University of Rochester EPA

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1 R University of Rochester EPA PM Center ULTRAFINE PARTICLES: CHARACTERIZATION, HEALTH EFFECTS AND PATHOPHYSIOLOGICAL MECHANISMS PROGRESS REPORT YEAR 4 (Project period: Years 6/99 5/2; 6/2 5/3) July, 23

2 TABLE OF CONTENTS Page Introduction iii Research Core 1: 1 Characterization of the Chemical Composition of Atmospheric Ultrafine Particles Research Core 2: 18 Inflammatory responses and cardiovascular risk factors in elderly subjects with angina pectoris or COPD in association with fine and ultrafine particles Research Core 3: 3 Clinical studies of ultrafine particle exposure in susceptible human subjects Research Core 4: 38 Animal models: Dosimetry, and pulmonary and cardiovascular events Research Core 5: 56 Ultrafine particle cell interactions: Molecular mechanisms leading to altered gene expression Appendices: I Literature Cited in Progress Report II. Listing of Enrichment Program Activities III. Visiting Scientist Project: J. Veranth Ultrafine oil aerosol generation for inhalation studies ii

3 YEAR 4 PROGRESS REPORT ROCHESTER PM CENTER Ultrafine Particle Characterization, Health Effects and Pathophysiological Mechanisms This progress report summarizes the PM Center accomplishments of Years 1-3 of the Center s existence, and in more detail progress made in Year 4. The focus of the Center, research on ultrafine particles (UFP), is pursued in a multidisciplinary team approach, spanning topics of physicochemical characterization of UFP in different locations of the U.S. and in Europe and their source apportionment to epidemiological, clinical and toxicological animal effect studies and mechanistic in vitro studies. An integrated research agenda involves continuous interactions among the 5 Research Cores with important feedback for designing subsequent studies based on significant findings in the individual cores. These Research Cores and their members are listed below, and the progress report is structured along these cores. Important Facility Cores assure the availability of additional expertise in specific fields, as listed below. Our Enrichment Program has been very successful with respect to inviting outside speakers to initiate discussions and start collaborations, selecting pilot projects and supporting visiting scientists to complement specific PM Center studies. These activities are listed in Appendix 2. The multidisciplinary team approach has resulted in important interactions among the Research Cores. Several examples include: (1) The truck study, a project housed in Core 4 has utilized expertise from the Characterization Core to monitor ambient particles as well as the Epidemiologic, Clinical and In Vitro Cores to either design complementary endpoints to ongoing human studies or to perform in vitro analyses. (2) Investigators in Core 1 have worked with investigators in Cores 3 and 4 to characterize ultrafine particle size and composition both from the UFP generator and the UFP concentrator. In addition, we are collaborating with the Harvard PM Center and the EPA Center to characterize particle composition from their concentrations. (3) The Epidemiologic, Clinical and Animal Cores have worked in parallel to develop both hematologic and cardiac endpoints. For many of our studies, the same assays are performed using a common core facility; for example, the cardiac monitoring in both humans and animals uses a common system facilitated by the Cardiac Core to perform the EKG analyses. A major emphasis of our program has been integration of our five Research Cores and these are merely a few examples of the continuing team effort within the Center. iii

4 ROCHESTER PM CENTER RESEARCH CORES Research Core 1: Characterization of the chemical composition of atmospheric ultrafine particles Principal Investigator: Philip K. Hopke, Clarkson University Co-investigators: Kimberly Prather, University of California-San Diego Ann Dillner, Arizona State University Glen Cass (deceased), Georgia Institute of Technology Research Core 2: Inflammatory responses and cardiovascular risk factors in elderly subjects with cardiopulmonary disease in association with fine and ultrafine particles Principal Investigators: H-Erich Wichmann and Annette Peters, GSF Institute, Germany Co-investigators: Angela Ibald-Mulli, GSF Institute, Germany Regina Rückerl, GSF Institute, Germany Gabriele Wölke, GSF Institute, Germany Josef Cyrys, GSF Institute, Germany Joachim Heinrich, GSF Institute, Germany Wolfgang Kreyling, GSF Institute, Germany Wolfgang Koenig, University of Ulm, Germany Research Core 3: Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects Principal Investigators: Mark W. Frampton, University of Rochester Mark J. Utell (co-p.i.), University of Rochester Co-Investigators: William Beckett, University of Rochester Günter Oberdörster, University of Rochester Paul Morrow, University of Rochester, Emeritus Wojciech Zareba, University of Rochester Christopher Cox, NIH Anthony Pietropaoli, University of Rochester iv

5 Research Core 4: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events Principle Investigator: Günter Oberdörster, University of Rochester Co-Investigators: Alison Elder, University of Rochester Jacob Finkelstein, University of Rochester Robert Gelein, University of Rochester Jean Phillippe Couderc, University of Rochester Wojciech Zareba, University of Rochester Christopher Cox, NIH Mark Frampton, University of Rochester Mark Utell, University of Rochester Wolfgang Kreyling, GSF Institute, Germany Paul Morrow, University of Rochester (Emeritus) Zachary Sharp, University of New Mexico Research Core 5: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression Principle Investigator: Jacob Finkelstein, University of Rochester Co-Investigators: Richard Phipps, University of Rochester Michael O Reilly, University of Rochester Günter Oberdörster, University of Rochester R. Gelein, (Particle Generation Core), University of Rochester ***** FACILITY CORES Particle Generation/Analytical Robert Gelein, University of Rochester Biostatistics: David Oakes, University of Rochester Immunology: Richard Phipps, University of Rochester Vascular: Victor Marder, University of California/Los Angeles Cardiac: Wojciech Zareba and Jean-Phillippe Couderc, University of Rochester v

6 RESEARCH CORE 1: Characterization of the Chemical Composition of Atmospheric Ultrafine Particles Principal Investigator: Philip K. Hopke Co-Investigators: Kimberly Prather the late Glen Cass Ann Dillner Objectives: The objectives of this core are to provide improved understanding of the chemical and physical nature of the ultrafine ambient aerosol. There is relatively little data available that provides distinct information on particles in the size range <1 nm. Because of the relatively small amount of particle mass in this size range, sampling and chemical analysis is difficult. However, such physical and chemical data provide critical information to the epidemiological and toxicological studies to help guide their studies of the relationships of the ultrafine particles and adverse health effects. Initially the focus of this core has been on the development of effective methods to sample and analyze ultrafine particles. Now, these methods are being applied to characterize the ultrafine aerosol in a number of locations across the country to assess the variations that exist in the nature of the ultrafine particles. Summary of Progress to Date (Years 1-3) In the early stage of this project, the Cass/Dillner group collected ultrafine particle samples in field experiments in a south central U.S. city (Houston, TX) and in a west coast city (Riverside, CA) and automated equipment that measures ultrafine aerosol size distributions. The Prather group completed the development of an improved aerosol time of flight mass spectrometry instrument to measure the chemical composition of single atmospheric particles smaller than 1 nm in particle diameter. An ultrafine particle aerosol time of flight mass spectrometry instrument has been constructed incorporating an aerodynamic lens system, which allows transmission of ultrafine particles into the instrument. An effective method for detecting ultrafine particles in the systems is being developed. Year 4 Activities: Instrumentation Development and Characterization An improved ATOMFS equipped with an aerodynamic lens inlet and an enhanced light scattering system was developed to provide higher detection efficiencies for fine and ultrafine particles. The particle sizing efficiency (product of particle transmission efficiency and particle scattering efficiency) was determined to be ~.5% for 95 nm PSL particles and ~47% for 29 nm PSL particles, while the particle detection efficiency (product of particle sizing efficiency and particle hit rate) was measured to be ~.3% for 95 nm PSL particles and 44% for 29 nm PSL particles, respectively. In addition, the beam profiles for PSL particles of various sizes were measured in the ion source of the ATOFMS and follow a Gaussian distribution with a full width at half maximum (fwhm) of ~.35 mm. A publication describing the optimization and characterization of the improved UF-ATOFMS instrument has been submitted for publication in Analytical Chemistry. Results from ambient sampling in San Diego, CA are detailed. In this 1

7 progress report, the capabilities of this improved ATOFMS for characterizing single fine and ultrafine ambient particles represent ambient measurements made in Rochester, NY and Atlanta, GA. Under typical ambient sampling conditions with particle number concentrations between ~1 and 1 particles/ cm 3, ~3, particles with aerodynamic diameter of 5-3 nm were detected with 24 hour hit rates varying between 1%-35%. Benefiting from higher sizing and detection efficiencies, this ATOFMS instrument is capable of detecting single fine and ultrafine particles with 3-6 minute temporal resolution even at low number concentrations of < 1 particles/cm 3. This advancement, allowing the rapid determination of particle composition at smaller sizes with higher efficiency, opens new research opportunities for ultrafine analysis in a number of areas including environmental and material sciences, health effects studies, industrial hygiene, and national security. Ambient Ultrafine Aerosols and Homogeneous Nucleation Beginning at the end of November 21, the number concentrations of ultrafine particles have been measured at the NYS Department of Environmental Conservation (DEC) monitoring site on the central fire station in downtown Rochester, NY. Particle size distributions are being measured using a Scanning Mobility Particle Sizer (SMPS) comprising of a differential mobility analyzer (DMA) and a condensation particle counter (CPC). In the diameter range of 1 to 5 nm, ambient particles are classified by a DMA (TSI 371) and counted with a CPC (TSI 31) every five minutes. This work was originally supported by the New York State Energy Research and Development Authority, but at the end of that support, we have continued this work with Center support. We have 1.5 years of data providing information on the number distributions of particles between 1 and 5 nm. In addition, the DEC site monitors SO 2, CO, PM 2.5, and meteorological variables. Summarizing the results from the past year of measurements, more than 7% of total number concentration was associated with particles in the size range 11 to 5 nm, and 2% was associated with particles 5 to 1 nm. The comparisons of number concentrations of ultrafine particles in three size ranges between weekdays and weekends during the study period are shown in Table 1-1. The differences of ultrafine particles in the size range 11 to 5 nm between weekdays and weekends clearly suggest that the sources of ultrafine particles are strongly associated with human activities, such as the traffic hour, whereas there was no relationship between fine particle in the size range1 to 47 nm and human activities. Figure 1-1 shows the monthly variations of the number concentrations of particles and ambient temperature. It suggests that the mean concentrations were inversely proportional to ambient temperature and ambient temperature can be act as a critical factor which affects the dispersion and formation of fine particles. Figure 1-1. Monthly variations of fine particle number concentrations measured in Rochester, NY. 2

8 The total number concentrations during the winter, December to February, tended to be higher than those during the summer. This difference is probably due to the expansion of the mixing heights during hot summer days and dilution of ultrafine particles. Diurnal variations of number concentrations of particles during winter and summer days are shown in Figure 1-2 and Figure 1-3, respectively. On average, there are two peaks in particle number concentrations in the size range 11 to 5 nm. The first peak occurred around 9 a.m., and the second peak appeared around 3 p.m. as shown in the results for the measurement period of December 22. However, during summer days as shown in Figure 1-2, the first peak dramatically diminished even though the second peak remained. It is probably due to the additional nucleation that can occur as hot exhaust gases mix with colder ambient air during the winter months. Thus, although the first peak was strongly related with morning rush-hours, the nucleation was much less prominent in the mornings during the summer months. The number concentrations measured from 7 a.m. to 1 a.m. during weekdays were compared with ambient temperature (Figure 1-4). The number concentrations in the morning tended to increase as the ambient temperature decreased. In addition, the second peaks of particles in the size range 5 to 1 nm tended to occur after the second peak of ultrafine particles in the size range 11 to 5 nm. It might be suggested that the formation of fine particles is associated with the coagulation of ultrafine particles after the evening rush-hour. The hourly variations of the relatively large particles in the size range 1 to 47 nm were negligible indicating a more regional source. Table 1-1. Average number concentrations of ultrafine particles in the three size ranges measured from December 21 to December 22 in Rochester, NY. 11~5 nm 5~1 nm 1~47 nm Weekday Mean Std. Dev Saturday Mean Std. Dev Sunday Mean Std. Dev

9 Figure 1-2. Diurnal variations of particle number concentrations measured during the winter of 22 in Rochester, NY. One of the surprising findings in recent studies of the urban aerosol is the discovery of relatively frequent homogeneous nucleation events in urban areas. For many years, it was believed that there was too much surface area in the existing aerosol to permit the formation of new particles in the urban atmosphere by vaporliquid nucleation. Figure 1-5 shows a typical pattern for the 24 hour period on February 18, 22. In these plots, the particle size as measured by a scanning mobility particle sizer (SMPS) is plotted on the vertical axis while time-of-day is plotted on the horizontal axis. The color indicates the observed concentrations for each size interval for each time interval. Temporal patterns can be seen in the plot. For example the morning rush hour traffic can be seen as a concentration peak at about 9 AM. This peak tends to correlate well with the CO concentration measured at the same location. There are several small concentration peaks during the morning into the early afternoon. Then, evening traffic can be observed between 6 and 9 PM. Figure 1-4. Correlation between particle number concentration and temperature. Figure 1-5. Plot of the particle size distributions measured on February 18, 22 in downtown Rochester. Scale is in units of particles/cm -3. Figure 1-3. Diurnal variations of particle number concentrations measured during the summer of 22. Temperature (ºC) 4

10 A second type of pattern of particle size distributions is shown in Figure 1-6, which presents a nucleation and growth event. Following the morning rush hour peak, there are a large number of very small (~1 nm) particles appearing just after noon in this figure. These particles grow rapidly through the early afternoon into particles in the 5 to 75 nm size range. The evening rush hour traffic shows some additional peaks in the late afternoon. Thus, the shift toward early afternoon in the prior figure is the result of the onset on more frequent nucleation events during the summer when photochemical processes are more intense. In addition to these nucleation events where there are sufficient particles to observe subsequent growth, there can be nucleation events without growth as shown in Figure 1-7. Thus, there are two distinct forms of nucleation events that have been seen to occur in Rochester. Important questions then are what is nucleating and what is the source of this material. In order to examine this question, the time variation of the other measured variables can be compared. Figure 1-6. Time evolution of the particle size distributions in Rochester, NY showing a nucleation with growth event. Scale is in units of particles/cm -3. Figure 1-7. Time evolution of the particle size distribution in Rochester, NY on a day with a nucleation event for which there is not significant subsequent growth. Scale is in units of particles/cm -3. In the case of the second type of event (Figure 1-7), there is a strong correlation between the occurrence of the events and the SO 2 concentration that is also measured at the same site. There is also a strong relationship with wind direction. These events appear to be the result of the plume from a local coal-fired power plant on the northwestern side of the city. However, for the nucleation with growth events, it appears that these events are more widely spatially distributed based on results from Pittsburgh, Florence and Philadelphia, PA in July 21. The nature of the nucleation and growth processes are not well understood in terms of the species that are nucleating and the source of the substantial amount of condensable mass needed to support the observed growth. These nucleation and growth events produce particles over a wide geographical area in sizes that could be a source of adverse health effects. These data are similar to those that have been previously accumulated in Erfurt, Germany by Core 2 researchers. We anticipate developing a comparable data set that will permit a 5

11 % % collaborative project to be developed examining the role of ultrafine particles in causing adverse health effects in Rochester. Particle Characterization Measurements Peroxide Measurements During this year, we have developed the methodology for determining the total peroxide content of aerosol samples using the method described by Hung and Wang (21). A fluorimeter has been procured and initial laboratory experiments have shown that we can make these measurements. Initial training for the graduate student in electron microscopy has begun, but we do not yet have any results to report. Field Studies in Rochester, NY Bulk ultrafine aerosol composition A coordinated measurement program was mounted at the University of Rochester Medical Center to characterize the composition of ultrafine and fine particles. The sampling systems included particle size distribution measurements using an SMPS, several Micro-Orifice Uniform Deposit Impactors (MOUDI) including nanomoudis to extend the measurement range to 1 nm. The ATOFMS with the aerodynamic lens was brought to Rochester. In addition, several semi-continuous measurements of PM 2.5 mass and composition were made PM.1 PM PM.1 PM Jun 12-Jun 14-Jun 16-Jun 1-Jun 12-Jun 14-Jun 16-Jun Figure 1-8. Percentage of trace elements in fine and ultrafine particles. Figure 1-9. Percentage of EC in fine and ultrafine particles. 6

12 Size segregated, speciated ambient and concentrated aerosol data was obtained in Rochester, NY during June of 22. On each day from June 4 to 8, one 6 hour sample of concentrated ultrafine aerosol was collected in three size bins (1-56 nm, 56-1nm and 1-18 nm) along with ambient and concentrated number size distributions. From June 11 to 17, four 48-hour ambient MOUDI samples (six size bins with sizes :m) and PM1.8 samples were collected along with number size distribution data. In addition, one 8 day ambient sample for ultrafines with three size bins (1-56 nm, 56-1nm and 1-18 nm) were collected during this time. All substrates and filters were analyzed for OC/EC, inorganic ions and trace elements. The 48-hour ambient PM1.8 concentrations ranged from 17 to 32 µg/m 3 with over half of the mass composed of organic compounds with an additional significant contribution from sulfate. A comparison of fine (PM1.8) and ultrafine (PM.1) was conducted to discern the relative potential toxicity of fine and ultrafine aerosol. Although the concentration of EC in the ultrafine (.32 ±.11 µg/m 3 ) is much less than the concentration of fine EC (.63 ±.35 µg/m 3 ), the percentage to total mass is much higher for ultrafine than for the fine aerosol (Figure 1-8 ). A similar patterns holds true for trace metals (Figure 1-9) indicating that potentially toxic materials, trace metals and EC, are found in greater abundance in the ultrafine than in the fine aerosol. 1% 8% 6% 4% 2% % Pb 75As 66Zn 63Cu 56Fe 55Mn 52Cr 51V 47Ti Figure 1-1. Percentage of trace metal species as a function of size. ICP-MS quantified 29 elements in the ambient samples. The composition of the trace metals varies by particle size. Figure 1-1 show the first row of transition elements plus arsenic and lead (potentially highly toxic material). The figure shows that both the smallest (.56.1 µm) and the largest ( µm) particles sizes have similar percentages of iron and copper. Ultrafine aerosol has a higher percentage of chromium and arsenic while the µm particles have a higher percentage of zinc and titanium. Studies of trace metals in ultrafine aerosols in two other cities, Los Angeles, CA and Houston, TX show that iron is one of the most abundant trace metals in urban areas. Table 1-2 lists the trace metals measured in three urban areas in order of their abundance. These results suggest that iron is commonly observed in the ambient ultrafine aerosol and thus, it is useful to examine its health effects in the toxicological studies being conducted in Core 4. 7

