2000 Progress Report: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events

EPA Grant Number: R827354C004
Subproject: 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: Airborne PM - Rochester PM Center
Center Director: Oberdörster, Günter
Title: Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
Current Investigators: Oberdörster, Günter , Elder, Alison C.P.
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, 2000 through May 31, 2001
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air


Earlier studies in our laboratory and others have shown that ultrafine particles elicit significantly greater pulmonary inflammatory responses than larger particles of the size of the accumulation mode when administered at the same mass to the respiratory tract. Normal background levels of atmospheric ultrafine particles are very low, about 1-2 µg/m3, although their number concentration at these background levels is high, up to 2-4 x 104 particles/cm3. Episodic increases as high as 5 x 105 particles/cm3 and 1 x 106 particles/cm3 have been observed. Our hypothesis is that ultrafine particles in the urban atmosphere contribute to the adverse health effects associated with PM. The objectives of the animal studies are to evaluate pulmonary and cardiovascular effects of inhaled laboratory-generated ultrafine and fine carbon particles using animal models of increased susceptibility and to obtain data on the deposition and subsequent fate of inhaled ultrafine particles.

In recent studies, we have developed a model of respiratory tract priming in rats and mice using very low doses of inhaled LPS. This model should mimic the early stages of a respiratory infection with gram-negative bacteria, and we continue using this model in combination with host factors such as old age and cardiovascular conditions. With respect to the latter, spontaneously hypertensive rats (SHR) and hypertensive rats that are prone to heart failure at 10-12 months of age (SHHF) are commercially available for use in our studies.

Very few data on the deposition of inhaled ultrafine particles in the respiratory tract of experimental animals have been reported. Preliminary data from our laboratory and others using inhaled ultrafine metal particles in rats indicated some translocation to the liver. However, solubilization of ultrafine metal particles, even those of very poorly soluble platinum and iridium, cannot be excluded and may have accounted for distribution of the metal to extrapulmonary sites. Because elemental carbon is insoluble, we have developed a technique to generate ultrafine pure 13C particles for use in these studies.

Progress Summary:

A major effort in this Core was devoted to improving the generation system for ultrafine carbon particles with respect to eliminating organic contaminants and setting up a separate system for generation of ultrafine organic particles and of mixed ultrafine carbon/iron particles. Exposures of young and old mice to the mixed carbon/iron ultrafine particles are now in progress, with the evaluation of pulmonary and cardiovascular endpoints. A continued focus of this Core also was the development, characterization, and use of compromised animal models and improving the analysis of rat ECG recordings from radio-transmitter implants. Furthermore, we continued our dosimetry studies using 13C ultrafine particles and have performed preliminary experiments using ultrafine fluorescent beads. Finally, we have initiated a collaborative study with the Harvard PM Center using their ultrafine ambient particle concentrator to expose hypertensive old rats (SHR rats) implanted with radio-transmitters for recording ECG, blood pressure, and body temperature. A summary of these studies is provided below.

A. Studies With Mixed Inorganic and With Organic Ultrafine Particles

We pursued our initial plans to add a transition metal into our ultrafine carbon particles to investigate the impact on pulmonary inflammation, first in animal and in vitro (Core 5) studies as the basis for subsequent controlled clinical studies (Core 3). Generation of mixed carbon/iron ultrafine particles involved the mixing of carbon black powder and metallic iron powder and adding glucose and distilled water to form a paste. This mix was extruded through a 3 mm ID glass tube. The resulting cylinders were first dried and then graphitized by slowly raising temperatures up to 2,300ºC.

The graphitized cylinders of the C/Fe mix were inserted into the PALAS soot generator for generation of mixed ultrafine particles. Particle size distribution was about 25 nm (count median diameter) with a geometric standard deviation of 1.7. At a particle number concentration of 1 x 107 particles/cm3, the mass concentration was approximately 100 µg/m3. Results from an in vitro citrate assay to determine bioavailability (performed by Dr. Aust, Utah State University) showed that these particles exhibited very high biological activities (370 nmol of bioavailable iron/mg of particle). ESR spin trapping analysis of the ultrafine particles also was performed (in collaboration with Dr. Castronova, NIOSH, Morgantown, WV) and revealed that the addition of iron to the ultrafine carbon particles resulted in the generation of OH radicals in the presence of H2O2.

