2006 Progress Report: Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics

EPA Grant Number: R832415C004
Subproject: this is subproject number 004 , established and managed by the Center Director under grant R832415
(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: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
Investigators: Oberdörster, Günter , Elder, Alison C.P.
Current Investigators: Oberdörster, Günter , Couderc, Jean-Philippe , Elder, Alison C.P. , Gelein, Robert , Kreyling, Wolfgang , Oakes, David , Phipps, Richard
Institution: University of Rochester
Current Institution: University of Rochester , GSF-National Research Center for Environment and Health
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2012)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

The objectives are to correlate physico-chemical particle characteristics (from Core 1 measurements) with pulmonary and cardiovascular endpoints following exposure of diabetic rats (JCR rats) to inhaled ambient concentrated ultrafine/fine particles; inhaled diesel exhaust particles on road from low and ultralow diesel fuel use; and intratracheally administered ultrafine and fine ambient particles from different sites and sources. Effect measurements will take into account endpoints determined in the epidemiological (Core 2) and clinical (Core 3) studies and coordinate mechanistic evaluations with Core 5 in vitro studies. In addition, effects on the CNS will also be assessed.

Progress Summary:

Exposure of JCR Rats to Concentrated Ambient Ultrafine Particles

Humans with type II diabetes have been shown in recent epidemiological studies to be susceptible to the adverse health effects related to ambient particulate matter exposures. There are several animal models of diabetes, but the JCR:LA-cp rat is one that combines insulin resistance with obesity, hyperlipidemia, hyperinsulinemia, and the atherosclerotic and ischemic lesions that are hallmark features of human type II diabetes. By the age of 6 months, 100 percent of the JCR cp/cp rats have atherosclerotic lesions. One important point about the JCR cp/cp rats is that they are not particularly hyperglycemic, so are probably more correctly termed “pre-diabetic.” The heterozygous and homozygous wild-type rats are neither diabetic nor obese. To coordinate with Core 2 and 3 studies and to address specific aspects of the core hypothesis of the Rochester PM Center, we exposed male JCR cp/cp (obese, diabetic) and +/? (lean, non-diabetic) to concentrated ambient ultrafine particle-containing aerosols (count median diameter ~ 75 nm; number concentration ~ 0.05-1.3 x 106/cm3) using the Harvard system (HUCAP). Exposures lasted for 6 hrs per day and were for 1 or 3 days. We obtained bronchoalveolar lavage fluid, blood, and various tissues (lung, heart, aorta, brain regions, kidney, spleen, liver, pancreas) from aerosol- and clean, filtered air-exposed rats ~18 hrs after exposure. (Chemical characterization of the filer samples is still on-going.)

Many parameters were elevated in the obese, diabetic rats as compared to the lean, non-diabetic controls. The data have not yet been analyzed statistically, so the following comments and those in the subsequent paragraph should be considered preliminary in nature. The parameters that were elevated in cp/cp as compared to +/? JCR rats included total lavage cell number; lavage fluid protein concentration and LDH and β-glucuronidase activities; plasma fibrinogen; total white blood cell number; and the number of blood leukocyte aggregates.

Several parameters were altered in the HUCAP-exposed in either JCR cp/cp and +/? rats. Total lavage cell numbers, the concentration of total protein (cytotoxicity, epithelial membrane integrity), and the activities of LDH and β-glucuronidase (cytotoxicity) appeared to be increased in HUCAP-exposed cp/cp and +/? rats. These changes occurred in the absence of increases in the percentage of lavage fluid PMNs, which is considered a sensitive indicator of lung inflammation; increases in PMNs may have explained the other changes we observed in other lavage parameters. HUCAP exposure also caused a slight increase in plasma fibrinogen in +/? rats, but this response was blunted in the obese diabetic rats. The number of peripheral white blood cells was increased by HUCAP exposure in both cp/cp and +/? rats. Platelet (hematology, flow cytometry measurements) and platelet microparticle (flow cytometry) numbers were slightly decreased by exposure and could be due to the increased aggregations with leukocytes that we observed in HUCAP-exposed rats. However, these platelet-leukocyte as well as leukocyte-leukocyte aggregates seemed to be blunted in the obese diabetic rats. These data are shown as an example below (Figure 1). We plan to further analyze the flow cytometry data to look at the intensity of ICAM-1 staining on blood leukocytes, to quantitate the platelet-platelet aggregates, and to assess the size distribution of platelets themselves. Examining this battery of endpoints will allow us to gain information about the activation status of circulating leukocytes and platelets. Comparisons can then be made with similar information obtained in human diabetics that are planned to be exposed to the HUCAP aerosols. Results will also be compared to those from the Core 2 (epidemiology) studies and mechanistic pathways explored using in vitro methods (Core 5).