13 Table 1-2. Relative abundance of elements measured in urban areas in the U.S. as part of the Rochester PM Center. Rochester, NY Los Angeles, CA Houston, TX Fe Fe Ni Cu Ti Fe Cr Cr V V Zn Cu Single Ultrafine Particle Characterization The size and chemical composition of individual concentrated and non-concentrated ambient fine (1-3 nm) and ultrafine (<1 nm) particles were obtained in Rochester, NY during June 22. During a 17-hour sampling experiment on June 6 and June 7, 14,166 particles with an aerodynamic diameter of lower than 3 nm, including 2,964 ultrafine (<1 nm) particles, were sized and chemically analyzed. After the concentrator experiment, the ATOFMS was used to sample ambient particles directly (non-concentrated). In 8-days of continuous ambient measurements from midnight June 1 to midnight June 18, 2,296 non-concentrated particles with aerodynamic diameters < 3 nm, including 1,489 ultrafine particles (Da < 1 nm), were sized and chemically analyzed. The improved ATOFMS was located in the annex of the University of Rochester medical center, and used to directly sample ambient fine and ultrafine particles about 2.5 m from Elmwood Avenue, a moderate traffic road located ~ 3 miles away from downtown Rochester. EC rich type particles are dominant in the ultrafine (< 1 nm) range while OC rich particles are the most significant in the fine (1-3 nm) mode. The major source of the EC and OC particles is vehicular emissions from cars during this study. The temporal variations of the major particle types observed in Rochester and apportionment to sources is currently being conducted. Continuous or semi-continuous concentrations of PM 2.5, organic carbon (OC), elemental carbon (EC), black carbon (BC), and sulfate were measured. Quartz filter samples were analyzed for filter-based OC/EC and sulfate. Two-hour integrated and twenty four-hour integrated OC and EC were measured using a thermal-optical transmittance (TOT) methods. Sulfate was determined using a high-time resolution sulfate analyzer and ion chromatography(ic). BC was analyzed using a two-wavelength Aethalometer. Semi-continuous OC and continuous sulfate accounted for more than 57% and 2%, respectively, to the PM 2.5 mass with a mean of 14.9 µg m -3. Intercomparison of the continuous sulfate analysis with the filter-based IC analysis showed very good correlation between the two measurements. Good agreement was found between the reconstructed chemical composition mass based on the filter samples and the real-time PM 2.5 mass. Field Studies in Atlanta, GA Three modified Nano-MOUDIs, a PM1.8 sampler and SMPS were used during a ten day period at the Atlanta Supersite in parallel with an ultrafine ATOFMS (Kim Prather). The modified Nano-MOUDIs were used to collect two five-day samples of fine and ultrafine aerosol. The two Nano-MOUDI sampling events were midnight to midnight August 6-1 and August 11-8

14 15. The PM1.8 sampler was used to collect PM1.8 filters every 24 hours period during the ten day sampling event. Filters and substrates were analyzed for inorganic ions, EC/OC and trace elements. The SMPS ran continuously to obtain number size distribution data. a) b) Figure Unscaled ATOFMS particle counts (a) ultrafine (b) fine in Atlanta. For the Atlanta study, 212,16 particles in the fine size mode (particles with aerodynamic diameters between 1 and 3 nm) and 21,859 particles in the ultrafine size mode (particles with aerodynamic diameters between 5 and 1 nm) were analyzed. The ATOFMS sampled approximately 5-2 particles per hour at sizes below 3 nm. The temporal profiles of the major particles types are displayed in Figure 1-11 with high temporal resolution (1 minutes). The ultrafine particles (5-1 nm) observed consisted of elemental carbon (EC), organic carbon (OC), and a mixture of OC and EC (EC/OC) as a result of vehicular, industrial, and biogenic emissions which a high correlation to relative humidity. EC was found to be dominant in the ultrafine mode, whereas OC (most likely coated on EC particle cores) was dominant for particles between 1 nm and 3 nm. Representative mass spectra of the major particle types are shown in Figure The presence of a greater fraction of particles containing OC compared to EC with increasing size is shown in Figure These ultrafine particles grow into the fine mode through agglomeration and/or condensation of organic species on the particles and contribute to the fine mode where particles with organic species (masking smaller EC cores in some cases most likely) dominate the particles observed by the ATOFMS. 9

15 Figure Typical mass spectra of major particle types observed in Atlanta, GA. Upper left, elemental carbon; upper right, organic carbon; lower left, K-rich elemental carbon coated with organic carbon; lower right, elemental carbon coated with organic carbon.2 A large fraction of aged particles as indicated by nitrates and sulfates markers was previously observed for fine particulate matter in an intensive Atlanta Supersite study in 1999 due to the high summer concentrations of SO 2 and NO x. The temporal variations and size distribution of similar transformed particles detected by the ATOFMS during this study is shown in Figure

16 Raw Counts (a) EC ECOC Aerodynamic Diameter (nm) (b) Figure EC vs. OCEC. (a) EC and OCEC size profiles. (b) Number of OC coated EC particles to pure EC particles Figure Ammonium, nitrate, and sulfate-containing particles (a) temporal plot (b) size histogram from Atlanta Figure 1-15 shows a comparison in the chemical composition of ultrafine and fine particles observed in Atlanta and Rochester. For ultrafine particles, there is a smaller fraction of elemental carbon particles were detected in Rochester. The greater contribution of EC particle found in Atlanta can be attributed, in part, to the diesel emissions originating from the Greyhound bus maintenance facility located near the sampling site and other nearby diesel traffic. Since the combustion process from diesel engines is less efficient, it is expected that diesel emissions produce a greater fraction of soot particles. Roadside measurements taken in Rochester were mostly coming from spark engine vehicles which could also account for the greater fraction of OC, PAH rich, and amine rich particles in the ultrafine particles. In both cities, OC dominates the fine mode. There is also a greater fraction of vanadium rich particles in Rochester observed in the fine mode. Transition 11

17 metals such as vanadium and iron were observed in association with organic particles as shown in Figure This particle type is indicative of vehicular traffic. In Atlanta, throughout the entire size range of particles detected, there was a greater fraction of potassium-rich particles. The source of these particles is unknown. Figure Comparison of chemical composition of ultrafine and fine particles in Atlanta and Rochester. Figure Single particle mass spectrum of a vanadium and iron rich organic particle. Concentrator Experiment A prototype Harvard Ultrafine Particle Concentrator, located at the University of Rochester Medical Center, was designed for particle health effects and toxicological studies. An important general question about the functionality of ultrafine particle concentrators relates to their performance with respect to maintaining the particle chemistry during the concentrating process. For example, are particle surface chemistry and the ratio of organic to elemental carbon of UFP altered during the hydration-dehydration process? This will be important to decide about the usefulness of UFP concentrators for controlled clinical and animal studies. After completing the ambient aerosol studies described above, additional measurements were made of the particles entering and exiting the concentrator. During the study, the concentrator was not operating optimally and the exit air stream was at high relative humidity rather than having been returned to typical ambient level. The ambient particles thus experienced saturation, condensation and desolvation/evaporation processes inside the particle concentrator, but may not have been fully restored to their original condition. Since the toxicity of ambient particles is closely related to their size, number and mass concentrations, and chemical composition, the improved ATOFMS and the nano-moudi were used to characterize the concentrated particles. Results from the concentrator experiments were compared with those obtained by direct (non-concentrated) ambient measurement to evaluate whether the animal and human models were exposed to real-world ambient particles and reported in several posters at the 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth 12

18 Colloquium on PM and Human Health. The results of this preliminary study have led us to develop additional studies of the more developed form of the ultrafine concentrator described below. ATOMFS Studies with laboratory-generated UFP: The size and individual particle composition of ultrafine mixed metal particles produced by the PALAS generator were directly analyzed by the ATOFMS. These particles are used as models for exposure studies of ultrafine metal aerosol particles in Core 4 studies. The metals used to form the rods for the generator were composed of iron, manganese, and chromium. A representative single particle mass spectrum of the particles produced 8 is shown below in Figure Fe The chemical composition of the 6 particles produced was very 5 homogeneous. This is shown in 4 Figure 1-18 which shows the 3 Mn relative number fraction of 2 Cr 1 different particle types obtained. From this figure, one can see the 92% of the particles contained almost identical species in the Relative intensity mass/charge Figure 1-17: Positive ion mass spectrum of the most common particle type observed from the metal particles generated from the PALAS generator using a welding rods. same relative abundances, demonstrating the ability of this method to produce homogeneous ultrafine particles. The types are Fe-Mn-Cr: 92.4% Cr-Fe-Mn-Ti: 2.4% Amine-Fe-Mn-Cr: 1.6% K-Fe-Mn-Cr: 1.2% OC-Fe-Mn-Cr-Ti:.9% EC-Fe-Mn-Cr-Ti:.6% K-Na-Fe-Mn-Cr:.5% Amine-Fe-Ti:.4% Figure 1-18: Fractions of particle types produced by PALAS generator. listed with the metals given in order of decreasing ion intensity. The remaining particle types (8%) showed different compositions which could have been due to slight heterogeneous spots in the rod, as well as the presence of minor impurities (amines, organic carbon, potassium). It is possible the amines (and other organics) coated some of the particles and came from the air being used to transport the particles to the ATOFMS system for analysis. This study demonstrates the ability of ATOFMS for assessing the chemical variability of model aerosols used for exposure in health effects studies. It would be impossible to obtain this level of detail on the homogeneity of composition from filter based techniques. For one, filter techniques would require much more sample for the analysis. Using filter based techniques, one has to 13

19 assume an average composition of all particles. For example, certain model aerosol particles in the sample could contain 9% of a toxic substance, whereas another set of particles (i.e. produced towards the end of the experiment) in the same exposure test contained only 1%. The filter based method would conclude each particle was composed of 5% of a toxic substance upon averaging the results of all particle compositions together. Furthermore, the ATOFMS provides the composition in real-time so any changes in composition due to the generation method are immediately detected. The ability to immediately detect changes in particle composition is extremely important for understanding acute health effects. Exposure System for Truck Study Recent studies have shown particle number concentrations over urban roadways are higher than roadside and much higher than downwind. In fact, on-road number concentrations are often higher than provided by concentrator systems. People in passenger cars and trucks are directly exposed to these particles that are present on highways at high concentrations. A pilot type toxicology study was designed to expose laboratory rats directly to freshly-generated highway aerosols almost all ultrafine particles involving PM Center investigators of Research Cores 1, 3, 4 and 5. For this purpose, an on-road exposure system was developed in which laboratory animals were transported on active roadways in order to provide exposures to fresh ultrafine aerosols. The University of Minnesota\'s Mobile Emissions Laboratory has been used extensively to make on-road emission measurements. It was adapted for use as both an emission laboratory and exposure system. An inlet brings the on-road aerosol from in front of the truck into the laboratory in the cargo container in the truck bed where measurements and animal exposures are performed. In these experiments, some of the equipment was removed to make room for the animal exposure system in the air conditioned compartment of the truck. An air delivery system was built to provide exposures for 3 groups of animals: whole highway aerosol; filtered air with the gaseous pollutants present; and clean air without particles, CO, VOCs, or NOx. The cages held 1 animals each so thirty animals could be used for a given experiment. As described in the Research Core 4 progress report, old rats were used, with and without preexposure to endotoxin or human influenza virus, as well as spontaneously hypertensive rats, with telemetric EKG and blood pressure implants. The truck was then driven on the NY Thruway between Rochester and Buffalo for 6 hours/day over a period of 1 days under varying conditions to provide a range of exposures to the animals. The results of this experiment are described in the Core 4 report later in this document. QA/QC The SOPs and QMP have been completed and submitted. FUTURE PLANS For the ATOFMS data, the mass spectral signatures will be compared to ion markers identified in ATOFMS source characterization studies to determine the particle emission sources in the ambient studies. In order to have representative number mass concentrations, the data will be scaled to number concentrations obtained by a scanning mobility particle sizer and to mass concentrations obtained by MOUDI measurements taken along side in Atlanta by Arizona State University. These results will be developed into publications based on the Rochester and Atlanta field measurements. Further data analysis on these two data sets from the east Coast and 14

20 comparison to the results from west Coast field measurements will provide signatures of fine and ultrafine particles on the east and west coasts of the United States. Measurements of peroxide concentrations will be made and samples will be collected for morphometric examination in Rubidoux, CA during July 23 in conjunction with experiments being conducted by Prof. Delbert Eatough of BYU. We also anticipate making measurements in January 24 in Philadelphia and/or New York City in conjunction with winter intensive studies being conducted in those locations. We will be conducting studies of the current generator of Harvard Ultrafine Concentrator during August 23. Given our initial results suggesting that there were observable changes in the aerosol characteristics between the inlet and the outlet of the concentrator, it is important to determine exactly what is happening in the later designs of these systems. There is substantial use of these concentrators to provide exposure of animals and humans to concentrated ultrafine airborne particles and it is necessary to fully understand the nature of that exposure and its relationship to actual ambient aerosol exposures. Experiments will be conducted at Harvard School of Public Health using both the improved ATOFMS, semicontinuous instruments and a nano-moudi. These systems will be used to collect and characterize the aerosol composition at the inlet and exit of the concentrators. The semicontinuous instruments will include a Sunset field OC/EC analyzer, a two-wavelength aethalometer, and a sulfate analyzer. The nano- MOUDI will use collection media that will permit the analysis of OC and EC. Subsequently in conjunction with Dr. Robert Devlin of NHEERL/EPA, we will examine the concentrator that EPA has in its human exposure chamber at the University of North Carolina. At the present time, we are trying to arrange additional tests of ultrafine concentrators to be able to better characterize the exposure being provided to a variety of animal and human subjects. We have begun increased collaboration with the Epidemiological Core (Core 2). In May, Prof. Hopke visited Prof. Wichmann at the GSF in Munich and during the first week of June, Matthias Stotzel of the GSF visited Prof. Hopke s lab at Clarkson to learn the use of positive matrix factorization for particle source apportionment. During the coming year, we will be looking to expand this collaboration to examine the use of apportioned source contributions in epidemiological studies. PUBLICATIONS AND PRESENTATIONS Publications Source Identification of PM2.5 in an Arid Northwest U.S. City by Positive Matrix Factorization, E. Kim, T.V. Larson, P.K. Hopke, C. Slaughter, L.E. Sheppard, C. Claiborne, Atmospheric Res. (In press, 23). Analysis of Ambient Particle Size Distributions using UNMIX and Positive Matrix Factorization, E. Kim, P.K. Hopke, T.V. Larson, and D.S. Covert, under revision to respond to reviews by Environ. Sci. Technol. (June 23). Measurement of Real-Time PM 2.5 Mass, Sulfate, and Carbonaceous Aerosols at Multiple Monitoring Sites, C.-H. Jeong, D.-W. Lee, E. Kim, and P.K. Hopke, submitted to Atmospheric Environ., June 23. Development and characterization of an ATOFMS with improved detection efficiency for individual fine and ultrafine particle analysis, Su, Y.X.; Sipin, M. F.; Furutani, H.; Prather, K.A., Submitted to Analytical Chemistry, June

21 Presentations Long-Term Measurement of Ultrafine Particle Number Concentration in Rochester, NY, C.-H. Jeong, P.K. Hopke, M. Utell, D. Chalupa, and H.D. Felton, presented to 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. Effects of Gaseous Pollutants and Meteorological Parameters On Nucleation and Growth of Ultrafine Particles in Urban Ambient Air, C.-H. Jeong, P.K.. Hopke, D. Chalupa, and H.D. Felton, presented to 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. Characterization of Chemical Compositions in Fine Particulate Matter During the Rochester Particulate Matter Study, C.-H. Jeong, D.-W. Lee, E. Kim, P.K.. Hopke, R. Gelein, P.K.presented to 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. On-Road Exposure to Highway Aerosols: 1. Particle Chemistry and Rat Exposure System, P.K.. Hopke, C.-H. Jeong, W. Liu, W. Zhao, L. Zhuo, D. Kittleson, W. Watts, R. Gelein, and G. Oberdoerster, presented to 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. On-Road Exposure to Highway Aerosols: 2. On-road Aerosol and Gas Mesurements, D.B Kittelson, W.F Watts, J.P Johnson, G. Oberdorster, R. Gelein, and P.K Hopke, presented to 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. On-Road Exposure to Highway Aerosols. 3. Exposures of Aged and Compromised Rats, A. Elder, R. Gelein, J. Finkelstein, C. Johnston, R. Phipps, M. Frampton, M. Utell, D. Kittelson, W. Watts, P. Hopke, and G. Oberdörster, presented to 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. The Comparison Between Thermal Optical Transmittance Elemental Carbon and Aethalometer Black Carbon Measured at Multiple Monitoring Sites, E. Kim, C.-H. Jeong, D.-W. Lee, and P.K. Hopke, presented to 23 AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. Speciated Size Distributions Using MOUDIs, A.M. Dillner, Invited Presentation at AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. Chemical characterization of fine and ultrafine aerosols during the Rochester, NY summer intensive, 22. A.M. Dillner, X. Su, X., J.J. Schauer, M. Zheng, and the late G.R. Cass, presentation at AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4,

