2007 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 , Elder, Alison C.P. , Oakes, David , Couderc, Jean-Philippe , Phipps, Richard , Gelein, Robert , Kreyling, Wolfgang
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, 2006 through September 30, 2007
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Human Health , Air


The objective of the Core 4 studies is 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 freshly-generated exhaust particles from low and ultralow diesel fuel, and intratracheally administered ultrafine and fine ambient particles from different sites and sources. Effects 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:

Exposures of Rats to Freshly-Generated On-Road Aerosols

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, of which one is the JCR:LA-cp rat. Although these rats are not hyperglycemic (according to published literature and our results), the JCR cp/cp rats are obese, hyperlipidemic, hyperinsulinemic, and have atherosclerotic and ischemic lesions that are hallmark features of human type II diabetes. Heterozygotes or homozygous normals (designated by JCR +/?) are not obese or hyperinsulinemic and do not exhibit the arteriosclerotic lesions that the cp/cp rats do. Females of the same strain are similar to the +/? rats, which is why males are used exclusively in our studies.

One of the hypotheses being investigated in our studies in coordination with the Cores 2 and 3 projects is that some of the adverse health effects associated with exposure to ambient particulate pollution are causally related to inhaled ultrafine particles (UFP) and their gaseous co-pollutants, especially when aerosols are freshly-generated.  Our previous studies using an on-road tractor-trailer exposure system have been conducted in old F-344 rats and spontaneously hypertensive (SH) rats.  Since our first study showed that there was high variability in particle number and gas concentrations when “chasing” other vehicles, we made modifications to the system (i.e. use of telescoping exhausts inlets mounted on the rear of the trailer) to achieve more continuous exposures by taking in the exhaust produced by the mobile laboratory’s engine (primary particle size, 13-19 nm; mass concentration, 9-27 μg/m3; number concentration, 1.6-4.3 x 106/cm3). This design allowed different fuels to be used to create specific exhausts under realistic environmental dilutions. In the most recent truck study, we exclusively used the JCR rats, focusing on our mechanistic hypothesis relating to the sensitivity imparted by vascular lesions.

Unlike our earlier studies, the rats did not receive any pre-treatments, e.g. inhaled endotoxin or instilled influenza virus. Due to the limitation on the total number of animals that could be exposed, we focused on genetic background and exhaust atmosphere as being the response modifiers.  Exposures (6 hr/day; 1 or 4 days in a row) to low- and ultralow-sulfur Diesel fuel exhaust emission aerosols were conducted in compartmentalized whole-body chambers while the truck was driven between Rochester and Utica (NY I-90). Endpoints related to lung inflammation, inflammatory cell activation, acute phase responses, platelet activation, and atherosclerotic lesion progression were measured after exposure.  As in the past, groups of rats were also implanted with radiotransmitters to continuously monitor changes in heart rate, blood pressure, temperature, and activity associated with exposure to exhaust emissions or clean, filtered air.

There was an obvious effect of obesity and insulin resistance on baseline lavage inflammatory parameters, namely that the JCR cp/cp (obese) rats had higher total cell numbers, percentages of PMNs, and lavage fluid protein content and LDH and Β­glucuronidase activities than their lean litter mates.  However, the emission aerosols did not have any consistent effects on these parameters.  We also measured several parameters in serum and lavage fluid related to inflammation (PAI-1, IL-1Β, IL-6, MCP­1, TNF-α) and metabolism (insulin, leptin) using bead array technology (rat adipokine panel, Luminex detector system).  There was a lot of variability in the results, more so in the JCR cp/cp than in +/? rats, but the obese rats had higher levels for most of the parameters we measured.  MCP-1 was not detectable in lavage fluid, whereas IL-6 was not detectable in serum. The only strikingly obvious effect of exposure was in serum leptin levels, which were increased in JCR cp/cp rats in low- and ultralow-sulfur fuel-exposed rats as compared to gas-phase only and filtered air-exposed rats.  We also measured, through collaboration with Dr. Petia Simeonova (CDC/NIOSH), the levels of mitochondrial DNA (mtDNA) in aortae as a marker of oxidative injury.  Although we have previously observed decreases in mtDNA amplification and there was slightly lower amplification in exhaust emission-exposed rats, there were no significant differences between exposure groups.

Based on our previous studies showing that solid UFP are translocated to the brain and, in one case, that glial fibrillary acidic protein (GFAP) was increased (a marker of astrocyte activation), we sampled tissues from several distinct brain regions for an analysis of GFAP protein content.  While there was a trend that JCR cp/cp rats had lower levels of GFAP in the sampled brain regions (olfactory bulb, olfactory tract, cortex, striatum, cerebellum, hippocampus), there was no consistent pattern of response of this protein to the exhaust emission aerosols.  CCl2 (MCP-1) and TNF-α mRNA levels were also quantitated via PCR in olfactory bulb tissue; the JCR cp/cp rats had higher transcript levels as compared to JCR +/?, but, again, there was no consistent effect of treatment on inflammatory mediator gene expression.

Those analyses that are on-going include the evaluations of blood pressure, heart rate, and heart rate variability in the JCR rats that were exposed to filtered air or whole exhaust (low and ultralow sulfur fuel) as well as chemical analyses of filter samples that were collected during the exposures.