In parallel with in vitro studies of Research Core 5, we have started 6-hour exposures of groups of aged (18 months) and young (8 weeks) mice to the mixed C/Fe ultrafine particles at approximately 100 µg/m3, with and without prior priming by inhaled low dose LPS and with and without additional exposure to ozone (0.5 ppm). The following endpoints are determined in this study: indicators of inflammatory responses from lung lavage analyses and histopathology; extraction of RNA from lung, heart, spleen, and liver for subsequent analysis of gene expression; blood leucocyte analysis for adhesion molecule expression; and blood plasma analysis for acute phase proteins. Results are being evaluated now.

A new research initiative also was started based on the suggestion by SAC members to investigate effects of organic ultrafine particles, which are a major fraction of urban ultrafines. This was a concerted effort of investigators in all five Research Cores of the Center. It involved at first the characterization of our spark discharge-generated ultrafine carbon particles prompted by an observation of investigators at the GSF (Munich, Germany) that these particles contain significant amounts of organics. Secondly, we began to develop methodologies for generating an ultrafine condensation aerosol from C2O and C3O compounds and from fresh as well as used motor oil.

Measurements (Dr. G. Cass's laboratory of Research Core 1) of the composition of the ultrafine particles generated with the electric spark discharge soot generator (PALAS generator) revealed that these particles consisted of more than 30 percent organic compounds. Additional collaborative efforts with investigators from Lovelace Respiratory Research Institute (Albuquerque, NM) and from GSF in Germany led to further characterization of the organic compounds. One source for these appeared to be off-gassing from plastic materials. A major effort of our Center was subsequently devoted to "cleaning up" the PALAS generator's plastic components inside the generator by replacing them with either stainless steel, Teflon®, or ceramic parts. Despite these efforts, there are still significant amounts of organic carbonaceous materials on the emitted ultrafine particles. These could be coming from small contaminants in the diluting air, which may adsorb rapidly onto the large surface area of the ultrafine carbon particles. The surface area of the electric spark discharge-generated ultrafine carbonaceous particles was determined in collaboration with Dr. B. Fubini (Turino, Italy) to be 580 m2/g.

Development of an ultrafine condensation aerosol generator for pure organic compounds is in progress. Methods include use of an electrospray nozzle, heating in a tube furnace, and subsequent cooling with and without seed nuclei. This resulted so far in ultrafine particles of C30-alkane (Triacontane) below 100 nm, and of used motor oil of approximately 100 nm. Pilot studies of exposures of rats to these particles did not show significant changes in lung lavage parameters. These efforts to generate organic ultrafine particles are presently continued by Dr. John Veranth, University of Utah, who is a Visiting Scientist supported by our PM Center's Visiting Scientist Program.

B. Development and Characterization of Compromised Animal Models

1. Analysis of HRV in unrestrained rats using a telemetry system. In our preliminary analysis of heart rate variability (HRV) in rats, we confirmed that short-term ECG recordings obtained using a telemetry system with implantable transmitters can provide reliable information about HRV in unrestrained rats. We designed software for measuring HRV from rat ECGs using both time- and frequency-based methods. Both approaches require accurate detection of the R peak from the ECG signal to extract the tachogram (RR intervals across time) from which the HRV is estimated. The frequency domain parameters were computed based on the power spectral density of the tachogram.

We implemented two frequency domain parameters: the energy of the high frequency band (HF: 0.7-2Hz) to estimate the vagal control (parasympathetic tone) and the low-frequency band to estimate sympathetic tone (0.1-0.7 Hz). The time domain parameters were SDNN and RMSSD. SDNN is the standard deviation of the normal-to-normal RR intervals over a specific recording period; RMSSD is the root means square of successive differences between normal-to-normal RR intervals.

In a preliminary study, we evaluated the validity of our algorithm for short-term ECGs (<10 minutes). Because the optimal length of short-term ECG recordings has not been determined for the analysis of HRV in rats, we studied the stability of HRV parameters according to the length of the ECG signal. This analysis led us to conclude that at least 1,500 beats are needed to obtain reliable and reproducible estimations of HRV parameters. Also, we found that the stability of the HRV parameters is dependent on the average heart rate. Less than 5 percent variation between averaged heart rate from continuous ECG segments is needed to ensure reliable HRV measurements.

Our investigation offers preliminary concepts about how to approach the analysis of HRV in unrestrained rats based on short-term ECG recordings. Stability of HRV using either time or frequency domain parameters depends on the activity of the rat during the recording. The stability of the HRV parameters, especially from the frequency domain, is higher when the identification of a stable period can be done. In our study, we propose a set of criteria to ensure stability of the HRV estimation when measured on ECGs shorter than 5 minutes. This set of criteria will be used in our future studies to analyze HRV in rats after particle exposures.

