Grantee Research Project Results
2002 Progress Report: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
EPA Grant Number: R827354C004Subproject: this is subproject number 004 , established and managed by the Center Director under grant R827354
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Rochester PM Center
Center Director: Oberdörster, Günter
Title: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
Investigators:
Current Investigators: Oberdörster, Günter , Elder, Alison C.P.
Institution:
Current Institution: University of Rochester
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2002 through May 31, 2003
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air
Objective:
The overall objectives of this research project are to: (1) identify factors that are causally associated with adverse pulmonary and extrapulmonary responses to inhaled laboratory-generated and real-world ultrafine particles (UFPs); and (2) develop rodent models of human disease to test our central hypothesis that UFPs contribute to the increased morbidity and mortality of susceptible people in association with small increases in urban particles. The specific objectives of this research project are to perform animal studies that will complement the field and controlled clinical particulate matter (PM) studies and form a link to the mechanistic in vitro studies. Our experiments focus on factors such as particle size; dosimetric aspects (lung deposition and disposition); host susceptibility (advanced age, cardiovascular disorders, respiratory tract sensitization); and pollutant coexposure (ozone).
Progress Summary:
Progress for Years 1-3 of the Project
Years 1-3 of the project focused on the: (1) generation and use of UFP-containing atmospheres in toxicology studies that are relevant for human ambient air exposures; and (2) achievement of concordance for the animal studies with the other research projects (R827354C002, R827354C003, and R827354C005) in terms of endpoints measured. We also have focused a great deal of attention on the uptake and tissue-specific distribution of laboratory-generated inhaled solid ultrafine carbon and metal particles (13C, MnO2). First, several studies have been completed in which ozone was used as a copollutant. In addition, we have completed studies in young and old rodents using two different relevant priming agents: inhaled low-dose endotoxin and human influenza virus. The particle types used in our studies have included laboratory-generated ultrafine carbon particles (+/- Fe); laboratory-generated organic UFP; concentrated Rochester ambient fine/UFPs; and freshly-generated vehicle exhaust emission fine/UFPs on highways (results described in Year 4 report). The parameters measured in research projects R827354C002, R827354C003, R827354C004, and R827354C005 are very similar in terms of markers of pulmonary inflammation, cell adhesion molecules, coagulation and acute phase factors, and heart rate and blood pressure variability.
UFP-Containing Atmosphere Generation
We have developed the ability to generate ultrafine carbon particles (~26 nm count median diameter [CMD], 15-20 x 106 particles/cm3) that contain, for example, transition metals (Fe, V) for toxicology studies or 13C UFP for use in dosimetry studies. The multigroup studies described below were performed with ultrafine carbon particles that were mixed with Fe (25%). After completing several of these multigroup studies, we have been able to compare responses in animals inhaling ultrafine carbon versus 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 (Dr. Ann Aust, Utah State University), and they generate more hydroxyl radicals in the presence of peroxide than UFPs without Fe (Dr. Vincent Castranova, National Institute for Occupational Safety and Health). We also found that the laboratory-generated UFPs initially contained up to 30 percent organic material because of 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 percent organic carbon.
An increasing number of studies and our R827354C001 measurements are showing that smaller UFPs (<~20 nm) consist of organic compounds, a significant component of which 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 (University of Utah). Used motor oil was utilized in this system, which was capable of generating stable organic UFP (30-50 nm; 106 particles/cm3) aerosols for 6 hours.
Toxicology Studies with Ambient UFPs
Through our collaboration with the Harvard PM Center (Dr. Petros Koutrakis), we have performed studies with ambient PM using a prototypical UFP concentrator that samples air from a moderately busy road and concentrates the ultrafine fraction with some overlap into the fine mode (7-316 nm; CMD=35 nm, geometric standard deviation [GSD]=1.9; ~2 x 105 particles/cm3). Results from studies with young and old F-344 rats show that the concentrated UFPs have significant main effects, but that these effects are different in the two age groups. Specifically, the polynuclear leukocyte (PMN) response to the combination of concentrated UFPs and lipopolysaccharide (LPS) in young rats is significantly lower than to LPS alone; these 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. Tissues taken from the exposed F-344 rats were screened for gene expression changes via microarray analyses and these results are presented below.
Development and Characterization of Compromised Animal Models
Several “multigroup” studies have been completed, in which young and old rats and mice were exposed to combinations of laboratory-generated UFPs and ozone with respiratory tract cell priming. 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. In agreement with our earlier studies in rats (Elder, et al., 2000), one set of studies showed that all four factors (UFPs, ozone, LPS, and age) had significant main effects for most of the respiratory and cardiovascular endpoints examined in aged and young mice. 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 adhesion molecule [AM] surface intercellular adhesion molecule-1 [ICAM-1] expression), the UFP effect was dependent on the presence of LPS. Our findings that inhaled UFP can alter blood PMN surface ICAM-1 expression are in agreement with results from R827354C003 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 UFPs and ozone, but the data also suggest an imbalance in old animals between pro- and antioxidant species production.