Exposure of JCR Rats to Concentrated Ambient Ultrafine Particles

We continued our studies in male JCR cp/cp (obese, insulin-resistant) and +/? (lean) rats that were exposed 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 (HUCAPS).  We have now completed a study in which rats were exposed for 6 hrs/day, 5 days/week for 4 weeks (total of 20 exposure 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 the end of the 4 week exposure.

As was true from the truck study with JCR rats, many baseline parameters were elevated in the obese, insulin-resistant rats (JCR cp/cp) as compared to the lean controls (JCR +/?). 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 Β­glucuronidase activities; plasma fibrinogen; total white blood cell number; and the number of blood leukocyte aggregates.

After three days of exposure, we found statistically significant increases in lavage fluid protein concentration and LDH activity in the HUCAPS-exposed JCR cp/cp rats as compared to filtered air-exposed and +/? rats.  These changes occurred in the absence of increases in the percentage of lavage fluid PMNs.  These changes were not observed following 4 weeks of exposure. Platelet (hematology, flow cytometry measurements) and platelet microparticle (flow cytometry) numbers were slightly decreased by three days of exposure and could be due to the increased aggregations with leukocytes that we observed in HUCAPS-exposed rats.  However, these platelet-leukocyte as well as leukocyte-leukocyte aggregates seemed to be blunted in the obese diabetic rats.  We did not observe these changes after 4 weeks of exposure and, rather, found that the platelet number decreased following HUCAPS exposure in both the cp/cp and +/? rats.  An intriguing piece of data from the 3-day exposure was that aortic mtDNA amplification was blunted in HUCAPS-exposed JCR cp/cp rats relative to air-exposed controls and +/? rats, suggesting that oxidative DNA damage had occurred.  However, this finding could not be confirmed with samples from the 4-week study, as the long fragment from the extracted mtDNA was degraded due to technical issues. 

We also examined the same brain tissues as listed above for GFAP protein content and inflammatory cytokine/chemokines message levels.  As we also described above, the GFAP levels in all of the brain regions tended to be lower in the JCR cp/cp rats as compared to the +/? rats.  However, there were no changes in the levels of GFAP protein or TNF-α or MCP-1 mRNA levels that were related to exposure atmosphere.

The JCR cp/cp rats are the animal model of choice for the Core 4 studies of this PM Center due to their obesity and insulin resistance-related atherosclerotic lesions. Humans with NIDDM have been shown to be more susceptible to the effects of ambient air pollution and they also have such lesions.  However, our data thus far does not indicate that the cp/cp rats are more sensitive than their non-obese litter mates and, in fact, are perhaps less sensitive than F-344 rats.  These findings, then, require some pursuit. We are collaborating with Dr. Andrew Ghio (US EPA), who is interested in iron homeostasis in humans and animal models used in air pollution research.  Samples from the HUCAPS-exposed cp/cp and +/? rats were sent to him for evaluation.  Dr. Ghio’s findings suggest that iron homeostasis is disrupted in the cp/cp rats such that they have higher loads of lavage fluid, liver and lung tissue Fe as well as higher levels of transferrin and ferritin in lavage fluid.  These findings imply that the cp/cp rats may have higher baseline levels of oxidative stress and may, therefore, compensate by synthesizing higher levels of antioxidants. We plan to measure key antioxidant species to test this hypothesis.  In addition, we found that leptin levels were high in cp/cp rats, both in the serum and lavage fluid.  Recent in vitro studies have shown that leptin induces surfactant exocytosis from type II alveolar epithelial cells, which may impart greater resistance to the effects of freshly-generated exhaust emission and concentrated ambient ultrafine particle-containing aerosols.

Future Activities:

We just completed a HUCAPS exposure study in JCR rats that were implanted with radiotelemetry devices to monitor blood pressure and ECG waveforms.  Lean and obese rats were exposed for 4 days in a row to HUCAPS aerosols and, after a minimum of 4 weeks, the same rats were exposed to clean, filtered hospital air.  Telemetry data was collected for 48 hrs prior to each exposure and for three days after exposure.  The analysis of this data is on-going.  In addition to the telemetered rats, additional sets of non-implanted JCR rats were exposed to HUCAPS aerosols or filtered air.  Twenty-four hrs after the last exposure, these rats were euthanized, blood was collected, and tissues were fixed via whole-body perfusion.  Heart, lung, abdominal aorta, aortic arch, and whole brain were removed and saved for later analyses.  Of particular interest is the GFAP protein expression in various brain regions and this analysis is being done via immunohistochemistry by our collaborator, Dr. Kim Tieu.

We will continue work to learn more about the JCR model and why it may be less sensitive to the effects of exhaust emission aerosols as compared to other strains we have used in the past. Specifically, we will carry out measurements on pulmonary surfactant levels and baseline antioxidant status.

Secondly, we will continue our analyses of the heart rate variability data from the telemetered JCR rats that were exposed to freshly-generated emission aerosols and concentrated ambient PM.  We have begun discussions with other investigators both here in the US and in Europe regarding standardization of recording, measurement, and analysis techniques so that results from various laboratories can be more easily compared.

Lastly, in September/October, 2008, we will carry out a fourth on-road exposure study using MEL and JCR rats. We will test the specific hypothesis that exhaust filter technology will cardiovascular, pulmonary and CNS responses in rats with compromised cardiovascular systems.

Journal Articles on this Report : 2 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 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
  • Abstract: Taylor and Francis-Abstract
  • 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, PHYSICAL ASPECTS, Scientific Discipline, 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

    Progress and Final Reports:

    Original Abstract
  • 2006 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