk, for 13 weeks) on the development of HFr-diet-induced cardiometabolic syndrome in mice. Animals will be fed a normal chow or an HFr diet for at least 13 weeks during the O3 exposures.
- Sympathetic Neural Recordings in Rodents Exposed to Ozone.
We continue to work on refining the technique for applying telemetric microneurography to future studies, with an emphasis on determining which specific sympathetic nerve (renal, lumbar, splanchnic) would be the most informative.
- Inhalation Toxicology Studies in Dexter, MI.
In the spring and summer of 2013, we will initiate inhalation toxicology studies in our rural Dexter, MI (predominantly regionally transported air pollution) to determine the cardiopulmonary, airway and metabolic effects of acute PM2.5±O3 exposures on normal- and HFr-fed rats. These studies will complement and extend the acute human exposure studies conducted in Years 1 & 2 of Project 1.
Collaborative Research Effort with Other Clean Air Research Centers.
Toxicity of Traffic-Based Air Pollution in Rats with Diet-Induced Cardiometabolic Syndrome:
In the Fall of 2012, investigators from GLACIER and Harvard University CLARC will initiate an intraCLARC collaborative toxicology study under the principal direction of Drs. Jack Harkema (GLACIER) and John Godleski (Harvard University). Using our established high fructose-diet-fed rat model of CMS, the proposed collaborative study is designed to determine if this dysfunctional cardiometabolic condition predisposes to the toxic effects of traffic-related air pollution and to identify underlying toxicological modes of action by which this may occur. We will be able to discern if CMS renders the laboratory animal more susceptible to the cardiovascular, autonomic and airway toxicity of a multipollutant mixture of primary particles and secondary organic aerosols derived from traffic emissions in the Boston Tunnel. The results of this study will be compared to similar studies in GLACIER’s Project 2 using the same animal model but exposures to concentrated PM2.5 (urban/industrial site in Dearborn, MI) with or without ozone.
A brief description of the proposed study design is presented below.
Twelve 200 gram Sprague-Dawley rats will be obtained from Taconic Farms with implanted DSI telemeters capable of monitoring blood pressure, heart rate, and temperature. Another 36 animals will be obtained from Taconic Farms without telemeters. All rats will be fed a high fructose diet (Harlan TD.89247; 60% of calories comes from fructose) for 8 weeks prior to use in any experiments. Diet will be provided by GLACIER CLARC. Inhalation exposures will be conducted at Harvard’s Boston Tunnel site (see below). Rats will be weighed weekly, and 4 hrs of continuous data will be collected from the telemeters in the animals at two week intervals until used in experiments. Once the study begins, rats are without food or water during the 5 hours of daily exposure, but when returned to their housing during nonexposure hours they will be fed their specified diets.
Traffic-Related Urban Aerosol Particles (TRUAP) exposure protocol:
Rats will be continuously exposed to TRUAP or filtered air (FA) in single-animal plethysmographs for 5 hrs/day. TRUAP inhalation exposures are derived from the real-time ventilation exhaust of a moderate traffic density tunnel (with small positive road grade, approximately 2°), in the northeast United States. TRUAP consists of primary and secondary traffic-derived fine and ultrafine particles (1nm to 2.5 μm (PM2.5)). Twelve (12) animals (6/group TRAUP; 6/group filtered air all with implanted telemeters continuously monitoring blood pressure, heart rate, and temperature) will be exposed each day, four days/week for three consecutive weeks. Another group of animals without telemeters, will also be exposed to filtered air or TRAUP for studies as described below. In prior TRUAP studies, concentrations average approximately 50 μg/m3 (combined primary and secondary particles), with standard deviations less than 25% of the mean for any component; the exposure is highly stable and reproducible. Exposures will be initiated at the same time each day, limiting variability due to diurnal traffic patterns.