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. Old F-344 (23 months) and spontaneously hypertensive (SH) (15 months) rats were used in these studies. Results suggest that: (1) the carbonaceous core of ambient PM is not solely responsible for cardiovascular effects; or (2) the ambient PM-induced alterations in heart rate variability (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 research projects R827354C002 and R827354C003 evaluations of electrocardiogram (ECG) recordings, our cardiac core has developed a Windows-based algorithm for the analysis of ECG and blood pressure signals from rats recorded using the data sciences international system (St. Paul, MN). The software includes noise reduction and filtering steps and then analyzes HRV based on time and frequency domain approaches to quantify autonomic changes (standard deviation normal-normal, root mean square of successive differences, low frequency [LF], high frequency [HF], and LF/HF ratio). The validation of our algorithm for the quantification of HRV has been implemented employing pharmacological blockade to dissect the relationship between heart rate, HRV, and arterial blood pressure. A first paper was published in the proceedings of the Institute of Electrical and Electronics Engineering, Inc. Computers in Cardiology Annual Conference in 2002 (Couderc, et al., 2002) describing the limitations and difficulties encountered in a first protocol. We later developed an algorithm for measuring HRV from the blood pressure signal from which we obtained higher stability and reproducibility. Greater morphological stability of the QRS waves was resolved by changing the ECG lead placement such that wave morphology was less affected by animal movements. The software was applied to various data sets to assess the effect of PM on the autonomic nervous system. Preliminary results are currently submitted (Couderc, et al., submitted, 2003).
Dosimetry Studies
Nine F-344 rats were exposed to ultrafine 13C particles for 6 hours to determine if ultrafine elemental carbon particles translocate to the liver and other extrapulmonary organs following inhalation as single particles by rats (Oberdörster, et al., 2002). 13C progressively accumulated in the liver from 0.5 to 24 hours postexposure, at which time the 13C concentration was more than one-third the 13C concentration found in the lung (by tissue weight, the 13C amount in the liver was approximately fourfold greater than in the lung). These results demonstrate effective translocation of ultrafine elemental carbon particles to the liver by 1 day after inhalation exposure, possibly via direct input into the blood following deposition in the respiratory tract or by uptake into the blood from the gastrointestinal tract.
In a subsequent pilot study with a prolonged 7-day post-exposure period, 13C of lung and extrapulmonary organs was analyzed on day 1 and day 7 post ultrafine 13C particle exposure. Results showed again significant amounts of added 13C in the liver on day 1, but no longer on day 7. However, on day 7, significant increases in added 13C in the heart, brain, and olfactory bulb were found.
Other studies performed within our PM Center’s Pilot Programs used ultrafine 192Ir particles. Dr. Kreyling of the GSF München found, in contrast to our results with ultrafine 13C particles, that only minimal amounts of ultrafine 192Ir particles were translocated to extrapulmonary organs after intratracheal inhalation exposure (Kreyling, et al., 2002). However, it also was found that larger (80 nm) 192Ir particle translocation was tenfold slower than that of smaller 15 nm 192Ir particles. Collectively, our studies suggest that particle size as well as the material and surface properties (structure, composition) may influence the efficiency of UFP translocation.
Summary of Year 4 Progress
HRV Studies
The final HRV analysis program of the cardiology research project has been implemented and tested using pharmacological interventions (atropine, propranolol) to test if our method can identify sympathetic and parasympathetic blockade. The algorithm has now been applied to data from 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 UFPs and ozone would induce greater lung injury and oxidative stress than either component by itself in young and old mice that were preexposed to influenza virus, a common respiratory pathogen, to prime respiratory tract cells. Young and old male C57 mice (10 weeks, 21 months) were exposed to ultrafine carbon particles containing 25 percent Fe (CMD ~26 nm, ~140 µg/m3) and ozone (0.5 ppm) for 6 hours, alone and in combination. Lung inflammation was induced with intratracheally instilled human influenza virus (X-31; H3N2; 104 EID50) 48 hours prior to exposures. UFPs were found to have consistent and independent effects on pulmonary inflammation and inflammatory cell activation. Ozone, influenza virus, and age had significant main effects for all endpoints examined. In addition, the interactions with UFP that were consistently significant involved ozone, influenza virus, and age. Using microarray analyses, we also have screened lung and heart tissue for changes in gene expression. There was a trend toward higher proinflammatory and lower antiinflammatory gene expression in tissues from old as compared to young animals. In addition, we found evidence of significant gene changes in heart tissue. The consistency of the results from this study with those from the LPS “multigroup” study is remarkable and strengthens the causality of associations that were found in the statistical analyses.