Figure 1. Flow Cytometric Determination of Platelet and Leukocyte Aggregation State

Figure 1. Flow Cytometric Determination of Platelet and Leukocyte Aggregation State

Acellular Detection of Ultrafine Particle-Associated ROS

We have adapted for our Core 4 studies an assay developed by Core 1 investigators to measure the activity of reactive oxygen species (ROS) that are associated with ambient ultrafine and fine particles (UFP). Core 1 is using the assay to test field samples, whereas we are using it to measure ROS activity of laboratory-generated and commercial surrogate, freshly-generated highway particles, and concentrated UFP. The assay is based on the oxidation of deacetylated (via NaOH) 2’,7’-dichlorodihydrofluorescin diacetate to its fluorescent product, 2’,7’-dichlorodihydrofluorescein (DCFH), in a phosphate buffer containing the radical electron acceptor, horseradish peroxidase. Results obtained with particulate samples are compared to hydrogen peroxide standards and activity is expressed in peroxide equivalents. Figures 2a and b show the results of ROS measurements performed with ultrafine and fine carbon-based particles (commercially-available and laboratory-generated). In Figure 2a, the dose metric is particle mass; in Figure 2b, it is particle surface area. When dose is expressed as surface area, the responses become more similar. However, differences remain that cannot be explained by surface area alone. Chemical composition is likely to regulate ROS activity and may be another important consideration in this response, as in biological responses. For example, we have shown that Printex-90 and Sterling V have surface areas that differ by a factor of ~8 and that, based on mass dose, the higher surface area particle (Printex-90) has greater inflammatory potential in rats exposed via inhalation for 90 days. When dose was normalized to surface area, though, inflammatory and proliferative responses were more similar. Nevertheless, the Sterling V had slightly greater inflammatory potential than would have been predicted by surface area dose alone. The results presented in Figure 2b could partially explain those results; specifically, Sterling V has slightly greater ROS activity than does the Printex-90 for a given surface area dose. This may be related to the fact that the Sterling V has more particle-associated PAH as compared to the Printex-90.

Figure 2. Particle-Associated ROS in Terms of Mass (a) and Surface Area (b) Dose

Figure 2. Particle-Associated ROS in Terms of Mass (a) and Surface Area (b) Dose

Exposures of Rats to Freshly-Generated On-Road Aerosols

We have continued to analyze data collected at our previous on-road exposures in a Mobile Exposure Laboratory (MEL). We hypothesized that the adverse health effects in elderly individuals with cardiopulmonary diseases associated with exposure to ambient particulate pollution is causally related to inhaled ultrafine particles (UFP) and their gaseous co-pollutants, especially when aerosols are freshly-generated. To test this hypothesis, we had exposed old rats (20-22 mo. F-344; 15 mo. spontaneously hypertensive [SH] rats) to freshly-generated highway aerosols (particles [< 1 μm]/gas phase, gas phase only, or filtered air) using an on-road tractor-trailer exposure system. An earlier set of studies using the MEL system demonstrated that there was daily variability in particle number and gas concentrations. Thus, we sought to produce more continuous particle/gas-phase mixture exposures under conditions that were realistic with respect to aerosol dilution and photochemical oxidation processes. In order to accomplish this, MEL was modified to allow sampling of its own exhaust plume. Inlet pipes at the end of the trailer allowed sampling of the diluted engine exhaust plume emitted from the front of the tractor (primary particle size, 13-19 nm; number concentration was 1.6-4.3 x 106/cm3). Rats were treated with inhaled endotoxin or instilled influenza virus either before or after on-road exposures to prime respiratory tract cells. Controls were treated with saline. Additional pre-treatments included intraperitoneally-injected endotoxin or saline for those SH rats that underwent telemetry recordings. Exposures (6 hr/day; 1 or 3 days in a row) in compartmentalized whole-body chambers occurred while driving between Rochester and Utica (NY I-90). Endpoints related to lung inflammation, inflammatory cell activation, and acute phase responses were measured after exposure. In addition, SH rats were implanted with radiotransmitters to continuously monitor heart rate, blood pressure, temperature, and activity. Stable blood pressure data were later analyzed for changes in heart rate and heart rate variability.

The particle number, NO, and CO2 concentrations in the in-coming aerosol indicated that the engine exhaust plume was sampled (dilution ~ 400:1). The expected significant changes in response due to the priming agent (endotoxin, virus) were observed. Exposure atmosphere had no effect on lavage PMN percentage, cell oxidant release, or biochemical indices of cytotoxicity. However, a significant increase in total lavage cell number, increases in macrophage intercellular adhesion molecule (ICAM)-1 expression, a decrease in the percentage of blood PMNs, and changes in plasma fibrinogen were due to the particle/gas phase mixture. The small increases found in AM surface ICAM-1 expression due to particle/gas-phase mixture exposure were independent of priming agent, length of exposure, or length of recovery period. The particle/gas-phase mixture caused an increase in plasma fibrinogen within 24 hrs of exposure in influenza-exposed old rats. When the evaluation period was 3 days after aerosol exposure, the mixture caused decreases in fibrinogen. We also harvested selected tissues for subsequent microarray analyses and found greater message expression for TNF-α and its receptor in lung, heart, and olfactory bulb from those rats exposed to the particle/gas-phase mixture. Furthermore, there was a trend in response suggesting that the effects of the particle/gas-phase mixture were slightly greater and more persistent than those of the gas phase alone. In telemetered SH rats, the on-road aerosol caused a decrease in heart rate that became more pronounced over a 5-day post-exposure monitoring period. This was accompanied by small increases in high frequency power. These changes occurred in both endotoxin and saline pre-treated SH rats (Figures 3a and b, endotoxin [LPS] only).