22 Characterization of concentrated ultrafine aerosols, A.M. Dillner, X. Su, J.J. Schauer, M. Zheng, R. Gelein, P. Koutrakis, the late G.R. Cass, presentation at AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. Single particle chemical speciation of ambient ultrafine particulate matter in Atlanta, GA, Sipin, M.F., Su, Y.X.; Prather, K.A., Presentation at American Geophysical Union 22 Fall Meeting, San Francisco, CA, December 6-1, 22 Characterization of single fine and ultrafine particles in Rochester, New York using aerodynamic-lens laser desorption/ionization time-of-flight mass spectrometry, Su, Y.X.; Sipin, M.F.; Furutani, H.; Prather, K.A.; Gelein, R.M.; Oberdorster, G. Poster presentation at American Geophysical Union 22 Fall Meeting, San Francisco, CA, December 6-1, 22 ATOFMS Characterization of ambient fine and ultrafine particles from a versatile aerosol concentration enrichment system. Su, Y.X.; Sipin, M.F.; Prather, K.A.; Gelein, R.M.; Oberdorster, G., Poster presentation at AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4, 23. Size and chemical characterization of urban ultrafine and fine particulate matter in the eastern United States, presentation at AAAR PM Meeting on Particulate Matter: Atmospheric Sciences, Exposure and the Fourth Colloquium on PM and Human Health, Pittsburgh, PA, March 3 to April 4,

23 RESEARCH CORE 2: Inflammatory responses and cardiovascular risk factors in elderly subjects with cardiopulmonary disease in association with fine and ultrafine particles Principal Investigators: H.-Erich Wichmann and Annette Peters Co-Investigators: Angela Ibald-Mulli, Regina Rückerl, Gabriele Wölke, Josef Cyrys, Joachim Heinrich, Wolfgang Kreyling in collaboration with Wolfgang Koenig, University of Ulm, Germany Objective: The objective of the study is to characterize the association between ambient particle exposures and changes in biomarkers of inflammation in the airways and the blood of patients with stable coronary artery disease (CAD) as well as of patients with chronic obstructive pulmonary disease (COPD). Monitoring of the autonomic function of the heart will investigate how these changes in the inflammatory state relate to alterations in the autonomic control. In order to identify the mechanisms that lead from the deposition of particles in the lung to cardiovascular disease exacerbation, it is crucial to also show that the proposed mechanism is at play in diseased patients The analyses will address the specific questions with respect to the function of the heart: (1) Do fine and ultrafine particles affect heart rate variability? (2) Are signs of QTc reduction observed as in the human exposure studies of the Rochester Particle Center? (3) Are ventricular ectopic beats and arrhythmia more prevalent on days with high concentrations of fine and ultrafine particles? (4) Are signs of ischemia present in 24-EKG readings during exercise periods in association with fine and ultrafine particles? Furthermore, blood biomarker will be used to ask the following specific questions: (5) Do acute phase reactants increase in association with fine and ultrafine particles? (6) Do clotting factors also increase in association with fine and ultrafine particles? (7) Do blood cell counts change in association with fine and ultrafine particles? The first time-series study on mortality considering ultrafine particles in addition to particles showed relative risk estimates of comparable sizes in association with cumulated particle concentrations over the last four days (Wichmann et al. 2). In addition, the data suggests that respiratory mortality increased immediately after elevated concentrations of particles while cardiovascular disease mortality showed a delay up to four days. Therefore, we will investigate the lag-structure carefully in all analyses. Summary of Progress Year 1 to 3: Two panel studies were conducted to assess the health effects of fine and ultrafine particles on patients with cardiopulmonary disease. A panel of CAD patients was studied during the winter 2/1 and a second panel of COPD patients was recruited for the winter 21/2. The study protocol and outcome measures were designed to be as similar as possible to that of the clinical studies (Core 3). Blood biomarkers are analysed by the vascular core under the lead of Victor Marder and ECG recordings are analysed by the cardiac core lead by Wojciech Zareba. For the CAD patients it comprised a maximum of 12 bimonthly clinical 18

24 examinations with an interview, resting ECG, blood pressure measurement, urine sample and blood sample. Further, 6 monthly 24-hour holter recordings and daily blood pressure measurements for a period of 1 month were taken. Throughout the whole study period subjects were recording symptoms and medication use in a diary. For the COPD patients additional baseline characterisation of the health status by bodyplethysmogaphy and spirometry before and after bronchodilator use was added to the initial baseline examination. Further pulseoximetry and an exercise challenge during the ECG recording as well as lung function testing by Impulse-Oscillation-Spirometry were added to the clinical protocol. The selection criteria were based on the recommendations of the scientific advisory board in collaboration with researchers from Rochester. All subjects gave informed consent to the study. The study protocol was based on written SOPs. It was reviewed and approved by the Bayerische Ärztekammer which is the responsible review board and which was accepted by the Rochester RSRB. The contracted field staff was trained before each field period. A pilot study was conducted before main phase of the CAD and COPD study to test the feasibility of the protocols and assess the training of the field nurses. The quality was carefully monitored throughout the study period by the quality assurance officer. Data was double entered and plausibility checks were preformed. Table 2-1. Summary statistics of the panel studies CAD-Panel COPD-Panel Data base Study period 1/ 4/1 1/1 4/2 Participants (n) Clinical Examinations (n) 683 (98%) 46 (98%) 2 min-ecg (n) 678 (97%) 46* (98%) 24-h ECG (n) 279 (8%) 181 (77%) Blood samples (n) 659 (95%) 439 (94%) Lung function tests (n) not in the protocol 459 (98%) Patient characteristics and disease history Age range (yrs) History of (n) Angina pectoris Myocardial infarction By-pass surgery/ballon dilatation Chronic bronchitis COPD Asthma Emphysema Hay fever Current Smoker (n) Non-Smoker (n) BMI Range (kg/m 2 ) Mean (kg/m 2 ) percent of scheduled examinations * 89 had a 2-min ECG without exercise challenge # percent of participants 38 (66%)# 43 (74%) 5 (86%) 2 (3%) 4 (7%) 1 (2%) 2 (3%) - 58 (1%) (38%) 8 (21%) 4 (1%) 29 (74%) 39 (1%) 21 (54%) 11 (28%) 7 (18%) 32 (82%)

25 Table 2-1 shows a summary statistic of the panel studies on cardiac patients and patients with COPD. 58 CAD patients were recruited who performed 98% of the scheduled clinic visits. Similarly, 98% of the scheduled clinic visits were during the second study with COPD patients. Compliance with 24 ECG recordings and exercise tests were lower (around 8%. The CAD patients had little evidence for COPD, but nearly half of the COPD patients also had CAD. We were able to recruit only non-smoking CAD patients, but decided to include 18% smokers into our COPD panel. The amount of cigarettes smoked were recorded both in the protocol at the clinic visit and in the diary. CAD Panel Study For the CAD panel all questionnaire based data was entered and has been checked for plausibility. The potential role of ECG parameters in air pollution epidemiology was established (Zareba, Couderc, and Nomura, 21) and based on these concepts ECGs obtained in Erfurt during the first field phase were analysed by the cardiac core. The ECGdata have been transferred and were to be analysed in association with the air pollution data. Analyses of the blood parameters by the vascular core were still ongoing because shipment of the blood samples from Germany was postponed after the September 11 th terrorist attack. The same blood parameters and EKG parameters will be determined by the same core facilities for both the epidemiological studies and the human exposure studies. Therefore, the results will be directly comparable to those of the human exposure studies. Particle Measurements The field study with the CAD patients was conducted during the winter 2/1. Concentrations during the field phase are given in Table 2-2. Unfortunately, no aerosol spectrometer data are available between January 2th and February 13th due to a technical defect of the measuring device. Data will be imputed by calculating the ultrafine particle fraction based on total number counts measured with the Condensation Particle Counter (CPC) during the same period. The ultrafine particle number concentrations are dominated by the very fine particles with diameters between 1 and 3 nm. The correlation coefficient between UFP (= NC.1-.1 ) and NC.1-.3 was.96. Ultrafine particles are correlated with accumulation mode particles (NC.1-1. ) with a correlation coefficient of.67. However, the two different fractions of the ultrafine particles do show different correlation coefficients with the accumulation mode particles (.9 for NC.3-.1 and.47 with NC.1-.3 ). The variation in exposure to particle number concentrations seems to be sufficient for epidemiological data analyses. Table 2-2: Distribution of the measured 24 hour average particle number concentrations and PM 2.5 during winter 2/21 when the CAD panel study was conducted. The indices give the size range of the particles counted in µm. N % Mean Median 95% Max NC.1-.1 [cm -3 ] 1) NC.1-.3 [cm -3 ] 1) NC.3-.1 [cm -3 ] 1) NC.1-1. [cm -3 ] 1) Total number concentrations [cm -3 ] 2) PM 2.5 [µg/m 3 ] 3) ) MAS data, UFP = NC.1-.1 [cm -3 ] 2) CPC data 3) Harvard impactor data 2

26 Preliminary Results Preliminary analyses of the blood cell counts and 24 hour averages of particle mass and number counts were conducted. Results indicate a decrease in red blood cells in association with fine particle mass (PM 2.5 ) and ultrafine particle counts. Similar effects of particulate matter on red blood cell counts have been observed by Seaton and colleagues (1999). All estimates are given for 1 µg/m 3 PM 2.5 or 1, ultrafine particles per cubic centimetre of ambient air. The decrease in red blood cells was strongest for the ultrafine particles at lag 4. The hematocrit showed results consistent with those observed for the red blood cells. However, the results were not statistically significant. Total white blood cell counts also decreased in association with ambient particle concentrations. For ultrafine particles a significant decrease is observed for lag 1 and lag 4 and fine particle mass shows a significant effect for lag 4 and 5. The lag structure observed in these analyses are partly consistent with those found by Wichmann and colleagues analysing the impact of ambient particle number concentrations on mortality (Wichmann et al. 2). Cardiovascular disease mortality showed the strongest evidence of an association with ultrafine particles with a lag of 4 days (RR: 1.51 (95% CI:.99 to 1.115) while respiratory disease mortality showed an increase in association with ultrafine particles lagged 1 day. However, the relative risk was strongest for lag 1 (RR: (95% CI: 1.55 to 1.264). Platelet counts also decreased in association with ambient particle concentrations. The strongest association was observed with the pollutant concentrations delayed by two days. For an increase of 1, particles a reduction of 4.6 platelets * 1 9 /l was observed (95% CI: to 7.87). The consistency and plausibility of these effects need to be further evaluated. In particular it will be assessed whether the results are in accordance with findings from the analysis of more specific biomarkers such as clotting factors or soluble ICAM-1. COPD Panel Study Recruitment of patients proved to be difficult. Lung specialists and general practitioners were contacted. In addition, news paper advertisements were issued every month during the recruitment period. The field phase of the COPD panel has been completed as of the end of April. Patients compliance with the protocols seemed to be poorer and more quality assurance efforts were needed to achieve the same overall compliance as with the CAD panel. All data is currently entered in the database and checked for plausibility. All ECGs have been shipped to the cardiac core for analysis. Blood samples will be shipped in the near future. The COPD panel study took place between October 21 and April 22. Due to an irreparable defect of the LAS-X from the aerosol spectrometer at the beginning of the field phase for the COPD panel study the aerosol spectrometer was replaced by a differential mobility particle sizer (DMPS), which measures size fractioned particle counts in the size range of 1 to 5 nm. Further ultrafine particle counts from a scanning mobility particle sizer (SMPS, TSI) counting particles in the size range from 3 to 64 nm are available for the whole COPD study period. Total number counts were measured by a Condensation Particle Counter (CPC, TSI) just like in the CAD panel study and can be used to impute the missing DMPS data at the beginning of the study period (October 15 th to November 28 th ). Currently the air pollution data is checked for plausibility and quality controlled. Summary of Progress Year 4: CAD Panel Study Statistical analyses considering objectives 1-3 as well as 5-7 have been conducted. In addition, a pilot study on the use of proteomics in epidemiological data has been preformed in collaboration with Robert Devlin and Lucas Neas at NHEERL, USEPA. Here we report in 21

27 detail on the results addressing objective 1 and 2 and on the pilot study applying proteomics approach. Cardiac rhythm disorders are the leading cause of the hospital admissions for cardiovascular diseases in the United States. More than half of the deaths due to ischemia, myocardial infarction and cardiomyopathies are directly related to cardiac arrhythmias, and these deaths are usually sudden. Myocardial substrate (myocardial damage due to coronary disease, infarction, or cardiomyopathy), the autonomic nervous system (sympathetic activation or/and parasympathetic withdrawal), and myocardial vulnerability (ventricular arrhythmias, repolarization dynamics) are believed to be key factors that contribute to the mechanism of arrhythmogenic conditions and arrhythmic death. Autonomous nervous system Heart rate variability (SDNN, rmssd, HF,LF, HRH) Myocardial Substrate QRS-Complex, QT-time, ST-Segments, Heart rate turbulance Myocardial vulnerability Arrhythmias, T-wave variability, T-Alternans Objective (1): Do fine and ultrafine particles affect heart rate variability? Analysis of heart rate variability The ECG recordings were analyzed with the H-Scribe 12-Lead Digital Holter System at the University of Rochester Medical Center. For the analysis of the periods of spontaneous breathing in supine position, paced breathing in supine position and spontaneous breathing in upright position the first minute of the 6minute recording was discarded to avoid a carry over effect from the previous period. For the 24 hour recordings the entire period was included in the analysis. For the calculation of heart rate variability parameters only normal QRS intervals were used, artifacts and ectopic beats were excluded after the scanning and manual editing of the QRS complexes Based on each set of normal to normal beat intervals the time domain parameters PNN5 (percentage of NN intervals with a difference greater than 5ms), RMSSD (square root of the mean square of successive differences of NN intervals (ms)), SDNN (standard deviation of NN intervals (ms)), and for the 24 hour recordings in addition SDANN (deviation of averaged 5-minute NN intervals (ms)) and SDNNIX (averaged SD from all 5-minute intervals (ms)) were calculated. Frequency domain parameters were calculated based on Power Spectral Density obtained trough Fast Fourier Transformation (FFT) of the NN intervals. The following parameters were calculated: total power (TP), very low frequency power (VLF, <.3 Hz), low frequency power (LF, Hz), high frequency power (HF,.15.4 Hz) and the LF/HF ratio. All parameters were given as normalized units which represents the relative value of each power component in proportion to the total power minus the VLF component. LF, HF and LF/HF and RMSSD were also log-transformed since their distribution was highly skewed. 22

28 According to the recommendation of the task force of the European Society of Cardiology and the North American Society of Pacing and Electrophysiology (Circulation, 1996) out of all time domain parameters given SDNN and RMSSD were used to assess heart rate variability in the 24 hour recordings. In the short term recordings only RMSSD was used in addition to the frequency domain parameters. For the analyses of frequency domain HRV normalized units of LF, HF and the log-transformed LF/HF ratio were used. LF and HF components were only applied to the short term recordings. Statistical Analyses Data were analysed using the statistical package SAS Version 8 (SAS Institute Inc., Cary, NC) and S-Plus 2 Professional Release 1 (Math Soft Inc). A descriptive analysis of the characteristics of the study participants was based on data obtained through the baseline questionnaire. To avoid uncertainties about GAM generalized mixed models were used to model the association between air pollutants and heart rate variability parameters. In order to explore the shape of the association between confounders such as trend and weather variables and the dependent variable non-parametric smooth functions based on locally weighted least squares within the gam function in S-Plus were used. Based on the shapes of the associations appropriate parametric functions of the covariables based on lowest AIC were included as fixed effects in the final linear mixed model and a random effect was used for each subject. Calculations were done using PROC MIXED in SAS. In order to adjust for other time-varying confounders, day of the week was included in the model. Results For the 24hr recordings the results show a significant decrease in overall heart rate variability measured as SDNN (Figure 2-1a) in association with PM 2.5 and a borderline significant decrease in association with accumulation mode particles The effects were strongest for the 5 day cumulative average exposure to particles and exposure -24 hours prior to the recording period. RMSSD was not associated significantly with ambient air pollution in the 24 hour recordings. %change of average SDNN from 24 hr recordings per increase in IQR Pollutant UFP ACP PM2.5 %change of average SDNN from 24 hr recordings per increase in IQR Pollutant UFP ACP PM2.5-1 concurrent -24hrs 25-48hrs 49-72hrs 73-96hrs 5 day average -1 concurrent -24 hrs 25-48hrs 49-72hrs 73-96hrs 5 day average Exposure to average concentrations of ambient air particles concurrent and prior to the 24 hr recording Exposure to average concentrations of ambient air particles concurrent and prior to the 24 hr recording Figure 2-1a: Heart rate variability in association with particulate air pollution in 24 hour ECG-recordings in patients with CAD Figure 2-1b: Heart rate variability in association with particulate air pollution in 24 hour ECG-recordings in patients with MI Particle effects were more pronounced in subjects with past myocardial infarction (Figure 2-1b) Short term recordings (5 min of spontaneous breathing at rest) With respect to the short term recordings effects on heart rate variability were observed for the period of 5 minutes spontaneous breathing at rest. The strongest effects of particles 23