2. Studies with endotoxin priming. Endotoxin (LPS) priming involves two different models, administration by inhalation and administration by i.p. injection, followed by inhalation exposure to the ultrafine particles within 30-minutes after LPS dosing. The former should serve as a model of the early stage of a respiratory tract infection with gram-negative bacteria, the latter as a model of extrapulmonary systemic infection.

The methods described above for heart rate variability (HRV) analysis were applied to data from a crossover study on the effects of inhaled ultrafine particles (carbon/20 percent Fe) with and without inhaled endotoxin priming. Six SH rats (18-20 months) with radiotelemetry implants were exposed to all combinations of ultrafine particles and endotoxin, following which ECG, body temperature, activity, and blood pressure signals were collected through the fifth post-exposure day. Preliminary analyses have not revealed any changes in HRV associated with exposure. However, the criteria described above currently are being applied in a reanalysis of the data. As a positive control, these same rats were exposed systemically (i.p.) to endotoxin prior to sacrifice in a pilot study. Preliminary analyses suggest dramatic HRV changes, and the results will be used in future experiments to: (1) target the appropriate postexposure times for analyses; and (2) gauge the magnitude of expected changes in HRV.

The endotoxin inhalation model also was used to examine the effects of ultrafine carbon/Fe particles in combination with ozone in young and old mice (see section A above). Separate ongoing studies are being done to assess the effect that systemic priming of lung cells with endotoxin has on the response to inhaled ultrafine particles. Initial results show that, aside from the independent effects of endotoxin itself, there are some significant interactions between particles and endotoxin. Specifically, the effect of the combination of particles and endotoxin tends to go in the opposite direction compared to the endotoxin response alone. This was true for BAL and blood cell intracellular oxidant generation, fibrinogen release, IL-6 production, and peripheral blood PMN number and percentage. Interestingly, the particles had independent effects on blood cell DCFD (2',7'-dichlorofluorescindiacetate) oxidation. These results suggest that inhaled ultrafine particles can have effects outside the lung, whether they are direct or indirect.

3. Influenza mouse model. We are developing a model of influenza A virus infection in mice as a priming agent for subsequent ultrafine particle exposures. The inflammatory response in the lung after intranasal instillation of influenza virus into mice was measured over several postexposure days and compared to results obtained after intratracheal instillation. The peak of the inflammatory response in terms of appearance of PMNs in lung lavage occurs at approximately 48 hours after instillation, at which timepoint a lymphocytic infiltration also occurs. Individual variability after intranasal instillation appears to be much greater compared to intratracheal instillation and subsequent studies will, therefore, be done using intratracheal instillation of influenza A. Studies are presently ongoing in influenza-primed mice with additional instillation of ultrafine TiO2 particles to test the concept that influenza priming increases sensitivity to a subsequent second particulate stimulus.

4. TNF-alpha transgenic mice. We also are testing a mouse model that demonstrates increased sensitivity to inflammatory stimuli (i.e., TNF-alpha transgenic mice). These mice express the human TNF-alpha gene, yet baseline levels of inflammatory mediators are not different from those in control mice. However, after an inflammatory stimulus in these primed mice, responses are significantly greater, particularly for TNF-alpha production, compared to wild type mice. Studies to evaluate the usefulness of this mouse model for inhaled particle exposures are underway, comparing initially the responses to ultrafine vs. fine TiO2 particles. Once this model is characterized, it will be used with exposures to ultrafine carbon particles.

C. Dosimetry Studies

Our present studies evaluating the fate of inhaled ultrafine particles in the lung are focused on their translocation to extrapulmonary tissues. For this purpose, we have developed a method to make 13C graphite electrodes using 13C amorphous carbon and 13C glucose as binder. Our initial results following inhalation of these ultrafine 13C particles in rats and mice had indicated that there seems to be a rapid transport of the ultrafine carbon particles to extrapulmonary sites, most notably the liver, shortly after deposition. Subsequent analyses of the data have shown that there is significant variation in the results upon repeated measurements. We spent much of our efforts on resolving this issue of variability.

Variability depends largely on the size of the tissue sample used for 13C analysis. Small differences in baseline levels of 13C in organs also can occur. A major improvement, therefore, was achieved by increasing the amount of lyophilized tissue for analysis to 1 mg, which reduced variability significantly. Rats were exposed to ultrafine 13C particles for 6 hours, and lung and extrapulmonary organs were excised at 30 minutes, 18 hours, and 24 hours postexposure for analysis. Results of these improved analyses confirmed our initial interpretation of the translocation of the ultrafine 13C particles from the lung to the liver. This also was found in a second experiment; however, the exposure concentration of 13C was only 1/4 to 1/2 the concentration of the first study and 13C increases in the liver were only significant at 18 and 24 hours postexposure. These studies will be repeated using the same higher concentration of inhaled 13C that was used in the first study.