We will compare filtered air vs TRUAP exposures in rats on a high fructose diet which produces hypertension and other facets of the metabolic syndrome (e.g., insulin resistance, hyperglycemia, dyslipidemia). From the telemetered animals, we will assess the following outcomes: (1) Cardiovascular parameters, including blood pressure, heart rate, heart rate variability, and bodytemperature all derived from the telemetry system; Respiratory parameters, including respiratory rate,times of inspiration, expiration, inspiratory pause, expiratory pause and relaxation, peak air flows during inspiration and expiration, average air flow during expiration, tidal and minute volumes, inspiratory dutycycle, and minute ventilation. From the non-telemetered rats, we will assess differences in in vivo chemiluminescence of the heart and lungs after 1 day of exposure in 6 animals/TRUAP or filtered air group. In another group of non-telemetered animals, we will also assess bronchoalveolar lavage fluid (BALF) parameters after 4 days of exposure, including total cells and cell types as well as total protein and ß-N-Acetyl-Glucuronidase determinations in the lavage fluid using 6 animals/ TRUAP or fitered air group. From the rats on which BAL is done, we will also collect heart blood from which complete blood count and differential as well as platelet counts will be done, and blood chemistry (chem 17), including blood glucose, electrolytes, insulin, triglycerides, and hepatic function assessment. The third group of non-telemetered 6 animals per TRUAP or Filtered air group will be exposed for a length of time to be determined for specific outcomes to be assessed by collaboratoring investigators at Havard University CLARC, GLACIER, and the University of Washington Center for Clean Air Research.
All aspects of the study protocol are now proceeding per the slightly modified study design outlined in the last progress report. We anticipate completing our Dearborn (industrial multi-pollutant atmosphere) exposure studies by the end of year 2 and will start acute animal toxicology studies at our Dexter rural site (transported multi-pollutant atmosphere) in year 3, and continuing through year 4. In light of our initial mechanistic findings in regard to TRP receptors, we will continue to explore the role of specific TRP receptors (e.g., TRPV4, TRPA1) in cardiovascular, airway and autonomic responses to acute inhalation exposures of concentrated PM2.5 ± O3 in rats (and/or mice) fed normal and high fructose diets. In future studies, we will use specific TRP antagonists in rats or genetically modified mice deficient for specific TRPs (e.g., TRPV4-knockout mice) to further explore their role in diet- or toxicant-induced alterations (e.g., hypertension, bradycardia). Based on the findings of our long-term exposure of high fructose-fed mice exposed to ozone, we will decide how to proceed with this animal model in targeted mechanistic studies using multipollutant exposures. Based on our project results in years 1-3, and those from studies in projects 1 and 3, we will either extend our acute rat inhalation studies in year 5 to urban sites in Columbus, OH (Project 3) or conduct long-term inhalation exposures of mice in Dearborn (or Dexter) using protocols outlined in Project 3.
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Supplemental Keywords:Inhalation toxicology, acute multipollutant exposures, high-fructose diet, rats, CAP, ozone, cardiometabolic syndrome, Scientific Discipline, Air, ENVIRONMENTAL MANAGEMENT, air toxics, Health Risk Assessment, Biochemistry, Biology, Risk Assessment, ambient air quality, particulate matter, aerosol particles, susceptible populations, acute cardiovascualr effects, human exposure, physiology, cardiopulmonary, cardiotoxicity, human health
Progress and Final Reports:Original Abstract
Main Center Abstract and Reports:R834797 Great Lakes Air Center for Integrative Environmental Research
Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R834797C001 Cardiometabolic Effects of Exposure to Differing Mixtures and Concentrations of PM2.5 in Obese and Lean Adults
R834797C002 Cardiometabolic, Autonomic, and Airway Toxicity of Acute Exposures to PM2.5 from Multipollutant Atmospheres in the Great Lakes Region
R834797C003 Long Term Metabolic Consequences of Exposures to Multipollutant Atmospheres in the Great Lakes Region