29 were seen in association with a decrease in normalized units of high frequency power which mainly reflects a withdrawal in parasympathetic tone (Figure 2-2). Significant effects were seen in association with average exposure of PM hours prior to the recording. However, ultrafine and accumulation mode particles also point at a decrease in high frequency power. %change of average HF.NN from 5 min spontaneous breathing per increase in IQR Pollutant hrs UFP ACP PM hrs 49-72hrs 73-96hrs 97-12hrs 5 day average Exposure to average concentrations of ambient air particles prior to the recording Figure 2-2: High frequency power heart rate variability in association with particulate air pollution in 5 minutes of spontaneous breathing in patients with CAD Concentrations of elemental and organic carbon could only be associated with heart rate variability for the short term recordings since EC/OC measurements were implemented later during the panel study. Significant effects were seen with exposure to elemental carbon as shown in Figures 2-3a + b in association with high frequency power and RMSSD. %change of average per increase in IQR Elemental Carbon SDNN spontaneous breathing HF.NN spontaneous breathing -24hrs 25-48hrs 5 day average -24hrs 25-48hrs 5 day average Means ratio of RMSSD and LF/HF per interquartile increase in Elemental Carbon 1,4 1,3 1,2 1,1 1,,9,8,7 RMSSD spontaneous breathing LF/HF Ratio spontaneous breathing -24 hrs 25-48hrs 5 day average -24 hrs 25-48hrs 5 day average Time of Exposure to Elemental Carbon prior to recording Figure 2-3a: SDNN and HF.NN in association with elemental carbon in patients with CAD Time of Exposure to Elemental Carbon prior to recording Figure 2-3b: RMSSD and LF/HF Ratio in association with elemental carbon in patients with CAD Although no effects on heart rate variability were seen in the clinical core this analysis is consistent with recent epidemiologic findings on the effects of ambient air pollution on heart rate variability. Overall the previously published findings suggest a decrease in heart rate variability measured as SDNN and RMSSD as well as HF associated with particulate air pollution in healthy elderly subjects. In these studies effects were seen with either ambient or personal exposure to PM 2.5 and rather immediate (within hours or on the same day of 24

30 exposure). Our analysis shows effects not only with the average exposure -24 hours prior to the recording but also a cumulative effect of the 5 day average exposure to particulate air pollution can be seen. Our results support the hypothesis that exposure to particulate air pollution can alter the autonomic function of the heart, a potential risk factor for cardiac morbidity and mortality. Objective (2): Are signs of QTc reduction observed as in the clinical human exposure studies of the Rochester Particle Centre? Analysis of repolarization The purpose of this study was to assess associations between daily variations in particulate air pollution and repolarization ECG parameters representing abnormalities in the myocardial substrate and increased vulnerability of myocardium to arrhythmias. The ECG recordings were analyzed with the research version of the H-Scribe 12-Lead Digital Holter System at the University of Rochester Medical Center. Mortara s automated algorithms provided measurements of QT interval, T wave amplitude and T wave complexity. QT was measured as the longest interval in any lead for each beat, then it was averaged over an 8- beat-segment adjusted for heart rate with Bazett s formula13 and subsequently the mean of these 8-beat averages from a 5-minute period was obtained (QTca). The measurements of corrected QT interval duration (QTc) were also performed manually in lead II. For T wave amplitude, original ECG leads I, II, V1 to V6 were used and the median value from those 8 original leads was taken for each beat and averaged over 5 minutes. T wave complexity was measured in each beat by principal component analysis (PCA) based on all 12 leads and averaged over the 5-minute period. T wave complexity describes global shape of the T wave with the advantage of not having the need for T wave end determination. The first component (eigenvector) accounts for most of the energy in repolarization in a normal T wave, whereas the second indicates a relevant contribution to the inhomogeneous repolarization. The average ratio between the second and the first component expressed in percent represents a measure of complexity and heterogeneity of repolarization and provides an estimate of the fatness of the T wave loop relative to its amplitude. Statistical Analyses Generalized additive models including pollutant and confounder variables were used for linear fixed effects regression with individual intercepts for each patient. Data were analyzed using the statistical package SAS Version 8.2 (SAS Institute Inc.) and S-Plus Version 6. (Math Soft Inc.). To adjust for confounders, model building was done for each ECG variable separately using non-parametric smooth functions based on locally weighted least squares. Model fit was based on the Akaike Information Criterion (AIC). The best non-parametric model was compared to parametric modeling (linear, polynomials of 2 nd or 3 rd order) for each confounder variable. As confounder variables were considered: an indicator variable for each subject, long-term time trend, temperature, relative humidity and barometric pressure each with lag to lag 3 and weekday of the visit. Results T wave complexity, computerized measure of repolarization morphology, increased in association with ACP and PM 2.5 on the same day and with a cumulative five-day average (Table 2-3). Consistently with this finding, the T wave amplitude showed a significant decrease with UFP and ACP and a borderline decrease with PM 2.5 for the same day. An increase in manually measured QTc could be also seen for the same day and the one-day lag for UFP, ACP and PM 2.5, although there was some inconsistency in the response of QTc over next few days. The variability of T wave complexity also showed a significant increase for the 25

31 same day and one-day lag with ACP and a borderline increase with PM 2.5. The effects on the myocardial substrate could clearly be ascribed to PM 2.5 as there were no significant effects for gaseous pollutants (Figure 2-4). In contrast to the results from of the human controlled exposure experiments, an increase in QTc associated with UFP was observed. As the EKG equipment and the procedures for EKG parameter generation were the same in both studies, the diverging results might be due to (a) the subjects studied or (b) the particle exposures considered. In the epidemiological study we investigated the repolarization changes in the patients with coronary artery disease. Therefore, their response might be quite different from the effects observed in healthy or mild asthmatics. Table 2-3. Effects of particulate air pollution on repolarization parameters reflecting myocardial substrate in patients with CAD QTc [ms] T wave complexity [%] T wave amplitude [µv] Variability of T wave complexity [%] Effect (95% CI) effect (95% CI) effect (95% CI) effect (95% CI) UFP lag 1.56 (-2.89;6.).41 (-.43;1.25) (-13.16;-.32).1 (-.12;.32) lag (.2;9.99).37 (-.57;1.3).4 (-7.12;7.19).14 (-.6;.34) lag 2.15 (-5.11;5.42).11 (-.92;1.15).7 (-7.45;7.59) -.4 (-.3;.22) 5 day ave 1.14 (-6.16;8.45).76 (-.64;2.16) (-14.37;6.44).7 (-.29;.43) ACP lag 3.25 (-.33;6.83).71 (.;1.42) (-1.32;-.4).19 (.1;.36) lag (-1.16;7.4).17 (-.66;1.) -.86 (-6.63;4.92).25 (.9;.41) lag (-5.76;2.6).48 (-.36;1.33) -.56 (-6.24;5.12).1 (-.18;.21) 5 day ave (-7.;3.91) 1.2 (.2;2.39) (-1.19;5.6).15 (-.12;.41) PM 2.5 lag 2.95 (-.23;6.12).78 (.14;1.42) (-8.57;.19).15 (-.1;.3) lag 1.17 (-3.44;3.78).2 (-.64;.68) (-5.95;3.58).12 (-.1;.25) lag (-4.57;1.77).37 (-.31;1.5). (-4.75;4.75).5 (-.11;.22) 5 day ave (-7.11;1.78) 1.3 (.5;2.1) (-8.26;4.42).13 (-.8;.35) Confounder-models: all models included the categorical variables patient number and weekday and additionally: QTc: trend loess (span.3), temperature lag3 loess (span.3), relative humidity lag1 polynomial (2 nd order), barometric pressure lag1 linear T wave complexity: trend loess (span.7), temperature lag3 polynomial (2 nd order), relative humidity lag3 loess (span.3), barometric pressure lag polynomial (3 rd order) T wave amplitude: trend polynomial (3 rd order), temperature lag2 linear, relative humidity lag3 polynomial (3 rd order), barometric pressure lag3 linear Var. of T wave complexity: trend loess (span.5), temperature lag3 loess (span.4), relative humidity lag2 polynomial (2 nd order), barometric pressure lag loess (span.5) 26

32 15 Same day effects 5-day-average effects %change of average T wave complexity per increase in IQR pollutant UFP ACP PM 2.5 SO 2 NO 2 CO ambient particle exposure: same day (lag ) and 5-day average Figure 2-4. Same day and 5-day average effects of 24 hour mean particulate and gaseous air pollution on T wave complexity in patients with CAD. Additional Topic: Can proteomics be used in panel study data? The Ciphergen ProteinChip system allows the separation of proteins based on a ProteinChip Array that selectively binds proteins based on their chemical of biochemical properties. Bound proteins are determined by SELDI-TOF-MS (Surface-Enhanced Laser Desorption/ Ionization Time-of-Flight Mass Spectrometry). As a result a sample specific protein expression profile is generated. Based on the preliminary analyses conducted in year 3 of the study, two subjects were selected for a pilot project. These subjects were selected because they showed an increase in E-Selectin in association with PM plasma samples were available for each subject. Six different protein chips were selected for each sample, and four samples from each subject were done as duplicates. Figure 2-5 shows the results of the typical protein profiles in two selected chips for one subject (upper panels). Qualitatively were the profiles very well reproducible, while quantitatively a large variability was observed. This variability is probably due to the variability in lab work which involves several steps with very small quantities of samples. The typical pattern was changes substantially when the subject recorded bronchitic symptoms (middle panels) or on a day with elevated PM 2.5 exposures (lower panels). Therefore, it seems that not concentration changes of one protein might be observed using this technique, but it offers the ability to observe the up- or down-regulation of pathways. 27

33 Figure 2-5. Protein profiles using the Chipergen method on one selected subject with CAD H H H 2821 H16-A, H H H16-A, H H49-D, H 2 H38-C, H H 97.2+H H H H H H49-D,2 H38-C,2 COPD Panel Study All epidemiological data was entered in the database and checked for plausibility. All ECGs was processed by the cardiac core. Blood samples have been analysed for acute phase reactants and have been shipped for coagulation factors. Analyses will be conducted after the analyses of the CAD study have been completed and the observed associations have been understood. The air pollution data has been cleaned and is available for analyses. Future Plans: The statistical analyses of the CAD and COPD panel will be done based on the concepts outlined in the background section. This is a major task given the large database. Effort will be spent on the relationship between blood biomarkers and EKG parameters, as these relations have not been assessed well in the current medical literature. However, this knowledge will be crucial in order to understand the results with respect to air pollution. The CAD and the COPD panel will first be summarized in terms of internal consistency and plausibility and then compared between each other. Comparable or additional information obtained by the human studies of the Rochester Particle Centre and epidemiological as well as human studies by the other centres will be used to substantiate the results observed. Publications of the Core: Ibald-Mulli A, Wichmann H-E, Kreyling W, Peters A (22) Epidemiological Evidence on Health Effects of Ultrafine Particles. Journal of Aerosol Medicine 15 (2): Cyrys J, Heinrich J, Peters A, Kreyling WG, Wichmann H-E (22) Emissionen, Immission und Messungen feiner und ultrafeiner Partikel. Umweltmed.Forsch.Prax., 7: Pekkanen J, Peters A, Hoek G, Tiittanen P, Brunekreef B, Hartwig A, Heinrich J, Ibald-Mulli A, Kreyling WG, Lanki T, Timonen KL, Vanninen E. (22) Particulate air pollution and risk of STsegment depression during repeated submaximal exercise tests among subjects with coronary heart disease: the Exposure and Risk Assessment for Fine and Ultrafine Particles in Ambient Air (ULTRA) study. Circulation 16(8):

34 Peters A, Heinrich J, Wichmann H-E (22) Gesundheitliche Wirkungen von Feinstaub: Epidemiologie der Kurzzeiteffekte. Umweltmed.Forsch.Prax.: 7: Wichmann HE, Cyrys J, Stölzel M, Spix C, Wittmaack K, Tuch T, Peters A, Wölke G, Menzel N, Hietel B, Schulz F, Heinrich J, Kreyling W, Heyder J (22) Sources and elemental composition of ambient particles in Erfurt, Germany. Ecomed Verlag Landsberg (monograph). A. Henneberger, A. Ibald-Mulli, W. Zareba, R. Rückerl, J. Cyrys, J.P. Gouderc, B. Mykins, G. Woelke, H.E. Wichmann, A. Peters. (23) Effects of Particulate Air Pollution on Repolarization Parameters in Coronary Artery Disease Patients. Am J Resp Crit Care Med A332 A. Ibald-Mulli, W. Zareba, R. Rückerl, J.P. Couderc, B. Mykins, M. Pitz, H.E. Wichmann, A. Peters. (23) Effects of Particulate Air Pollution on Heart Rate Variability in Patients with Coronary Artery Disease. Am J Resp Crit Care Med A333 29

35 Research Core #3: Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects Principal Investigator: Mark W. Frampton Co-Investigators: Mark J. Utell (co-p.i.), William Beckett, Günter Oberdörster, Paul Morrow, Wojciech Zareba, Christopher Cox Background and Objectives Our objectives were to develop a facility for human studies of ultrafine particles and to utilize controlled human exposures to examine, in healthy and potentially susceptible subjects, the deposition of inhaled ultrafine carbon particles (UFP), and the role of UFP in inducing respiratory and cardiovascular health effects. Approval of all of the human studies has been obtained from our institutional RSRB; it is on file and current. QA is performed by Ms. Ellen Miles according to our QA Plan, with all SOPs being up to date and having been reviewed. Progress Report (Results & Discussion): We have developed a facility for experimental exposure of humans to ultrafine particles, which permits the quantitative determination of the exposure levels, respiratory intakes, and depositions of the aerosol. A detailed description of the facility has been presented at the Third Colloquium on Particle Matter and Human Health in Durham, North Carolina, in June Because our initial exposure mass concentrations are within the range of PM 1 measurements outdoors, it was important to know numbers and mass concentrations of particles within the Clinical Research Center and the Environmental Chamber where the facility is located, as well as in the intake air for the exposure facility. These measurements were performed, and the ambient measurement results were also reported at the Third Colloquium on Particulate Matter. For our initial studies, exposures were conducted at rest with a relatively low concentration of pure carbon UFP (~1 µg/m 3, ~2 x 1 6 particles/cm 3, count median diameter 26.4 nm, GSD 2.3). Twelve healthy non-smoking subjects (6 female) inhaled either filtered air or UFP by mouthpiece for 2 hours at rest, with a 1 min break after the first hour. Exposures were double blinded, randomized, and separated by at least 2 weeks. The total respiratory tract deposition fraction (DF) was determined for 6 different particle size fractions after correction for system losses, and the overall DF was calculated for both number and mass for each subject. Respiratory symptoms, spirometry, blood pressure, pulse-oximetry, and exhaled NO were assessed before and at intervals after the exposure. Sputum induction was performed 18 hours after exposure. Continuous 24-hour, 12-lead Holter monitoring was performed on the day of the exposure and analyzed for changes in heart rate variability and repolarization phenomena. The overall deposition fraction (DF) was.66 ±.12 (mean ± SD) by number, and.58 ±.14 by mass. The number of DFs during the first and second hours of exposure were similar within subjects, but the average overall DF varied from.43 to.79 among subjects. The DF decreased with increasing particle size in the UFP range, from.76 ±.11 at 7.5 nm to.47 ±.17 at nm. 3

36 We found no significant differences in respiratory symptoms, blood pressure, pulse-oximetry, spirometry, exhaled NO, blood markers of coagulation and endothelial activation, leukocyte activation, or sputum cell differential counts. There was no convincing evidence for significant effects on heart rate variability, repolarization, or arrhythmias. We concluded that exposure to 1 µg/m 3 pure carbon UFP at rest does not cause significant respiratory or cardiac effects in healthy nonsmokers. Ultrafine Particle Exposure-Response with Exercise We then initiated studies, also in healthy subjects, to examine concentration-response effects, and to incorporate exercise. Subjects received each of three exposures (air, 1, and 25 µg/m 3 UFP), which were separated by at least two weeks. A total of seven visits were required for each subject. Monitoring and measurements were identical to those for the resting exposures. The planned 12 subjects (6 male and 6 female) completed all phases of the study. Analysis of variance was performed by Dr. Cox, from the Biostatistical Core. Preliminary analyses indicated that exercise further increased the relatively high resting deposition of UFP (number deposition fraction at rest:.63±.4; exercise:.76±.6; means±sd). There were no particle-related effects on symptoms, spirometry, airway nitric oxide production, or sputum cell differential counts. There was a significant exposure-gender interaction for an effect on oxygen saturation (p=.2), with oxygen saturation decreasing slightly in females at 21 hours after exposure, only with the highest concentration of UFP (25 µg/m 3 ) (Figure 3-1). There was evidence for a concentration-related effect of UFP exposure on the percentage of blood monocytes, with the response differing by gender (Figure 3-2). In addition, monocyte expression of CD54 (ICAM-1) decreased after exposure in a concentration-response pattern (p=.1), with the greatest effect occurring at and 3.5 hours after exposure, and the differences resolved at 21 hours after exposure (Figure 3-3). Overall, the findings from UPDOSE appeared to provide evidence for modest effects of UFP exposure, with exercise, on blood monocyte number and leukocyte expression of surface markers. In general, surface marker expression decreased in association with UFP exposure, consistent with retention of higher expressing cells within the capillary bed. O 2 Saturation (%) O 2 Saturation (%) A Females -3 Baseline h 3.5h 21h O 2 Saturation (%) -3 Baseline h 3.5h 21h C Figure 13-1 Figure 23-2 O 2 Saturation (%) B Air Baseline h 3.5h 21h D Males UFP x Gender p=.2-3 Baseline h 3.5h 21h Monocytes (%) Monocytes (%) Baseline h 3.5h 21h A C Females -3 Baseline h 3.5h 21h Monocytes (%) Monocytes (%) B Air 1 25 Baseline h 3.5h 21h D Males UFP x Gender p=.15-3 Baseline h 3.5h 21h 31

