Final Report: Mechanisms of Toxicity of Particulate Matter Using Transgenic Mouse Strains

EPA Grant Number: R827355C005
Subproject: this is subproject number 005 , established and managed by the Center Director under grant R827355
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Airborne PM - Northwest Research Center for Particulate Air Pollution and Health
Center Director: Koenig, Jane Q.
Title: Mechanisms of Toxicity of Particulate Matter Using Transgenic Mouse Strains
Investigators: Luchtel, Daniel L. , Baker, Coralie , Ceballos, Diana , Kavanagh, Terrance J , Leaman, Susan , McConnachie, Lisa , Rosenfeld, Michael
Institution: University of Washington
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 30, 2004 (Extended to May 31, 2006)
Project Amount: Refer to main center abstract for funding details.
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air

Objective:

The working hypothesis of this project, Mechanisms of Toxicity of Particulate Matter Using Transgenic Mouse Strains, is that PM deposited in the lung causes release of inflammatory cytokines that adversely the cardiovascular system. In PM research, there is a need to identify potential health conditions that enhance susceptibility to adverse PM health effects. To address this, transgenic mouse strains with specific genetic alterations that mimic human cardiovascular disease (CVD) were used. The nature of such health conditions provide insight into the biological mechanisms by which PM mediates acute and chronic health effects.

Summary/Accomplishments (Outputs/Outcomes):

Aged apolipoprotein E deficient (apoE-/-) mice, a model of CVD, were implanted with miniaturized electrocardiogram radiotelemetry devices and intranasally exposed to 50 μl saline or 50 μg Seattle PM2.5 (PM having a mean aerodynamic diameter of ≤ 2.5 μm) or silica in 50 μl of saline. They were monitored for a one-day baseline prior to and for four days following exposure. After an initial increase in both heart rate and activity in all groups, there was delayed bradycardia with no change in activity of the animals in the PM and silica exposed groups. In addition, with PM and silica exposure, there was a decrease in the heart rate variability (HRV) time domain parameter standard deviation of the normal to normal beats (SDNN), and the frequency domain suggesting a decrease in parasympathetic tone which may lead to cardiac arrhythmia and mortality. Following the acute exposure, all values returned to baseline levels by the fourth day of measurement.

Microarray technology also was used to examine global genomic changes in the apoE-/- mouse. Mice aged to 11–12 months were exposed via oropharyngeal aspiration to 0, 50, 150, or 400 μg of ambient Seattle PM2.5 collected on Teflon filters. The mice were sacrificed 6, 24, and 72 hours post-exposure and lungs and hearts were aseptically removed and flash frozen. Total RNA was isolated from homogenized tissue using Trizol (Gibco, Rockville, MD) and tissue was pooled within each dose/time group to reduce the effects due to inter-animal variability. Gene expression was determined using the Amersham CodeLink 20K Mouse platform (Motorola, Northbrook, IL) and analyzed using MA-ANOVA (Jackson Labs, Bar Harbor, ME), Bioconductor (Harvard, Cambridge, MA), and GeneTraffic (Iobion, LaJolla, CA) software packages.

For the 72 hour post-exposure time point we found that, at all dose levels, more genes changed expression levels in the lung compared with the heart. There was a significant response at all dose levels in mice exposed to PM compared with saline, but no obvious dose response. Over-represented functional categories were apoptosis, cellular adhesion, immune response, oxidant response, and inflammation.