These results show that the inhalation of diesel exhaust-derived ultrafine particle/gas phase mixtures affects pulmonary inflammatory cell activation, acute phase responses, and alterations in heart rate and heart rate variability (HRV) in compromised, old rats. Some effects of the aerosol mixture were evident 3-5 days after the last on-road exposure. As compared to previous studies in MEL in which the particle number and gas concentrations were lower and less continuous, we observed here more pronounced effects of freshly-generated vehicle exhaust aerosols.

Figure 3. Changes in Heart Rate (a) and High Frequency Power (b) in Endotoxin Pre-treated SH Rats Exposed to Freshly-Generated On-Road Aerosols

Figure 3. Changes in Heart Rate (a) and High Frequency Power (b) in Endotoxin Pre-treated SH Rats Exposed to Freshly-Generated On-Road Aerosols

Future Activities:

Subchronic exposures (4 weeks) of JCR rats to concentrated ultrafine/fine ambient particles using the HUCAPs will be performed next. Endpoints will be the same as reported above for our 3 day exposures.

In September/October 2006, we will carry out a new on-road exposure, using the MEL described above. JCR rats will be exposed to the exhaust from using new ultralow diesel fuel in the truck and cardiovascular, pulmonary, and CNS responses will be compared to those from low sulfur fuel exhaust exposed rats.


Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other subproject views: All 62 publications 49 publications in selected types All 43 journal articles
Other center views: All 190 publications 156 publications in selected types All 143 journal articles
Type Citation Sub Project Document Sources
Journal Article Elder A, Couderc J-P, Gelein R, Eberly S, Cox C, Xia X, Zareba W, Hopke P, Watts W, Kittelson D, Frampton M, Utell M, Oberdorster G. Effects of on-road highway aerosol exposures on autonomic responses in aged, spontaneously hypertensive rats. Inhalation Toxicology 2007;19(1):1-12. R832415 (2010)
R832415 (2011)
R832415 (Final)
R832415C003 (2011)
R832415C004 (2006)
R832415C004 (2011)
R827354 (Final)
R827354C001 (Final)
R827354C003 (Final)
R827354C004 (Final)
R828046 (Final)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
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  • Other: University of Minnesota-Abstract
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  • Journal Article Oberdorster G, Stone V, Donaldson K. Toxicology of nanoparticles: a historical perspective. Nanotoxicology 2007;1(1):2-25. R832415 (2007)
    R832415 (2008)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2006)
    R832415C004 (2007)
    R832415C004 (2010)
    R832415C004 (2011)
  • Full-text: ResearchGate-Full Text PDF
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  • Abstract: Taylor and Francis-Abstract
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  • Journal Article Pui DYH, Qi C, Stanley N, Oberdorster G, Maynard A. Recirculating air filtration significantly reduces exposure to airborne nanoparticles. Environmental Health Perspectives 2008;116(7):863-866. R832415 (2007)
    R832415 (2008)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2006)
    R832415C004 (2010)
    R832415C004 (2011)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Journal Article Semmler-Behnke M, Takenaka S, Fertsch S, Wenk A, Seitz J, Mayer P, Oberdorster G, Kreyling WG. Efficient elimination of inhaled nanoparticles from the alveolar region: evidence for interstitial uptake and subsequent reentrainment onto airways epithelium. Environmental Health Perspectives 2007;115(5):728-733. R832415 (2007)
    R832415 (2008)
    R832415 (2010)
    R832415 (2011)
    R832415 (Final)
    R832415C004 (2006)
    R832415C004 (2007)
    R832415C004 (2010)
    R832415C004 (2011)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Supplemental Keywords:

    on-road exposure, diabetic rats, concentrated particles, ROS,, RFA, Health, Scientific Discipline, PHYSICAL ASPECTS, Air, particulate matter, Toxicology, Health Risk Assessment, Risk Assessments, Physical Processes, atmospheric particulate matter, atmospheric particles, acute cardiovascular effects, airway disease, exposure, animal model, ambient particle health effects, atmospheric aerosol particles, ultrafine particulate matter, PM, inhalation toxicology, cardiovascular disease

    Relevant Websites:

    http://www2.envmed.rochester.edu/envmed/PMC/indexPMC.html Exit

    Progress and Final Reports:

    Original Abstract
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R832415    Rochester PM Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832415C001 Characterization and Source Apportionment
    R832415C002 Epidemiological Studies on Extra Pulmonary Effects of Fresh and Aged Urban Aerosols from Different Sources
    R832415C003 Human Clinical Studies of Concentrated Ambient Ultrafine and Fine Particles
    R832415C004 Animal models: Cardiovascular Disease, CNS Injury and Ultrafine Particle Biokinetics
    R832415C005 Ultrafine Particle Cell Interactions In Vitro: Molecular Mechanisms Leading To Altered Gene Expression in Relation to Particle Composition