37 CD54 Monocytes (MESF x 1 3 ) A UFP p=.12 Figure 3-3 Baseline h 3.5h 21h Figure 3-4 CD54 Monocytes (MESF x 1 3 ) B Air 1 25 Baseline h 3.5h 21h ECG recording analyses showed that the response of the parasympathetic system (measured by normalized units of high-frequency [HF] components) was blunted during recovery from exercise immediately after exposure to UFP in comparison to air exposure. This diminished vagal response was not observed 3.5 hour later. The analysis of QT interval duration and T wave amplitude also showed a blunted response after UFP exposure in comparison to pure air exposure. Figure 3-4 shows that the QT and QTc shortened during exercise more substantially during UFP particle exposure than during pure air exposure, and that the QT and QTc interval remained shortened for several hours after UFP exposure but not after pure air exposure. These findings suggested that inhalation of UFP at both concentrations altered myocardial repolarization in healthy subjects. UFP Exposure in Subjects with Asthma Subjects with asthma may represent a group with increased susceptibility to the health effects of ultrafine particles, both because of the possibility of increased airways deposition of particles, and because of underlying airway inflammation. We have completed a clinical exposure study (co-funded by the Health Effects Institute) of subjects with mild asthma. Sixteen subjects (8 male, 8 female) were exposed to air and to 1 µg/m 3 carbon UFP for 2 hours with intermittent exercise. We measured effects on pulmonary function, symptoms, airway inflammation (exhaled NO and induced sputum), blood leukocyte activation, and cardiac electrophysiologic function. Because these studies were performed using mouthpiece exposures, we were able to measure ultrafine particle deposition during exposure, and to compare findings with our previous studies 32

38 in healthy subjects. Results were analyzed in collaboration with the Biostatistical Core, as with the previous studies. Ultrafine Particle Deposition In the asthmatic subjects, we found that total respiratory fractional deposition by particle number was high at rest (.77±.5) and increased during exercise (.86 ±.4). Rest deposition was significantly increased compared with our previous study in healthy subjects at rest pfigure y3-5 p 1 (.63 ±.3). We conclude that ultrafine p<.1 p<.1 Healthy particle deposition is increased in mild Asthma.8 asthmatic subjects compared with healthy subjects (Figure 3-5). Symptoms and Lung Function There were no significant changes in respiratory symptoms or pulmonary function in response to these exposures. Subjects did not report an increase in medication use following exposures. Number Deposition Fraction Blood studies revealed a particle-related decrease in blood eosinophils, basophils, and CD4 + T- lymphocytes. Blood monocytes showed a significant reduction in CD11b expression after exposure (p=.29). Expression of CD54 on PMN decreased in a time-related fashion, with the greatest difference from control at 45 hours after exposure, with a significant time-exposure interaction (p=.31). Expression of CD62L on PMN showed a significant exposure-gender interaction, with an increase in expression of CD62L in males only. The most significant effect on leukocyte surface molecule expression in subjects with asthma appeared to be on eosinophils. As noted previously, there was a small exposure-related reduction in eosinophil percentage from the blood leukocyte differential count. In addition to this early reduction in eosinophil number, there was a delayed reduction in eosinophil expression of CD32 (time-exposure interaction, p=.15), and CD11b (main effect, p=.15). In summary, data on leukocyte expression of adhesion molecules in subjects with asthma, similar to the findings for healthy subjects, suggest modest effects of UFP exposure with exercise. In general, the effect appears to be a reduction in expression, or a blunting of the post-exposure increase seen after air exposure. These findings are consistent with increased vascular retention of leukocyte subsets in the hours following UFP exposure, perhaps due to pulmonary vasoconstriction. Effects of UFP Exposure on Endothelial Function For the last six subjects studied in this project, we examined effects of UFP exposure on endothelial function by performing flow mediated dilatation of the forearm circulation before exposure and 24 and 48 hours after exposure. Figure 3-3 shows the change from pre-exposure in post-ischemic total forearm flow compared with the baseline measurement. After air exposure, flow-mediated dilation increased compared with pre-exposure baseline, an expected response to the exercise during the previous day s exposure. However, this response was blunted following Rest Exercise 33

39 UFP exposure. At 48 hours, there appeared to be a rebound, with the change in total flow greater after UFP than after air. Figure 3-6: Change in flow-mediated dilation and blood nitrates Flow-mediated dilation is mediated by endothelial NO, so we measured the plasma NO products nitrite and nitrate before and after exposure in all 16 subjects in this study. As shown in the inset in Figure 3-6, we found a significant increase in blood nitrates 48 hours after exposure. This finding is consistent with a rebound increase in nitric oxide production, which likely mediates the rebound seen in flow mediated dilation. These data support the hypothesis that exposure to very low concentrations of UFP alters systemic endothelial function. This is a novel finding, and we have now begun a study to determine if exposures in healthy subjects have similar effects on vascular function, and to determine the time course of the response. In summary, our study indicates that asthmatics have increased airway deposition of ultrafine particles, and that exposure to even low mass concentrations of ultrafine particles alters circulating leukocyte subsets. These data are most consistent with an alteration in leukocyte retention in the pulmonary circulation. In addition, our preliminary results suggest there are also effects on systemic endothelial function. As a result of these findings, we have undertaken discussions with other Center Research Cores to develop additional strategies to investigate the vascular effects of exposure to UFP. Progress Report, Year 4: In our Progress Report for years 1-3, we described the development of an ultrafine particle exposure system for use in human studies, particle generation and characterization, measurements of ultrafine particle deposition in healthy subjects at rest and exercise, and effects on induced sputum cellularity, alterations in blood leukocytes, and heart rate variability and repolarization. The studies summarized in that report indicated that exposure to ultrafine carbon particles at low mass concentrations (1 and 25 µg/m 3 ), in both healthy subjects and subjects with asthma, may cause sub-clinical effects on pulmonary ventilation/perfusion matching, circulating leukocytes, and cardiac repolarization, particularly in females. Although these effects are not likely to be clinically important in healthy subjects, the fact that there are changes at all at these very low concentrations is quite striking, and is consistent with the hypothesis that these particles have the potential to elicit cardiovascular effects. If these findings are confirmed, they will represent the most convincing support for the ultrafine hypothesis to date, and would suggest that females experience increased susceptibility to UFP exposure relative to males. Interestingly, our data did not confirm our original hypothesis that ultrafine particle exposure with exercise would induce airway inflammation or an acute phase response. 34

40 It is imperative that we confirm these preliminary findings. We have therefore designed a study to confirm and extend these observations in a larger group of healthy men and women, using a higher, yet still environmentally relevant, concentration of UFP. Our hypothesis is that inhalation of UFP alters pulmonary vascular function, circulating leukocyte activation, and cardiac repolarization. We speculate that these alterations reflect mechanisms involved in the observed increase in cardiovascular morbidity and mortality associated with particulate air pollution. Furthermore, we postulate that these effects are influenced in part by gender. Objective #1: Perform a human clinical inhalation study of exposure to filtered air and to carbon UFP, 5 µg/m 3 for two hours, with intermittent exercise. Objective #2: Measure UFP effects on cardiac electrical activity, circulating leukocyte activation, and blood oxygenation. We have now initiated studies at the higher concentration of 5 µg/m 3. Aside from the higher concentration, the experimental protocol is identical to our previous studies. Sixteen subjects will be studied, eight men and eight women. The study design incorporates features to avoid bias and reduce the impact to inter-subject variability. Each subject undergoes both air and particle exposure in a randomized, double-blinded, cross-over design, so that each subject serves as his/her own control. Exposures are for two hours, with intermittent exercise, to filtered air and to 5 µg/m 3 carbon UFP (~1 x 1 7 particles/cm 3 ). Exposures are separated by at least three weeks to avoid the possibility of carry-over effects. Subject assessments and measurements are performed before, and at intervals after exposure: immediately, 3.5, 24, and 48 hours after exposure. Digital 12-lead high-resolution EKG recordings are obtained at each measurement time point before and after exposure, and 24-hour ambulatory cardiac monitoring is initiated at the start of exposure. Systemic endothelial function is assessed using flow mediated dilatation of the forearm, before and at intervals after exposure. Pulmonary vascular function is assessed using measurement of the diffusing capacity for carbon monoxide (DLCO). In addition, subjects undergo continuous digital pulse oximetry monitoring throughout the post-exposure period, including overnight at home. Other assessments at each time point include respiratory symptoms, blood pressure, heart rate, and phlebotomy. Blood samples are immediately sent to the clinical laboratory for complete blood count, leukocyte differentials, and platelet count. Fresh whole blood is anticoagulated and prepared for immunofluorescence staining with monoclonal antibodies to a variety of cell surface markers and adhesion molecules. These are analyzed using multi-parameter flow cytometry. Samples of blood plasma and serum are stored at 8 C for subsequent analysis of soluble cytokines and adhesion molecules, and for markers of endothelial activation. At the time of completion of this report, all 16 subjects have been enrolled in the study, and 12 subjects have completed both exposure to air and ultrafine particles. The investigators remain blinded to the exposures, so no analysis of the effects of ultrafine particle exposure is available. However, no subjects have experienced significant symptoms, cardiac arrhythmias, or other adverse effects from the exposures. 35

41 The particle count median diameter of the freshly generated particles at inhalation is ~3 nm. The count median diameter of exhaled particles (particles not retained in the lung) was ~37 nm. In the subjects completing exposures to date, preliminary calculations of fractional deposition in the respiratory tract showed a very high resting deposition of.73 ±.1, with the deposition fraction during exercise increasing to.8 ±.8. This finding confirms our previous observations of very high fractional deposition of ultrafine particles in healthy human subjects, with a further increase in the deposition fraction during exercise. For these studies, we have initiated a novel technique of continuous pulse oximetry for the entire 48 hours of observation. Subjects wear a finger pulse oximeter connected to a digital recording device, and all data are downloaded in ASCII format for subsequent analysis. This will allow us to detect very small changes in oxyhemoglobin saturation that would reflect subtle changes in pulmonary ventilation-perfusion matching in response to particle exposure. We have now performed a preliminary analysis of the first 12 subjects completing this exposure protocol. As in our previous studies, there were no symptoms associated with exposure, and subjects were unable to identify which exposure day involved inhaling particles. There were no significant changes in the forced expiratory volume in one second, or in the forced vital capacity in association with exposure. However, small reductions were seen in mid-expiratory flow rates, suggesting that particle inhalation may have induced very mild construction of the small airways of the lung. Findings from measurements of DLCO before and after exposure are shown in the Figure. There was a highly statistically significant decline in DLCO 21 hours after exposure to UFP, when compared with air exposure at the same time point. DLCO returned towards normal, but Inhalation of 5 µg/m 3 carbon UFP Decreases the Pulmonary Diffusing Capacity 1 DLCO (ml/min/mmhg) Air UFP remained somewhat reduced 45 hours after exposure. This finding, although preliminary, supports our hypothesis that inhalation of carbon UFP may affect the pulmonary circulation. The DLCO is influenced by pulmonary capillary blood volume, and pulmonary vasoconstriction with reductions in pulmonary capillary blood volume result in reductions in DLCO. This finding provides further support for the concept that low mass concentrations of ultrafine particles may have vascular effects, even in healthy subjects. * p=.1 Baseline 21h 45h 36

42 The ultrafine particles generated from elemental carbon that are used in these and our previous studies are relatively inert compared with ambient air particles, and do not contain reactive metals or aromatic hydrocarbons. If we confirm that inhalation of these relatively benign particles causes subclinical effects on pulmonary ventilation/perfusion matching, circulating leukocytes, and/or cardiac repolarization in healthy subjects, this would represent the most convincing support for the ultrafine hypothesis to date, and would provide a basis for subsequent studies with more complex laboratory-generated particles or concentrated ambient particles. Summary of Human Clinical Studies of UFP Exposure: We have developed and validated an exposure system for human studies of inhalation of ultrafine particles. We have demonstrated that the total respiratory deposition of ultrafine particles is relatively high, consistent with prediction models. However, UFP deposition increased further with exercise, and in the presence of mild asthma. Inhalation of ultrafine carbon particles at concentrations up to 25 µg/m 3 caused no symptoms, changes in lung function, or evidence for airway inflammation. Our hypothesis that inhalation of ultrafine carbon particles would cause pulmonary inflammation and an acute phase response was not confirmed. However, we observed subtle changes in blood leukocyte subsets and adhesion molecule expression that suggest there may be effects on endothelial function. Preliminary findings from our study of exposure to 5 µg/m 3 appear to confirm these observations. We also found evidence for effects on heart rate variability, and on cardiac repolarization in healthy subjects. The finding that these very low mass concentrations of particles have vascular effects would have important implications for future particulate matter regulatory strategies. Further studies are needed to confirm our observations, and to determine effects in people with underlying vascular dysfunction. Future Plans: We expect to complete the current exposure protocol, healthy subjects breathing 5 µg/m 3 UFP with exercise, in the next few weeks. The results will then be analyzed with the help of the Biostatistics Core. We are particularly interested in the results of forearm flow-mediated dilatation testing, in order to determine whether UFP exposure has effects on systemic endothelial function. We will also be focusing carefully on the results of the 48-hour ECG recordings, to determine whether our previous observation of effects on the repolarization interval is confirmed in the current studies. We then plan to initiate studies in healthy elderly subjects, and aged-matched subjects with Type II diabetes mellitus. Patients with diabetes have been identified in recent epidemiological studies as being particularly susceptible to the effects of PM exposure. We hypothesize that patients with diabetes, who are known to have underlying endothelial dysfunction, will show enhanced vascular responses to particle inhalation. Our findings will be interpreted in conjunction with ongoing and planned studies in the other research cores, examining effects of UFP on vascular endothelium. 37

43 RESEARCH CORE 4: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events Principle Investigator: Günter Oberdörster Co-Investigators: Alison Elder, Jacob Finkelstein, Robert Gelein, Jean-Philippe Couderc, Wojciech Zareba, Christopher Cox, Mark Frampton, Mark Utell, Wolfgang Kreyling, Paul Morrow, Zachary Sharp Summary of Progress to Date: Objectives The animal studies are designed to be complementary to the field and controlled clinical studies and to form a link to the mechanistic in vitro studies. They are designed to determine pulmonary and systemic responses to inhaled laboratory-generated and real world ultrafine particles (UFP) and to develop rodent models of human disease to test our central hypothesis that UFP contribute to the increased morbidity and mortality of susceptible people in association with small increases in urban particles. Thus, the overall objective of the animal studies is to identify factors which are causally associated with adverse pulmonary and extrapulmonary health effects after low-level exposures to UFP. These factors include particle size; dosimetric aspects (lung deposition and disposition); host susceptibility (advanced age; cardiovascular disorders; respiratory tract sensitization); and pollutant co-exposure (ozone). QA according to our QA Plan is carried out by Ms. Ellen Miles. She has reviewed all SOPs related to the animal studies, which are updated once a new methodology has been validated. Summary of Progress Years 1-3: Overview Two of the main focuses of the first three years of this project have been to 1) generate and use ultrafine particle (UFP)-containing atmospheres in toxicology studies that are relevant for human ambient air exposures and 2) achieve concordance for the animal studies with the other cores (#2,3,5) in terms of endpoints measured. Firstly, several studies have now been completed in which ozone was used as a co-pollutant, delivered by itself or with laboratory-generated UFP. In addition, we have completed studies in young and old rodents using two different relevant priming agents: inhaled low-dose endotoxin, as a model for pneumonia or exacerbations of COPD, and human influenza virus (described below), a common respiratory pathogen. The particle types used in our studies have included laboratory-generated ultrafine carbon particles and those particles with added Fe; laboratory-generated organic UFP; concentrated fine/ultrafine particles (ambient Rochester air); and freshly-generated vehicle exhaust emission consisting of fine/ultrafine particles on highways (results described in Year 4 report). As to the parameters measured in the core-specific studies, there are now many similarities in terms of markers of pulmonary inflammation, peripheral blood cell adhesion molecules, coagulation and acute phase factors, and heart rate and blood pressure variability used in Cores 2, 3, 4 and 5. We have also focused a great deal of attention on the uptake and tissue-specific distribution of laboratory-generated inhaled solid ultrafine carbon and metal particles ( 13 C, MnO 2 ). 38

44 Ultrafine Particle-Containing Atmosphere Generation As stated above, one of the main focuses of these Core 4 studies was to develop model particles to be used in exposures in animals. We now have the ability to generate ultrafine carbon particles (~26 nm count median diameter [CMD], 15-2 x 1 6 particles/cm 3 ; the same particles that are usedin the controlled clinical exposures and in the in vitro studies) that contain, for example, transition metals (Fe, V) for toxicology studies or 13 C UFP for use in particle dosimetry studies. The carbon particles have a high surface area (58 m 2 /g) as determined by the BET method (Dr. Bice Fubini, Turin, Italy). By aging these particles for 5 minutes they coagulate into accumulation mode particles with CMD of 28 nm. However, particle surface area remains as high as before; when rats were exposed to the coagulated particles at 2-fold higher concentration than UFP, the inflammatory response was slightly higher than that from the singlet UFP, confirming the importance of particle surface area for eliciting inflammatory effects. Because surface area remained as high for the aged particles as for the freshly-generated particles, we feel that these coagulated particles are not appropriate for use in future size comparison studies. The multigroup studies described below were performed with ultrafine carbon particles that were mixed with Fe (25%; i.e. generated from C/Fe graphitized rods). Having completed several of these multigroup studies now, we have been able to compare responses in animals inhaling ultrafine carbon vs. carbon/fe particles; we have found no evidence to support that in vivo inflammatory responses are different. This is intriguing given the fact that the Fe in these particles is bioavailable (measurements by Dr. Ann Aust, Utah State Univ.) and they generate more hydroxyl radicals in the presence of peroxide than do UFP without Fe (measurements by Dr. Vincent Castranova, NIOSH). Another interesting point regarding the laboratory-generated UFPs is that they initially contained up to 3% organic material due to a number of plastic components in the spark generator. Efforts to remove all possible sources of contamination in the spark generator were successful so that we now have less than 5% of organic carbon on the laboratorygenerated UFP of elemental carbon. The ambient smaller UFP (<~2 nm) consist to an increasing degree of organic compounds, as seen by our Core 1 measurements. A significant constituent in these is used and unused motor oil. Recognizing this, studies were conducted through our Visiting Scientist Program to develop a method for generating ultrafine organic particles. To this end, a vaporization-condensation aerosol generator was assembled and characterized by Dr. John Veranth (Univ. of Utah). Used motor oil was used in this system, which was capable of generating stable organic UFP (3-5 nm; 1 6 particles/cm 3 ) aerosols for 6 hrs. Briefly, the motor oil (in hexane and ethyl alcohol) was nebulized such that large droplets were removed by inertial impaction on an impinger, leaving a fine mist that was then directed into a furnace with seed nuclei of NaCl. The report from these studies is attached. The oil droplets were completely vaporized and ultrafine aerosols formed when the vapor began to cool at the exit of the furnace. Other studies done with paraffin oil and short-chain alkanes showed that the generation system is adaptable to other low-volatility hydrocarbons. 39