We also evaluated the possibility of using another surrogate ultrafine particle for these translocation studies, focusing on iridium as the least soluble among metals. After a number of attempts to dissolve iridium particles in spiked lung samples, we are now able to quantitatively recover iridium using dissolution under increased pressure at high temperature. The dissolution of iridium is accomplished using a high strength acid digestion bomb after prior tissue digestion in nitric acid. After addition of a mixture of HCl/HNO3, the sample is sealed in the bomb and heated to 230ºC for 4 hours. The digestate is then diluted with 18 Mohm water before analysis by AES or ICPMS. Recovery of Ir in spiked lung samples is greater than 95 percent.

Future Activities:

We have initiated collaborative studies with the Harvard PM Center (P. Koutrakis) to use their ultrafine particle concentrator for exposures of our aged SH rats prepared for telemetry recordings of ECG, blood pressure, and body temperature. These studies will be performed at Rochester after installing the Harvard ultrafine concentrator in a location where air from an adjacent busy road can be drawn in and concentrated for animal exposures. We will in these studies also use our LPS priming model. We also plan to use our other animal models described above for exposures. We focus on the use of models of compromised animals because the epidemiological studies have demonstrated that adverse responses to ambient particles are only seen in compromised hosts but do not occur in the healthy organism. Animal studies seem to confirm this unless very high concentrations or very long exposure times are used. Concentrating the ambient ultrafine particles in our planned studies should mimic episodic increases of those particles that have been reported in the literature.

We also will continue to characterize in more detail our compromised animal models, focusing mainly on the ones described above. A major effort continues to be the dosimetry studies where we plan to use animals with a compromised pulmonary system and advanced age (i.e., SHR rats) for comparison with normal healthy animals. A new focus will be on exposures to organic particles once the method of generating those particles has been established successfully. In addition, combined exposures to ultrafine carbon particles with added transition metals (Fe) in combination with ozone will continue.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Other subproject views: All 33 publications 31 publications in selected types All 27 journal articles
Other center views: All 104 publications 98 publications in selected types All 90 journal articles
Type Citation Sub Project Document Sources
Journal Article Oberdorster G. Pulmonary effects of inhaled ultrafine particles. International Archives of Occupational and Environmental Health 2001;74(1):1-8. R827354 (Final)
R827354C004 (2000)
R827354C004 (2001)
R827354C004 (Final)
R826784 (Final)
R832415 (2010)
R832415 (2011)
R832415 (Final)
R832415C004 (2011)
  • Abstract from PubMed
  • Full-text: Precaution.org-Full Text PDF
  • Abstract: SpringerLink-Abstract
  • Journal Article Riesenfeld E, Chalupa D, Gibb FR, Oberdo G, Gelein R, Morrow PE, Utell MJ, Frampton MW. Ultrafine particle concentrations in a hospital. Inhalation Toxicology 2000;12(Suppl 2):83-94. R827354 (Final)
    R827354C003 (2000)
    R827354C003 (2001)
    R827354C003 (2002)
    R827354C003 (Final)
    R827354C004 (2000)
    R827354C004 (Final)
    R826781 (2000)
    R826781 (2001)
    R826781 (Final)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C003 (2011)
    R832415C004 (2011)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
  • Supplemental Keywords:

    pollution prevention, atmosphere, particulates, metals, sensitive population., 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, biostatistics, health effects, particle size, particulates, risk assessment, sensitive populations, cytokine production, cardiopulmonary responses, fine particles, human health effects, lung, morbidity, ambient air monitoring, ambient air, cardiovascular vulnerability, pulmonary disease, susceptible populations, animal model, ambient monitoring, particle exposure, environmental health effects, pulmonary, lung inflamation, particulate exposure, coronary artery disease, tropospheric ozone, urban air pollution, inhalation toxicology, aerosol, cardiopulmonary, mortality, human health, urban environment, aerosols, cardiovascular disease, metals, ultrafine particles

    Relevant Websites:

    http://www2.envmed.rochester.edu/envmed/pmc/indexpmc.html Exit

    Progress and Final Reports:

    Original Abstract
  • 1999 Progress Report
  • 2001 Progress Report
  • 2002 Progress Report
  • 2003 Progress Report
  • 2004 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R827354    Airborne PM - 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