Translocation of Inhaled Solid UFP to the Central Nervous System (CNS)
Reports in the literature have identified the olfactory nerve axons acting as translocation pathways for soluble metals and we hypothesized that this route also may be a potential pathway for solid UFPs. We generated ultrafine elemental 13C particles (160 µg/m3; CMD = 36 nm; GSD = 1.66) and exposed rats for 6 hours. 13C concentrations were determined by isotope ratio mass spectroscopy (Dr. Zachary Sharp, University of New Mexico). There was a significant and persistent increase in added 13C in the olfactory bulb from 0.35 µg/g on day 1 to 0.43 µg/g on day 7, with respective 13C levels of 30-40 ng per organ. We conclude from this study that the CNS can be targeted by inhaled UFPs, and that a neuronal route of translocation of nasally deposited UFPs via the olfactory nerve may exist. This represents a previously unrecognized pathway for clearance of solid UFPs in the respiratory tract.
We confirmed the olfactory translocation route for inhaled, poorly soluble UFPs using ultrafine Mn-oxide. Mn can be sensitively detected by atomic absorption spectroscopy. We generated ultrafine Mn-oxide particles (CMD=31 nm; GSD=1.77) and exposed three groups of three young F-344 rats for 6 or 12 days. Progressive large increases (>threefold) in the Mn content of the olfactory bulb were found, and smaller increases also were seen in striatum and frontal cortex, which are close to the olfactory bulb. Lung Mn content slightly more than doubled, but there was no evidence of lung inflammation. The results are consistent with olfactory translocation of solid UFPs, implying that potential effects of ambient UFPs on CNS functions may occur, especially if there is continuous accumulation under even low environmental exposure conditions.
Studies with a Prototypical UFP Concentrator: Gene Expression Changes
We harvested tissues to examine gene expression changes in selected brain regions (cerebrum, cerebellum, trigeminal nerves) after exposure as analyzed via microarray analyses (J. Carter, Proctor and Gamble Co.). No changes were detected in any of the brain regions in those rats 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; two genes in the trigeminus increased as well (nerve growth factor [NGF], corticotropin-releasing factor). The expression of these two genes increased in olfactory tissue as well. Gene expression appeared to be the same in the two LPS-exposed groups (i.e., sham- and concentrated ambient UFP-exposed). These results will be confirmed using slot-blot or Northern analyses.
Effects of Exposure to Freshly Generated On-Road Fine/UFPs (Truck Study)
For toxicological studies with realistic UFPs, diluted exhaust from stationary engines, or concentrated ambient UFPs have been used, yet questions remain about how well these particles model those found in ambient air. Freshly generated UFPs are present at high concentrations on highways, and vehicle passengers are directly exposed. We exposed rats to such UFPs using a newly designed mobile environmental laboratory from the University of Minnesota (D. Kittelson) to test the potential of highway aerosols to cause effects. Because such exposures have not been conducted before, our objectives were to: (1) demonstrate the feasibility of an on-road exposure study; (2) determine if there are significant effects in aged rats; and (3) determine if priming modulates in the respiratory tract. This was a multidisciplinary team approach involving PM Center investigators from research projects R827354C001-R827354C005, the particle generation and cardiac facility research projects, and collaboration with University of Minnesota scientists (D. Kittelson and W. Watts). Old rats (21 month F-344) were exposed directly on highways to either the gas phase/particle mixture, gas phase only, or filtered air. Some were pretreated with a low dose of inhaled endotoxin or with instilled influenza virus to induce lung inflammation. The exposures consisted of 6 hour driving periods on I-90 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. Telemetry data was collected continuously 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., 2000); 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 filtered air chamber was 0.01-0.12 x 105 particles/cm3. The incoming sampled air had a number concentration of 1.95-5.62 x 105 particles/cm3, but some losses were experienced when that air was directed into the chambers. An effort was made to follow diesel-emitting trucks when present. However, exposure concentrations were not very consistent, with long periods of low particle number concentrations. No differences in response were observed in rats exposed to gas-phase components alone versus the gas-phase/particle mixture. We did observe the expected responses to the priming agents. In one part of the study, we also found a significant particle-associated increase in plasma endothelin-2 (Dr. R. Vincent, Ottawa, Canada), suggesting alterations in vascular endothelial function. We also observed some main effects of on-road particles and interactions between priming agents and particles relating to surface ICAM-1 expression on lavage AMs and blood PMNs as well as plasma fibrinogen. The changes in blood PMN ICAM-1 paralleled findings from research project R827354C003 studies in humans exposed to laboratory-generated UFPs. The data from the lavage cell and biochemical analyses are presented in a newly submitted manuscript (Elder, et al., 2003).