45 Toxicology Studies with Laboratory-generated UFP: The design for the toxicological studies, unless otherwise specified, involved 16 different groups of animals, representing all possible combinations of exposure components (UFP, ozone, priming agent) and age (young vs. old). The age factor is an important focus for the Core 4 and 5 studies and our results show that there is a striking difference between the age groups in the balance between pro-oxidant and antioxidant processes. This specific factor will be discussed below for individual groups of experiments. Eight groups of young (8-1 weeks) and old (2-22 months) mice and rats were exposed (n=5 per group) to UFP (carbon or mixed carbon/fe) with or without ozone for 6 hrs in compartmentalized whole-body chambers with and without prior priming by endotoxin or human influenza virus. Low-dose endotoxin aerosols were used to prime the respiratory tract (~5-1% PMNs in lavage fluid after 24 hrs); these priming exposures lasted for ~15 mins and were done immediately prior to UFP/ozone exposures. When human influenza virus (X-31, H3N2; 1 4 EID 5 ) was used as the priming agent, it was intratracheally instilled 48 hrs prior to UFP/ozone exposures. We collected all samples (lung lavage, blood, organ tissues) for our measurements 24 hrs after exposure. Since there were four factors involved in these studies, all data were analyzed via four-way analysis of variance (ANOVA) for main effects and interactions between factors. This type of ANOVA is not only the most appropriate one for the study design, but it also has high statistical power. The results from these analyses, particularly the consistent significant interactions, combined with results from inflammatory cell and gene expression analyses are critical to the design of more mechanistic-type studies. Toxicology Studies with Ambient UFP: In addition to these laboratory-generated UFP, we have also performed studies using ambient PM. Through our collaboration with the Harvard PM Center (Dr. Petros Koutrakis), we have performed studies using a prototypical ultrafine particle concentrator. The prototype sampled air from a moderately busy road and concentrated ultrafine particles with some overlap into the fine mode (7-316 nm; CMD=35 nm, GSD=1.9). The average number concentrations during the 6-hr exposures were ~2 x 1 5 particles/cm 3. We performed studies in young and old F-344 rats to measure lung inflammatory processes and lavage cell oxidant release 24 hrs post-exposure. Results shown in Figure 4-1 (analyzed by 2-way ANOVAs per age group) show that the concentrated UFPs have effects (i.e. significant main effects in ANOVA), but that these effects are different in the two age groups. Specifically, the response to the combination of concentrated UFPs and LPS in young rats is significantly lower than to LPS alone; the responses in old rats are not significantly different from one another. In old rats, the concentrated UFPs alone induced a small, but significant decrease in response as compared to sham-exposed rats; in young rats, the trend is the same, but the two groups are not different from each other. Other sets of studies have been done in telemetered SH rats for EKG recordings to assess changes in heart rate variability (HRV) following exposure; these data are currently undergoing analysis. Lung and heart tissues taken from the exposed F-344 rats were screened for gene expression changes via microarray analyses and these results are presented below. 4

46 Results from UFP Concentrator Study in Young and Old Rats with LPS Priming 25 Young Rats 25 Old Rats 2 p <.5 2 % PMNs 15 1 % PMNs 15 1 NS 5 5 p <.5 No LPS No UFP No LPS UFP LPS No UFP LPS UFP No LPS No UFP No LPS UFP LPS No UFP LPS UFP Figure 4-1. Lavage PMNs 24 hrs after 6-hr exposures to concentrated ambient ultrafine particles in combination with inhaled LPS priming in young (8-1 wks) and old (2 mos) F-344 rats. Data were analyzed via 2-way ANOVA followed by Tukey multiple comparisons (means were considered significantly different when p <.5). Development and Characterization of Compromised Animal Models: Several multigroup studies have been completed now in which young and old rats and mice were exposed to combinations of laboratory-generated UFP and ozone with respiratory tract cell priming. The last report described 6-hr exposures of aged (18 months) and young (8 weeks) mice to mixed C/Fe ultrafine particles (~1 µg/m 3 ) and ozone (.5 ppm) after priming with inhaled low-dose LPS. In agreement with our earlier studies in rats (Elder et al., 2), all four factors (UFP, ozone, LPS, and age) had significant main effects for most of the respiratory and cardiovascular endpoints examined. The striking age effect was such that inflammatory and cell activation responses in old mice were greater than in young mice. For some endpoints (e.g. lavage PMNs, lavage AM surface ICAM-1 expression), the UFP effect was dependent upon the presence of LPS. This draws attention to the fact that there were several consistent interactions involving inhaled UFPs, among them those involving LPS and age (response enhancement) and ozone (response suppression). Our findings that inhaled UFP can alter blood PMN surface ICAM-1 expression are in agreement with results from Core 3 clinical studies. Some of the most striking effects were observed in exposure-related lung and heart tissue gene expression changes. Not only were significant alterations in heart tissue gene expression observed, indicating extrapulmonary effects of inhaled UFP and ozone, but the data also suggests an imbalance in old animals between pro- and antioxidant species production. Examples for MIP-1α and IL-1 are shown in Figure 4-2 below. These findings have been extended in another series of studies using human influenza virus as the priming agent. Results from those studies are described below (Year 4 Progress Report). 41

47 8 MIP-1α Gene Expression using Slot Blot Analysis 14 IL-1 Gene Expression using Slot Blot Analysis 7 Young Old Young Old 14 1 relative intensity Control UFP Ozone UFP Ozone LPS Lung UFP LPS LPS Ozone C/Fe UFP Ozone Control UFP Ozone UFP Ozone LPS Heart UFP LPS LPS Ozone UFP LPS Ozone relative intensity Control UFP Ozone UFP Ozone LPS Lung UFP LPS LPS Ozone UFP LPS Ozone Control UFP Ozone UFP Ozone Figure 4-2. MIP-1α (left) and IL-1 (right) gene expression in lung and heart tissue after exposure to inhaled UFP in combination with ozone after LPS priming. The insets show line plots of the group means for reference. Responses in young mice are shown in the red bars and those from old mice are shown in green. LPS Heart UFP LPS LPS Ozone UFP LPS Ozone Other exposures with laboratory-generated ultrafine carbon particles were done after endotoxin priming via intraperitoneal (ip) injection to simulate the early phase of response to an inflammatory stimulus that would prime respiratory tract and circulating cells from the systemic compartment. These studies, however, did not involve ozone. Old F-344 (23 mos) and SH (15 mos) rats were exposed to UFP for 6 hrs, with or without ip LPS priming treatment, which immediately preceded inhalation exposures. Inflammatory lung lavage and blood parameters were determined, including measurement of intracellular ROS generation by inflammatory pulmonary and blood cells (oxidation of DCFD). Neither inhaled UFP nor ip LPS caused a significant increase in lavage PMNs or PMA-stimulated ROS release in either animal model, confirming what others have shown about the lung being somewhat protected from systemically-delivered LPS. In F-344 rats, the combination of UFP and i.p. LPS was suppressive in terms of lavage cell intracellular ROS activity; in SH rats this parameter was not altered by either factor. LPS significantly increased the number of circulating PMNs in both F-344 and SH rats. Interestingly, in F-344 rats, the combination of LPS with UFP led to an enhancement of response, whereas in SH rats, response was suppressed. Blood PMN intracellular DCFD oxidation was affected by exposure in both animal models; however, response was enhanced when UFP and LPS were combined in SH rats and somewhat suppressed in F-344 rats. Plasma fibrinogen was significantly increased by LPS in both animal models. Despite indications that the acute phase response (alterations in plasma fibrinogen) and blood cells were activated, blood viscosity, hematocrit, and coagulability (TAT complex increases) did not change. The results show that inhaled laboratorygenerated UFP did not consistently enhance the i.p. LPS response. Although these observations are somewhat limited in scope, they do suggest that 1) the carbonaceous core of ambient PM, as modeled in these experiments, may not be solely responsible for cardiovascular effects or 2) the ambient PM-induced alterations in HRV found in epidemiological studies can be independent of overt increases in coagulability or acute phase activation. HRV Analyses in Unrestrained, Telemetered SH Rats In parallel to the evaluation of the ECG recordings from the epidemiological and clinical PM Center studies, scientists of the Cardiac Core of our Center have developed 42

48 an algorithm for analysis of recorded ECG and blood pressure signals from rats. A Windows-based algorithm was developed that was compatible with electrocardiograms and blood pressure signals acquired using the Data Sciences International system (DSI; St. Paul, MN). The decryption and management of data files was done in collaboration with engineers from DSI. The company shared their proprietary binary file format so that we could implement our own analytic algorithm. The development of the software was done in Microsoft Visual C++ in order to insure compatibility of our software with all windows-based operating systems. The Graphics User Interface was designed in order to facilitate the use and access to the various features of the software including the visual check of both the tachograms and systograms and the ability to automatically analyze short and long-term ECG and BP signals. The software includes several pre-processing steps: 1) digital pre-filtering to reduce noise and artifacts (Oppenheim and Schafer, 1989), 2) reduction of baseline wandering due to animal movements (Badlini, 1991), 3) QRS detection and extraction of the tachogram for ECG and systolic BP detection and extraction of the systogram, and 4) filtering and interpolation of the non-sinus beats according to general methods. Based on time and frequency domain approaches (Akselrod et al., 1987; Kuwahara et al., 1994; Rubini et al., 1993), our assessment of HRV relies on a set of parameters known to provide quantification of autonomic changes. These parameters were SDNN, RMSSD, and the various frequency bands of the power spectrum computed from the tachograms and the systograms (described as very low frequency (VLF), low frequency (LF) and high frequency (HF) components). The validation of our algorithm for the quantification of heart rate variability has been implemented in a similar fashion as previous validation studies (Akselrod et al., 1987; Cerutti et al., 1991; Ramaekers et al., 22), employing pharmacological blockade to dissect the relationship between heart rate (HR), heart rate variability (HRV) and arterial blood pressure (ABP). Signals were acquired at 1 HZ sampling frequency with 16 bit amplitude resolution (Merri et al., 199). The preliminary analyses of variability of the HRV parameters led us to conclude that at least 15 beats are needed to obtain reliable and reproducible estimation of HRV parameters (Couderc et al., 22). Thus in our current experiments, we use this length of ECG recordings in order to insure better estimation of HRV parameters. The first version of our software was based on ECG signals only. We investigated the limitation of our approaches based on a first atropine-propranolol protocol. The results were unexpectedly unstable. A first paper was published in the proceedings of the IEEE Computer in Cardiology meeting in 23 (Couderc et al., 22) describing the limitations and difficulties encountered in this first protocol. The lack of stability of the method was attributed to the profound and transient changes of the QRS morphology occurring when the animal was moving in its cage. This led to a high level of R-peak misdetection. Consequently, the resulting tachogram was difficult to interpolate and analyze. We later developed an algorithm for measuring HRV from BP signal from which we obtained higher stability and reproducibility. Greater morphological stability of the QRS waves was resolved by changing the ECG lead placement. The DSI-recommended two-lead configuration involves placement of the negative lead in the right scapular region and the positive lead under the left side of the rib cage. The negative lead was, instead, placed in the musculature surrounding the trachea and the positive lead in the musculature on top of the sternum. With this new 43

49 lead placement, movement of the rats did not affect the morphology of the QRS complex and our analysis increased in stability. The software was applied to various data sets in order to assess the effect of particulate matter on the autonomic nervous system. Preliminary results are currently submitted (Couderc et al., submitted). We are also currently working on a new tool for the analysis of the repolarization interval from rat ECG signals. Recent experimental findings show that modifications of ionic channel functions by pharmacological agents, ischemia, or electrolyte abnormalities generate repolarization abnormalities with increased heterogeneity of repolarization. These repolarization abnormalities are associated with increased risk of ventricular arrhythmias leading to episodes of torsades de pointes with subsequent ventricular fibrillation (Burashnikov and Antzelevitch, 2; el Sherif and Turitto, 1999). Air pollution may affect the myocardium at the cellular level by modifying intrinsic electrical properties of the myocardial cell through direct (blood-born or reflex-based) or indirect (inflammatory) mechanisms (Aronow, 1981; Gold et al., 1998; Samet et al., 2). The program will measure QT and QT peak interval duration by identifying the beginning of the QRS complex and the peak and end of the T-wave (Zareba et al., 21). Normal QT and QT peak values will be determined based on a series of baseline recordings in normal rats and using heart rate-adjustment formulae. The T-wave area analysis will be used to quantify repolarization morphology by tracking the distribution of the amplitude along the time axis. The measurement of T-wave morphology has the benefits of being less noise-dependent and is also less dependent on accurate detection of the end of the T- wave. To conclude, our work focuses on the design of tools for the analysis of HRV and repolarization intervals in unrestrained rats based on long-term ECG recordings. The program is being applied in analyses that are currently underway to the ECGs recorded during several experiments. Statistical analyses of these data will follow. Dosimetry Studies As described in the previous progress report, we have developed a method to generate ultrafine 13 C particles for use in rat dosimetry studies. After several methodological improvements, we performed a study with the objective to determine whether ultrafine elemental carbon particles translocate to the liver and other extrapulmonary organs following inhalation as singlet particles by rats (Oberdörster et al., 22). We generated ultrafine 13 C particles as an aerosol with CMDs of 2-29 nm (GSD 1.7) using electric spark discharge of 13 C graphite electrodes in argon. Nine Fischer-344 rats were exposed to these particles for 6 hrs. in whole-body inhalation chambers at concentrations of 18 and 8 µg/m 3 ; three animals each were killed at.5, 18 and 24 hrs post-exposure. Six unexposed rats served as controls. Lung lobes, liver as well as heart, brain, olfactory bulb, and kidney were excised, homogenized and freeze-dried for analysis of the added 13 C by isotope ratio mass spectrometry. Organic 13 C was not detected in the 13 C particles. The 13 C retained in the lung at.5 hrs post-exposure was about 7% less than predicted by rat deposition models for ultrafine particles, possibly indicting some rapid initial elimination from the lung. Lung levels did not change significantly during the 24 hr. post-exposure period. Normalized to exposure concentration, the added 13 C per gram of lung on average in the post-exposure period was ~9 ng/g organ/µg/m 3. Significant amounts of 13 C had accumulated in the liver by.5 hr. post-inhalation only at the high exposure concentration, whereas by 18 and 24 hrs post-exposure the 13 C concentration of 44

50 the livers of all exposed rats was more than one-third the 13 C concentration found in the lung (Figure 4-3). Considering the ~1-fold greater weight of the liver compared to the lung, the 13 C amount in the liver was ~4-fold greater than in the lung by 18 and 24 hrs after exposure. No significant increase in 13 C was detected in the other organs which were examined. These results demonstrate effective translocation of ultrafine elemental carbon particles to the liver by one day after inhalation exposure. Potential translocation pathways include direct input into the blood compartment from ultrafine carbon particles deposited throughout the respiratory tract as well as uptake into the blood circulation of UFP particles from the GI-tract after swallowing. These studies also indicated a slight increase of 13C in the olfactory bulb. Normalized Lung and Liver Excess 13 C Concentration Following Ultrafine 13 C Particle Exposure in Rats 1 ng 13 C/g organ per µg/m Lung Hours Post-Exposure Liver Figure 4-3. Lung and liver 13 C concentration in rats at different times after exposure to ultrafine 13 C particles, normalized to the exposure concentration. In a subsequent pilot study with a prolonged 7-day post-exposure period, 13 C of lung and extrapulmonary organs was analyzed on day 1 and day 7 post ultrafine 13 C particle exposure. Results showed again significant amounts of added 13 C in the liver on day 1, but no longer on day 7. However, on day 7, significant increases in added 13 C in heart, brain and olfactory bulb were found. This prompted us to conduct a follow-up study focusing on uptake of inhaled solid UFP into the CNS. (Reported in Year 4 Progress Report). Other studies performed within our PM Center s Pilot Programs used ultrafine 192 Ir particles. Iridium is the least soluble of metals in the lung and is, therefore, best suited to study its disposition after inhalation. Dr. Kreyling of the GSF München, is the PI of this pilot study, and he found in contrast to our results with ultrafine 13 C particles that after intratracheal inhalation exposure only minimal amounts of ultrafine iridium particles were translocated to extrapulmonary organs (Kreyling et al., 22). However, it was also 45