The telemetry data from SH rats that were exposed to on-road particles or filtered air are currently undergoing analyses for HRV. Preliminary results suggest on-road particle-associated changes (e.g., a decreased heart rate). A significant exposure*time interaction was found for this endpoint that was present for both preexposure types (saline or LPS). These data will be compared to results obtained by research project R827354C002 investigators in human subjects exposed to traffic UFPs while driving in cars.
In summary, for the studies in the mobile laboratory, the results suggest that on-road exposures are feasible in an animal model. Future plans will, therefore, include more constant exposure by deflecting the exhaust of the mobile laboratory truck towards the sampling pipe, thus producing more continuous and source-specific exposures.
Future Activities:
Future activities will continue to use both laboratory-generated and real-world UFPs in our animal studies. Because organic carbon compounds constitute the bulk of the smaller UFPs, a major effort will be directed at generating organic UFPs from different organic materials for rodent inhalation studies in collaboration with research project R827354C001 investigators. Depending on the analysis of the Harvard UFP concentrator (collaboration between our R827354C001 and Harvard PM Center scientists), we also will perform animal studies using this concentrator. These studies with ambient concentrated UFPs will involve longer term exposures with endpoints of respiratory, cardiovascular, and CNS effects. With respect to extrapulmonary effects, in addition to the cardiovascular system we also will focus on the CNS based on our results showing UFP translocation to the brain and increased proinflammatory gene expression in CNS tissues. We plan to study extrapulmonary translocation pathways for UFPs across alveolar epithelium as well as uptake into and translocation along axons of sensory nerves. 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 UFPs. These studies represent an integrated team approach among Rochester PM Center investigators, including the aerosol generation, vascular, and immunological facility cores. We also are developing plans to conduct another on-road study involving scientists from all of the Rochester research projects in collaboration with Dr. Kittelson. 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 include respiratory, cardiovascular, and CNS parameters.
Journal Articles:
No journal articles submitted with this report: View all 33 publications for this subprojectSupplemental Keywords:
urban air pollution, atmosphere, air, health, atmospheric sciences, biochemistry, environmental chemistry, molecular biology, health risk assessment, risk assessments, incineration, combustion, combustion engines, air toxics, tropospheric ozone, PM2.5, particulates, ultrafine particles, particulate matter, particle exposure, particle size, aerosol, ambient air, ambient air monitoring, ambient air quality, animal model, atmospheric, cardiopulmonary, cardiopulmonary responses, cardiovascular disease, cardiovascular vulnerability, coronary artery disease, cytokine production, fine particles, human exposure, human health, human health effects, environmental health effects, inhalation toxicology, lung, lung inflammation, morbidity, mortality, pathophysiological mechanisms, pulmonary, pulmonary disease, stratospheric ozone, sensitive populations, susceptible populations,, RFA, Health, Scientific Discipline, Air, particulate matter, Toxicology, air toxics, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, Atmospheric Sciences, Molecular Biology/Genetics, ambient air quality, cytokine production, particle size, particulates, sensitive populations, biostatistics, health effects, risk assessment, cardiopulmonary responses, fine particles, human health effects, morbidity, ambient air monitoring, lung, cardiovascular vulnerability, pulmonary disease, susceptible populations, animal model, ambient air, environmental health effects, particle exposure, ambient monitoring, particulate exposure, lung inflamation, pulmonary, coronary artery disease, inhalation toxicology, tropospheric ozone, urban air pollution, mortality, urban environment, aerosol, cardiopulmonary, human health, aerosols, cardiovascular disease, ultrafine particles, pathophysiological mechanismsRelevant Websites:
Relevant Web Sites: http://www2.envmed.rochester.edu/envmed/ Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R827354 Rochester PM Center Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827354C001 Characterization of the Chemical Composition of Atmospheric Ultrafine Particles
R827354C002 Inflammatory Responses and Cardiovascular Risk Factors in Susceptible Populations
R827354C003 Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects
R827354C004 Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
R827354C005 Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
R827354C006 Development of an Electrodynamic Quadrupole Aerosol Concentrator
R827354C007 Kinetics of Clearance and Relocation of Insoluble Ultrafine Iridium Particles From the Rat Lung Epithelium to Extrapulmonary Organs and Tissues (Pilot Project)
R827354C008 Ultrafine Oil Aerosol Generation for Inhalation Studies
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2004 Progress Report
- 2003 Progress Report
- 2001 Progress Report
- 2000 Progress Report
- 1999 Progress Report
- Original Abstract
27 journal articles for this subproject
Main Center: R827354
106 publications for this center
91 journal articles for this center