51 found that larger, 8 nm, 192 Ir particle translocation was 1-fold slower than that of smaller 15 nm 192 Ir particles. Collectively, our studies suggest that not only the particle size but also the material and surface properties, like structure and composition, may influence the efficiency of UFP translocation. Differences in translocation of inhaled ultrafine particles based on their chemistry will be further investigated in a collaborative approach. Summary of Year 4 Progress: Heart Rate Variability Studies As summarized above, the Cardiology Core of our PM Center, has been developed several algorithms for the analysis of ECG and blood pressure recordings for changes in heart rate variability parameters. The final analysis program has been implemented and tested using positive control data from rats exposed via intraperitoneal injections to atropine or propranolol, similar to our first study. Two groups of 6 rats were randomized in a cross-over study to receive either of the two agonists or saline; thus the total n per treatment was 4. Treatments were separated by one day. Post-exposure recordings were done for 24 hrs. Various time and frequency domains parameters were measured to assess autonomic control when computed from ECG and blood pressure signals. Pharmacological interventions were used to testing whether our method is able to identify sympathetic and parasympathetic blockade. HRV parameters were computed from the tachogram and the systogram and correlations between parameters were computed in order to provide insights into the complex relationship between respiratory activity, heart rate and blood pressure (Saul, 1998). Another aim of this study was to better understand the autonomic changes in hypertensive rats. The literature provides controversial results on the autonomic impairment in hypertensive rats. Authors have reported an impaired control of sympathetic drive (Akselrod et al., 1987) in comparison to normotensive rats, whereas others revealed reduced vagal tones (Murphy et al., 1991). Our validation study will help to investigate levels of hypertension and autonomic impairment. Results from this study are presented in a manuscript by Couderc et al. that is in preparation. The algorithm has also been applied to data from exposures of telemetered SH rats exposed to on-road aerosols (described below). Effects of UFPs and Ozone in Influenza Virus-Exposed Mice: We tested our hypothesis that inhaled UFP and ozone would induce greater lung injury and oxidative stress than either component by itself in young and old mice that were pre-exposed to influenza virus to prime respiratory tract cells. The design of the study was essentially the same as that of the LPS priming study summarized in the previous section. Young and old male C57 mice (1 wks, 21 mos) were exposed to ultrafine carbon particles containing 25% Fe (CMD ~26 nm, ~14 µg/m 3 ) and ozone (.5 ppm) for 6 hrs, alone and in combination. Lung inflammation was induced with intratracheally instilled X-31 human influenza virus 48 hrs prior to UFP or ozone exposures. Parameters of inflammation in lavage fluid and blood as well as lavage cell oxidant release were measured 24 hrs after exposure. RNA was also extracted from lung and heart tissue for microarray analyses. As before, a 4-way ANOVA was used to analyze the results. Examining the endpoints from this study as a whole, UFPs were found to have consistent and independent effects on pulmonary inflammation and inflammatory cell activation. Ozone, influenza virus, and age had significant main 46

52 effects for all endpoints examined. In addition, the interactions with UFP that were consistently significant involved influenza virus, ozone, and age. Using microarray analyses, we have also screened lung and heart tissue for changes in gene expression. There was a trend toward higher pro-inflammatory and lower anti-inflammatory gene expression in tissues from old as compared to young animals. In addition, we found evidence of significant gene changes in heart tissue. The microarray screening results will be confirmed in future studies using slot blot-r Northern analyses. When the results from this study are considered as a whole with the studies involving LPS as the priming agent, a consistent pattern of main effects and factor interactions emerges (Table 4-1). Given the fact that there were so many endpoints analyzed in these two sets of studies, the consistency of these interactions (amplification vs. suppression of response was discussed in text above) is remarkable and strengthens the causality of associations that were found in the statistical analyses. Moreover, the results are also consistent with those of our earlier multigroup studies in young and old rats showing that effects are not species specific. Multigroup Studies: Synopsis of Effects Tests (both priming agents/all endpoints) Main Effects: UFP Ozone Priming Agent Age > 1/2 of the endpoints always always all but one endpoint Consistent Interactions: UFP interacting with Priming agent Ozone Priming agent * Ozone Priming agent * Age Table 4-1. Summary of 4-way ANOVAs from the multigroup studies involving either inhaled LPS or intratracheally instilled influenza virus given as the priming agent for subsequent exposures to combinations of inhaled UFP and ozone in young and old mice. Translocation of Inhaled Solid UFP to the CNS: Our earlier dosimetry studies with inhaled ultrafine 13 C (summarized under Year 1-3 studies) showed translocation to the liver at one day post-exposure, and indicated also significant translocation into the olfactory bulb at day 7 post-exposure in a pilot study. Reports in the literature have identified the olfactory nerve axons acting as translocation pathways for soluble metals, and we hypothesized that this route may also be a potential pathway for solid particles of ultrafine size. Our objective in a follow-up study, therefore, was to determine translocation of ultrafine 13 C particles to regions of the brain, expecting that UFP deposited on the olfactory mucosa of the nasal region will translocate along the olfactory nerve into the olfactory bulb thereby resulting in high increases in that region as 47

53 opposed to other areas of the CNS. We generated ultrafine elemental 13 C particles (CMD = 36 nm; GSD = 1.66) from 13 C graphite rods by electric spark discharge in an argon atmosphere at a concentration of 16 µg/m 3. Rats were exposed for 6 hrs. and lungs, cerebrum, cerebellum and olfactory bulbs were removed after 1,3,5 and 7 days. 13 C concentrations were determined by isotope ratio mass spectroscopy by Dr. Sharp, our collaborator in these studies at the University of New Mexico, and compared to background 13 C levels of sham-exposed controls (day ). The background corrected pulmonary 13 C added as ultrafine 13 C particles was 1.39 µg. Lung 13 C concentration decreased from 1.39 µg/g (day 1) to.59 µg/g (day 7) over the 7-day post-exposure period. There was a significant and persistent increase in added 13 C in the olfactory bulb of.35 µg/g (day 1) to.43 µg/g (day 7) throughout the 7-day post-exposure period with respective 13 C levels of 3-4 ng per organ. Day 1 13 C concentrations of cerebrum and cerebellum were also significantly increased but the increase was not always significant over the following days (Figure 4-4). We conclude from this study that the CNS can be targeted by inhaled ultrafine particles and that a neuronal route of translocation of nasally deposited ultrafine particles via the olfactory nerve may exist. This represents a previously unrecognized pathway for clearance of solid UFP in the respiratory tract. We wished to confirm the olfactory translocation route for inhaled poorly soluble UFP using another material and selected ultrafine Mn-oxide. Mn can be sensitively detected by AAS, it is a known neurotoxicant and is of importance for certain occupational exposures where these particles are generated as UFP (metal smelting, welding). 1.5 Ultrafine 13 C Particle Inhalation, Rat: Lung and Extrapulmonary Tissue Concentrations µg 13 C/gram organ * * Lung µg 13 C/gram organ 1..5 * * * Olfactory Cerebellum Cerebrum Control * Days after Exposure * * * * * * Figure 4-4. Translocation of inhaled ultrafine 13 C particles to tissues of the CNS over 7 days postexposure. * Significantly increased 13 C compared to control levels (p<.5, Dunnet s test). 48

54 We had already developed a system for generating relevant metal UFP and, based on this method, fabricated rods for use in the electric spark PALAS generator with Mn as the starting material. By introducing a small amount of O 2 into the spark chamber, we were able to generate ultrafine Mn-oxide particles (CMD=31 nm; GSD=1.77). Nine 1-week old F-344 rats were used in these studies. Mn tissue concentrations from sham-exposed rats were compared to these from rats exposed to the ultrafine MnO 2 particles for either 6 days or 12 days. Several tissues were harvested after exposure, including lung, olfactory bulb, trigeminal nerve at the base of the brain, striatum, midbrain, frontal cortex, cortex, and cerebellum. Progressive large increases in the Mn content of the olfactory bulb were found from 6 to 12 days of exposure (Figure 4-5), and smaller increases were also seen in striatum and frontal cortex which are close to the olfactory bulb. Lung Mn content was slightly more than doubled, but there was no evidence of lung inflammation, as assessed by cellular and biochemical lung lavage parameters. Several tissues (lung, heart, brain regions) are presently screened for gene expression changes via microarray analyses. These results of extraordinary high increases of Mn in the olfactory bulb are again consistent with olfactory translocation of solid ultrafine particles. This implies that potential effects of inhaled UFP on CNS functions may occur, in particular if there is continuous accumulation even if environmental exposure conditions are low. Preliminary results of respective CNS effects are summarized below. 2. * ngmn / mg wet wt * * * * * * * Control (NP+P) 6 days 12 days * * *. Lung Olfactory Trigeminal Striatum Midbrain Frontal Cortex Cortex Cerebellum Figure 4-5. Mn concentration in rat lung and brain regions following 6 and 12 days of exposure to ultrafine MnO 2 particles (means ± SD). * Indicates significant difference from controls (green bars). Effects of PTFE Fumes on Gene Expression in CNS: We used UFP of known high toxicity initially to determine changes in CNS gene expression in order to develop a basis against which results of subsequent studies with more benign UFP can be scaled. Our previous work has shown UFP from inhaled PTFE fumes translocate rapidly to interstitial sites of the lung (Oberdörster et al., 2 [HEI Report]). We conducted exposures with PTFE fumes in order to determine how the 49

55 translocation of highly toxic UFPs affects gene expression in extrapulmonary tissues. Four spontaneously hypertensive (SH) rats were exposed for 7.5 mins to PTFE fumes (1.32 x 1 6 particles/cm 3, 18 nm median diameter, 6.31 µg/l F - ) and four were shamexposed. This length of exposure resulted in a mild inflammatory response (3.74 ± 1.36 % lavage PMNs) 24 hrs after exposure. The lavaged lung as well as heart, liver, olfactory bulb, cerebrum, and cerebellum were removed for subsequent microarray analysis (J. Carter, Proctor and Gamble Co.). All of the tissues have not yet been analyzed, but results thus far show that ultrafine PTFE fume particles induced significant changes in gene expression in the cerebrum and the olfactory bulb. Genes involved in inflammation (e.g. IL-1, IL-6, TNF-α) are markedly increased (6- to 14-fold changes) relative to sham-exposed rats in both brain regions. Interestingly, a large decrease (25- to 33-fold) in glutamate transporter gene expression was found in both regions after exposure. These changes are remarkable given the fact that they resulted from inhalation of PTFE fumes and that the response in the lung (i.e. % lavage PMNs) was mild. However, we cannot deduce from these results that neuronal translocation of ultrafine PTFE particles via olfactory nerve caused the CNS changes since a systemic inflammatory state might be responsible as well. Respective results of our Mn UFP exposures described above are forthcoming and should be more revealing since altered gene expression can be correlated with measured increases in Mn levels in the different CNS regions. Studies with a Prototypical Ultrafine Particle Concentrator: Gene Expression Changes: A series of studies was described in the last project report in which groups of rats were exposed for 6 hrs to combinations of concentrated ambient UFPs and inhaled LPS as a priming agent. Through a collaboration with the Harvard PM Center (P. Koutrakis), we used their prototypical UFP concentrator for exposures with aged (2 mos) and young F-344 rats. Results from analyses of cellular and biochemical parameters in lavage fluid were previously reported (summary years 1-3). Newer data, however, is available on gene expression changes in selected brain regions (cerebrum, cerebellum, trigeminal nerves) as analyzed via microarray analyses (J. Carter, Proctor and Gamble Co.). Relative to controls (saline aerosols, sham exposures), no changes were detected in any of the brain regions in those rats (either age group) exposed to concentrated ambient UFPs alone. In the olfactory bulb and cerebellum, the expression of several genes (e.g. metallothionein, inos, IL-6) increased in response to inhaled LPS; these changes were present in both shams and concentrated UFP-exposed groups. Two genes in the trigeminus increased relative to the controls (nerve growth factor, corticotropin-releasing factor). The expression of the two genes increased in olfactory tissue, but not cerebellum, as well. Gene expression appeared to be the same in the two LPS-exposed groups (i.e. no change after concentrated ambient UFPs). These results will be confirmed using slot-blot or Northern analyses. Effects of Exposure to Freshly-Generated On-Road Fine/Ultrafine Particles (Truck Study): For toxicological studies with realistic UFP, diluted exhaust from stationary engines, or concentrated ambient UFP have been used, yet questions remain about how well these particles model those found in ambient air. Freshly-generated UFP are present at high concentrations on highways and vehicle passengers are directly exposed to them when 5

56 driving behind other vehicles on roads. We wished to expose rats to such UFP using a newly-designed mobile environmental laboratory from the University of Minnesota (D. Kittelson) for driving rats directly on highways and to test the potential of highway aerosols to cause effects. Since such exposures have not been carried out before, our objectives were to: (i) demonstrate the feasibility of an on-road exposure study; (ii) determine if there are significant effects in aged rats; and (iii) determine if priming modulates effects in the respiratory tract. This was a multidisciplinary team approach involving PM Center investigators from Research Core 1 (UFP characterization), Research Core 2 (input from epi. studies on HRV changes in people after driving cars in traffic), Research Core 3 (cardiovascular endpoints), Research Core 4 (animal exposures), Research Core 5 (in vitro studies of lavaged cells) as well as Particle Generation Facility Core and Cardiac Facility Core in collaboration with University of Minnesota scientists (D. Kittelson and W. Watts). Old rats (21 mo. F-344) were exposed directly on highways to either the aerosol + gas phase, gas phase only, or filtered air using the on-road exposure system in groups of 5 rats. Some were pretreated with a low dose of inhaled endotoxin or with instilled influenza virus to induce lung inflammation as developed in our studies with laboratory-generated UFP. The exposures in compartmentalized whole-body chambers consisted of 6 hr driving periods on I-9 between Rochester and Buffalo, once or 3 days in a row. Endpoints related to lung inflammation, inflammatory cell activation, and acute phase responses were measured after exposure. In addition, two experiments were conducted in telemetered SH rats, one using inhaled and the other injected LPS priming. Heart rate, blood pressure, temperature, activity, and ECG and blood pressure waveforms were continuously recorded for 5 days after exposure. We found that the exposures were well-tolerated by rats, as baseline values from sham-exposed animals did not differ from what has been previously published for old F- 344 rats (Elder et al., 2); in addition, there were no statistically significant effects of exposure on body weight. Animals were under the constant supervision of personnel in the trailer and no obvious signs of distress were noted during exposure. The daily average number concentration in the control (filtered air) chamber was x 1 5 particles/cm 3. The in-coming sampled air had a number concentration of x 1 5 particles/cm 3 ; however, losses were experienced when that air was directed into the chambers, resulting in exposure number concentrations of x 1 5 particles/cm 3. An effort was made to follow Diesel-emitting trucks; however, very little of this traffic was present during exposures. These results suggest that on-road exposures in mobile laboratories such as the one used here are indeed feasible. We observed the expected increases in response (inflammation, inflammatory cell activation) to the priming agents. In one part of the study, we also found a significant particle-associated increase in plasma endothelin-2 (collaboration with Dr. R. Vincent, Ottawa, Canada), suggesting alterations in vascular endothelial function (Figure 4-6). However, we did not observe any other consistent main effects of on-road particles or interactions between priming agents and particles. The results showed no differences in rats exposed to gas-phase components alone vs. the gas-phase + particle mixture. Given that exposure concentrations were not very consistent over the 6 hr. drive with longer periods of low concentrations, future plans will 51

57 consider more constant exposure by deflecting the exhaust of the mobile laboratory truck after normal dilution has occurred outside into the truck. 25 BEt1 Et1 Et2 Et3 2 pmoles/ml 15 1 * * 5 Saline + Air LPS + Air Saline + Particles LPS + Particles Figure 4-6. Endothelins in rat plasma as determined by HPLC. * indicates significant effect of on-road particles, i.e. groups are significantly different from air-exposed counterparts, p <.5. The telemetry data from SH rats that were exposed to on-road particles or filtered air is currently undergoing analyses for heart rate variability. Preliminary results, though, suggest on-road particle-associated changes (these data will be further analyzed by group and priming agent). As an example, Figure 4-7 shows the data for heart rate in SH rats exposed to particles or filtered air. A significant exposure * time interaction was found for this endpoint that was present for both pre-exposure types (saline or LPS). These data will be compared to results conducted by Core 2 investigators in human subjects exposed to traffic UFP while driving in cars. In summary, for the studies in the mobile lab, the results suggest that on-road exposures are feasible in an animal model, making it possible for future studies with more continuous and source-specific exposures (e.g. sampling directly from mobile laboratory s exhaust). The data from the lavage cell and biochemical analyses are presented in a newly submitted manuscript (Elder et al., 23). 52

58 Figure 4-7. Plot of heart rate (HR) from telemetered SH rats primed with either inhaled saline or LPS (data is combined in this graph) and exposed to on-road particles (exp 1, black line) or filtered air (exp 2, red line). Data was analyzed by repeated measures ANOVA, in which a significant exposure * time interaction was found. The analysis shows a significant decrease in HR for the first 4 time points for particle-exposed rats regardless of priming exposure (i.e. saline or LPS). Future Plans: We will continue to use both laboratory-generated UFP and real world UFP in our animal studies. Since organic carbon compounds constitute the bulk of UFP the smaller they are, a major effort will be directed at generating organic UFP from different organic materials for rodent inhalation studies. This will be done in collaboration with Research Core 1 investigators. Depending on the result of testing of the Harvard UFP Concentrator which will be done collaboratively by our Core 1 and Harvard PM Center scientists we will also perform animal studies using this concentrator. These studies with ambient concentrated UFP will involve longer-term exposures with endpoints of respiratory, cardiovascular, and CNS effects. With respect to extrapulmonary effects, we will in addition to the cardiovascular system also focus on the CNS based on our studies of UFP translocation to the brain and on findings of increased proinflammatory gene expression in CNS tissues following UFP exposure. We also plan to study extrapulmonary translocation pathways for UFP across alveolar epithelium as well as uptake into and translocation along axons of sensory nerves. 53

59 Evaluation of markers of endothelial function has been an endpoint in our epidemiological, clinical, animal and in vitro studies, and we are in the process of setting up an animal model to monitor in real-time the formation of thrombi in peripheral blood vessels following exposures to UFP. These studies represent an integrated team approach among Rochester PM Center investigators, including the aerosol generation, vascular, and immunological facility cores. We are also developing plans to conduct another on-road study involving scientists from all of the Rochester Research Cores in collaboration with Dr. Kittelson s group at the University of Minnesota. As pointed out in the progress report, the plan is to employ a more consistent exposure by deflecting the naturally diluted exhaust into the animal exposure chambers. Endpoints will be respiratory, cardiovascular and CNS parameters. Publications: Couderc, J.P., A. Elder, W. Zareba et al. (22). Limitations of Power-Spectrum and Time-Domain Analysis of Heart Rate Variability in Short-Term ECG Recorded using Telemetry in Unrestrained Rats. Comp. in Cardiol., IEEE Computer Society Press, Vol. 29, pgs Couderc, J.P., A. Elder, X. Xia, S. Eberly, C. Cox, W. Zareba, D. Kittelson, M. Utell, M. Frampton, R. Gelein, G. Oberdörster (23). Using Blood Pressure Signal to Assess the Effect of On-Road Particle Exposure on Heart Rate and Heart Rate Variability in Unrestrained Rats. Circulation Submitted. Elder, A.C.P., N. Corson, R. Gelein, P. Mercer, C. Cox, J. Finkelstein, J. Carter, K. Driscoll, G. Oberdörster. Influenza Virus, Ozone Exposure, and Age Modify the Inflammatory Response to Ultrafine Particles in Mouse Lung (23). Am. J. Respir. Crit. Care Med. 167(7): A761. Elder, A.C.P., R. Gelein, J. Finkelstein, M. Frampton, M. Utell, J. Carter, K. Driscoll, D. Kittelson, W. Watts, P. Hopke, R. Vincent, P. Kumarathasan, G. Oberdörster. Effects of Inhaled Fine/Ultrafine Particles Combined with Other Air Pollutants. INIS 23 Conference Proceedings. Elder, A.C.P., R. Gelein, J. Finkelstein, R. Phipps, M. Frampton, M. Utell, D. B. Kittelson, W. F. Watts, P. Hopke, R. Vincent, P. Kumarathasan, G. Oberdörster. On- Road Exposure to Highway Aerosols. 2. Exposures of Aged, Compromised Rats. Inhal. Toxicol. Submitted. Elder, A.C.P., R. Gelein, M. Azadniv, M. Frampton, J. Finkelstein, G. Oberdörster. Systemic Interactions Between Inhaled Ultrafine Particles and Endotoxin in Two Rat Strains. Inhal. Toxicol. Submitted. Elder, A.C.P., R. Gelein, J.N. Finkelstein, C. Cox, and G. Oberdörster (2). The Pulmonary Inflammatory Response to Inhaled Ultrafine Particles is Modified by Age, Ozone Exposure, and Bacterial Toxin. Inhal. Toxicol. 12 (Suppl. 4): Elder, A.C.P., R. Gelein, M. Azadniv, M. Frampton, and G. Oberdörster (22). Systemic Interactions Between Inhaled Ultrafine Particles and Endotoxin. Ann. Occup. Hyg. 46 (Suppl. 1):

60 Kittelson, D.B., W. F. Watts, J. P. Johnson, M. L. Remerowki, E. E. Ische, G. Oberdörster, R. M. Gelein, A. C. Elder, and P. K. Hopke. On-Road Exposure to Highway Aerosols. 1. Aerosol and Gas Measurements. Inhal. Toxicol. Submitted. Oberdörster, G., Z. Sharp, V. Atudorei, A. Elder, R. Gelein, W. Kreyling, C. Cox. Translocation of Inhaled Ultrafine Particles to the Brain. Inhal. Toxicol. Submitted. 55

61 RESEARCH CORE 5: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression Principle Investigator: Jacob Finkelstein Co-Investigators: Richard Phipps, Michael O Reilly, Günter Oberdörster, Robert Gelein, Facilities Cores: Particle Generation Core, Immunology Core Objectives The experiments proposed within this project are designed to address specific mechanistic hypotheses regarding the interactions between inhaled ultrafine particles and pulmonary cell populations. We have used a number of cell lines and primary cells derived from rats and mice to test the overall PM Center hypothesis that the unique physicochemical characteristics of ultrafine particles in comparison to accumulation mode particles of similar composition contribute to the observed increases in morbidity and mortality in susceptible populations exposed environmentally. We will define mechanisms that follow particle cell contact and test the specific hypothesis that many of the subsequent physiologic effects are the consequences of cellular oxidative stress. We further plan to examine host and environmental factors, including age, the influence of co-exposure to gaseous oxidants or prior priming or activation by pre-exposure to other inflammatory stimuli. A key component of the proposed studies is our plan to examine these particle cell interactions in individual cell populations to begin to assess the role of epithelial, inflammatory and interstitial cells in the systemic response to UP In our experiments, in collaboration with Core 4 we have begun to define susceptible populations on the basis of age as well as prior or concurrent infection. In this way the proposed in vitro experiments are intended to provide a link between the whole animal (Core 4) and controlled clinical (human) exposures (Core 3), described in the other programs of this PM Center. Using animal models developed in Core 4 and marker endpoints identified in Core 2 and Core 3 we are able to elucidate specific mechanism that are triggered following particle cell contact. Summary Years 1-3 The initial phase of our investigations progressed along two simultaneous tracks with the major effort in establishing the various cell models and beginning studies of their responses to particles in solution. During this year we attempted to establish an effective aerosol exposure system for cells, in collaboration with the Exposure Core. This system would have permitted exposure to particles in the airborne state. Although preliminary studies with ultrafine Pt particles were successful, with a measured deposition of ~3 ng per well. Issues of cell viability and uneven distribution made this approach unrealistic. Studies were begun looking at ultrafine carbon black particles as well as a number of studies combining exposures of particles, endotoxin and ozone. We established conditions for LPS induced chemokine expression in vitro. After 24 hours of exposure to either of these stimuli, production of MIP-2 was enhanced 2-8 fold over unexposed controls. Interestingly, culture of these cells at the air/liquid interface, suggested primary release of this chemokine into the airspace compartment. Similar experiments were performed with a macrophage cell line to determine the cellular specificity of this response. Having established appropriate culture conditions of media and adherence, we 56

62 PG/ML determined the appropriate dose response and time course relationship for LPS in these cells. Cytokine and chemokine expression was found to be stimulated 2-3 fold dependent on In Vitro Response of LPS and Carbon Black appropriate dose and on Epithelial Cells time. Similar to our studies with the epithelial cells ultrafine MIP-2 carbon alone did not O LPS TOP TRANSWELL lead to a stimulation of O LPS BOTTOM TRANSWELL 1 LPS TOP TRANSWELL chemokine production. 1 LPS BOTTOM TRANSWELL In fact addition of ultrafine carbon, either before, during or after exposure to LPS lead to a suppression of chemokine production. CARBON 5 CARBON 1 CARBON2 CARBON Figure 5-1 Directional secretion of cytokines by pulmonary epithelial cells We also evaluated the effect of age on the response of cells to particles. In our initial studies we compared macrophage production of cytokines following LPS and particles from month old rats to cells from 1-12 week old rats. As shown in Figure 5-2, when macrophages from young rats are treated with LPS, a clear dose response, PG/ML In Vitro Response of LPS on Freshly Isolated Control Macrophages from Young and 27 Month Rats RAT TNF LPS ( NG/ML ) 27 MONTH RATS YOUNG RATS with TNF as the endpoint, was obtained. A similar dose response relationship was observed with ultrafine carbon particles alone. When the two stimuli are combined, no enhanced effect is observed except at the highest dose of particles. When a similar study was performed with macrophages from old rats a number of clear differences were observed. Interestingly, baseline (unstimulated) production of MIP-2 Figure 5-2 Effct of age on cytokine production by rat (and TNF) was elevated macrophage 3-5% in these cells. In 57

63 addition, response to LPS was enhanced at every dose. Response to particles alone was similar to that observed in young cells. Most significant, in the context of our investigation of age effects and the ability of particles to induce effects at low dose, was the fact that in the aged animals co-administration of particles and LPS lead to synergistic effects at the lowest dose of particles. This result was similar to results obtained in the in vivo studies in collaboration with Core 4, in which enhanced response to combined insult was noted in aged rats. An important question that was addressed, in collaboration with Cores 2, 3 and 4, was the choice of appropriate endpoints. While production of TNFa or MIP-2 following interaction with particles may be well described, the role of these mediators in environmental particle induced systemic disease is less clear. Thus, some studies were carried out looking at additional endpoints. These were chosen on the basis of data obtained from the clinical studies and the possibility that measurements could be made in the in vivo studies. Among the cytokines measured the only one that showed some promise was IL-6. Production of IL-6 was observed when epithelial cells were cultured in the presence of silica, used as a positive particle control, and LPS. However no effect of the addition of ultrafine carbon particle on IL-6 protein or mrna was observed in our mouse experiments. We also developed reagents and approaches that would allow extension of our in vitro studies to human cells while also developing a test of our oxidant stress hypothesis. We developed a human lung cell line, A549, that was stably transfected with a reporter gene that other studies have shown was responsive to oxidant stress. During the past year we have made significant progress in developing in vitro models that will be useful in understanding the mechanism of ultrafine particle induced gene expression in various cell types. Additionally, these models should prove useful in studies of other size Cytokine Levels in Aging Mice fractions and we are using them to attempt to Plasma Cytokine (pg/ml) Young IL-6 Old IL-6 Young TNF Old TNF Figure 5-3 Cytokine Levels in Plasma of Young and Old Mice differentiate between particles of differing toxicity and activation potential. A goal of these experiments is to define mechanisms of cellular activation, the effects of age or prior activation on cytokine gene activation, and differential responses of epithelial cells and macrophages to particles of different sizes. 58

64 We had previously proposed using isolated cells from aged animals as a model to study age related particle induced gene activation. In collaboration with Core 4 we began to further characterize the aged mice and began to evaluate the utility of isolated cells from these mice as a model of aging. Since one of the outcomes being measured was changes in cytokine gene expression we first measured baseline cytokine levels in the plasma of aged mice. The cytokines we chose to measure were based on the recommendations of our advisory committee that we use similar markers that are used, or suggested for use in the in vivo animal experiments and the human clinical studies. As shown in Figure 5-3 both TNFα and IL-6 were significantly elevated in the aged mice. 1 CONTROL CARBON/IRON + 1 NG/ML LPS 8 MOUSE MIP YOUNG OLD We also continued to evaluate the effect of age on the response of cells to Figure 5-4 Expression of MIP-2 by Macrophages After particles. Results of Stimulation by Endotoxin : Effect of Age our most recent studies in mice, compared the effects of age on macrophage responses to particles and LPS. Similar to our data in rats, mouse macrophages showed an age dependent difference in cytokine production following stimulation with particles or LPS. (Figure 5-4 and 5-5) An important development during this year was the development and use of laboratory generated ultra fine particles containing various metals. The choice of the specific metal was based on the data provided by our Chemical UFP characterization Project (Core 1) that Iron is among the most abundant metal constituents. This material was produced by our particle generation Core. Our studies with this particle would be used to assess the likelihood of using this particle in both the animal studies (Core 4) as well as potentially in the Human Clinical Studies (Core3). We compared macrophage production of cytokines following LPS and particles (with C/Fe) incubation with cells from 2-22 month old and 8-1 week old mice (Figure 5-5). Baseline MIP-2 and TNF was significantly elevated in cells from old mice. After stimulation the old mice were also found to be more responsive. 59

65 MIP-2 pg/ml YOUNG MOUSE OLD MOUSE 5 CONTROL LPS LPS 1 NG/ML 1 NG/ML When particles and LPS were combined as a stimulus an enhanced effect is observed only in the old cells Figure 5-5 Expression of MIP-2 by Macrophages After Stimulation by Endotoxin and particles except at the highest dose of particles.. Most significant, in the context of our investigation of age effects and the ability of particles to induce effects at low dose, was the fact that in the aged animals co-administration of particles and LPS lead to synergistic effects at the lowest dose of particles. This result is somewhat similar to results obtained in the in vivo studies in which enhanced response to combined insult was noted in aged rats CONTROL CARBON/IRON + 1 NG/ML LPS YOUNG We also continued to examine the types of endpoints to be used. These were chosen on the basis of data obtained in collaboration with the clinical studies (Cores 2 and 3) and data from the animal experiments in Core 4 and the possibility that measurements could be made in the in vivo studies. Among the cytokines measured the only one that showed some promise was IL-6. Production of IL-6 was observed when human pulmonary epithelial cells (A549) were cultured in the presence of silica, used as a positive particle control, and LPS. However no effect of the addition of mixed C/Fe particle on IL-6 OLD Figure 5-6 Expression of PGE2 by Macrophages After Stimulation by Endotoxin and Particles 6

66 protein or mrna was observed in our mouse experiments. We will continue to consult with the in vivo animal studies (Core 4) to attempt to develop additional in vitro markers that could accurately predict effects of the inhalation studies. One marker that has proven useful is the production of prostaglandins. By measuring changes in PG s we could indirectly monitor activity of COX-2, the rate limiting enzyme and also determine the role of PG s in pulmonary and systemic inflammation. Stimulation of young and old cells with a combination of ultrafine C/Fe particles and LPS lead to an increase in PGE2 production (Figure 5-6). As with MIP-2 (and TNF) this was mainly observed in the cells from the old mice. This is consistent with our other age experiments an reinforces the theme of age related increased susceptibility. In order to pursue our mechanistic studies, testing the oxidant stress/ signaling hypothesis we have continued to use the cell lines we have developed. This cell line has proven to be responsive to particles and we have initiated studies looking at the possible signaling pathways that may be involve in particle induced stimulation. Using inhibitors of signaling 8 pathways (Figure 5-7)we MIP-2 have begun to Control 6 investigate the LPS mechanism of Particles C/Fe particle induced gene 4 expression. The addition of the p38 kinase inhibitor PD effectively inhibited both LPS and particle PG/ML DMSO PD 9859 SB 2358 Figure 5-7 Effect of Kinase Inhibitors on MIP-2 Expression by Epithelial Cells after addition of 5 ug/ml ultrafine C/Fe Particle induced MIP-2 expression. In contrast SB2358, which blocks p44 MAP kinase had little effect. We plan to continue to use this cell line as well as others to test the response of cells to particles and develop an understanding of the potential interactions between particles and endotoxin. In addition to the human cell line we have recently developed a stably transfected mouse epithelial line using the same reporter construct. We are currently evaluating the response to particles in these cells and their relative sensitivity to priming. 61

67 PG/ML YOUNG VS. OLD MOUSE MACROPHAGE CONDITIONED MEDIA IN PRESENCE OF CARBON/IRON MOUSE MIP-2 YOUNG MOUSE OLD MOUSE MAC CM MAC CM % OF CONTROL Luciferase activity Ultrafine CARBON/IRON.47 ug/cm ug/cm ug/cm ug/cm 2 Figure 5-8 Effect of Age on Particle induced cytokine production Summary of Year 4 Progress Recent work has continued the refinement of in vitro models of particle cell interactions with the goal to define mechanisms of cellular activation, the effects of age or prior activation on cytokine gene activation and differential responses of epithelial cells and macrophages to particles of different sizes. and the development of assays for the specific endpoints. We continue to maintain our main focus in the area of understanding how age effects the interaction of particles. Most significant, in the context of our investigation of age effects and the ability of particles to induce effects at low dose ( Figure 5-8), was the fact that in the aged animals coadministration of particles and LPS lead to synergistic effects at the lowest dose of particles of.47 ug/cm 2. Our current results show both an age dependent change in cytokine production as well as a response at low, environmentally relevant doses. We have continued our studies on dose effects by refining our indicator A549 LUCIFERASE CULTURES EXPOSED TO CARBON/IRON ug/cm 2 CARBON/IRON Figure 5-9 Luciferase Activity in Transfected A549 cells cell line to be useful in detecting particle induced effects at a wide range of doses. 62

68 Using the transfected A549 cell line developed by our laboratory we are able to detect changes in gene expression at particle doses below 1 ug/cm 2. This clearly puts us in the realistic range of nanoparticle mass burdens. We anticipate working with our particle generation core to determine if this relationship wopuld be maintained for particles of Cellular Bi-layer Light microscopy shows the presence of endothelial cells on the basal side of the membrane and epithelial cells on the apical side of the membrane measurements. Alveolar Epithelial Cells Endothelial Cells Apica l side Membrane Basal side Figure 5-1 Biculture model system different composition or with particle collected using the Harvard ultrafine particle concentrator that we have available for our use once Core 1 studies of the concentrator performance have been successfully completed. We are also working with our Immunology Core to attempt to make additional cytokine measurements on these cells to confirm this result. Our initial studies comparing cytokine analysis with luciferase activity show a reasonable correlation between these two During the past year we have worked closely with Core 4 (in vivo animal exposures) on a number of IL-6 Upregulation in HMEC-1 experiments. As described in that core a large real 7 time on road 6 exposure study 5 was carried on.5% Serum 4 exposing aged 3 7%Serum primed rats to the actual highway 2 particle aerosol. 1 After exposure, when animals untreated 1ug/ml LPS 1 ng/ml TNF-alpha were sacrificed, IL-6 in conditioned media (ug/ml) we recovered the Figure 5-11 IL-6 prodution by Human Microvascular Endothelial lavage cells and Cells (HMEC)Endothelial cells placed them in culture, in the presence or absence of a laboratory test particle ( mixed C/Fe). Our initial analysis of the 63