Another leg of this study analyzed changes in cytokine levels. Serum was collected in 1 ml serum separator tubes and centrifuged at 1000 rpm for 10 minutes. Bronchioalveolar lavage fluid was collected by washing the lungs three times with 0.8 ml sterile saline. The cells were pelleted by centrifugation and the supernatant was removed for cytokine analysis. The Bio-Plex Cytokine Assay (Bio-Rad Laboratories, Munich, Germany) was used to determine the serum levels of interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-10, granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-g (IFN-γ), and tumor necrosis factor-α (TNF-α). In the serum there were increased levels of IL-1β, IL-2, GM-CSF, IL-5, TNF-α and IFN-γ at the 150 and 400 μg dose levels. Whereas IL-1β and IL-2 levels reach their peak values at 24 hours, IL-5, GM-CSF, TNF-α and IFN-γ remain elevated at 72 hours. The anti-inflammatory cytokines IL-4 and IL-10 showed no trend following exposure to Seattle PM2.5. There was little change in pulmonary cytokine levels.

We concluded that inflammation plays an important role in the response of the apoE-/- mouse to Seattle PM in both lung and heart. The serum cytokine results suggest there is widespread and persistent inflammation. Modulation of genes associated with an immune response as shown in this study is of particular interest. The results of these studies indicate an involvement of a series of pathways in response to PM exposure, ranging from oxidative stress to inflammation to thrombotic events.

Ambient particulate matter (PM) in the Seattle area is heavily influenced by wood smoke in the winter months. To evaluate whether exposure to wood smoke is adverse, we exposed the apoE-/- mouse to PM with high or low wood smoke influence, focusing attention on inflammatory effects, such as changes in lung lavage cell populations, cytokines in serum and lung lavage fluid, and upregulation of genes involved in the inflammatory response in both the heart and the lung.

PM2.5 was collected from two Seattle neighborhoods with differing contributions of wood smoke on Teflon filters during winter months in order to evaluate the toxic effect of woodsmoke. Beacon Hill PM is considered low in wood smoke influence while Lake Forest Park is higher in wood smoke. ApoE-/- mice and its background strain C57BL6/J were exposed, via oro-pharyngeal aspiration to 50 μl sterile saline or PM2.5 from Beacon Hill or Lake Forest Park (50 or 400 μg suspended in 50 μl sterile saline). The mice were sacrificed at 72 hours post-exposure and the lungs lavaged with sterile saline. Lung lavage fluid was used to determine cell counts, cell differentials, and levels of the cytokines interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, GM-CSF, IFN-γ, and TNF-α. Cytokine levels were also measured in the serum. RNA from whole lung and heart homogenates was used to measure gene expression of heme oxygenase 1 (HO1) and interleukin-10 (IL-10), normalized to glyceraldehyde 3-phosphate dehydrogenase using quantitative RT-PCR.

There was a dose dependent increase in cell numbers in apoE-/- mice exposed to high wood smoke PM, but not low wood smoke PM. No change was seen in the C57BL6/J strain. This increase was due to a dose dependent increase in polymorphonuclear (PMN) cells in apoE-/- mice exposed to Seattle PM with high wood smoke. Levels of IL-4, IL-5, IL-10 and IFN-γ in the serum of apoE-/- mice were increased over saline controls with both doses of Seattle PM exposures, but there were no differences between high and low wood smoke exposures. No change was seen in BAL cytokine levels. There was a dose-dependent increase in HO1 expression in both heart and lung tissue following high wood smoke exposure that was not seen with low wood smoke exposure. IL-10 expression was upregulated in the heart in response to high wood smoke, but not low wood smoke PM. However, in the lung, IL-10 was upregulated in response to both high and low wood smoke PM.

Animals exposed to Seattle PM with either a high or low contribution from wood smoke showed an inflammatory response indicated by increases in total cell counts (mainly due to an influx in PMN cells) in the lung, increases in cytokines in the lung and in the systemic circulation, and upregulation of the HO1 and IL-10 genes in both lung and heart. Although many of these effects are present with high or low wood smoke exposures, the inflammatory effects are greater with PM from areas with higher wood smoke levels.

Subproject: Effect of PM on Glutathione Synthesis
Terry Kavanagh PhD, Dan Luchtel PhD, Mike Rosenfeld MD, MPH, Coralie Baker, MS, Lisa McConnachie ,PhD, Susan Leaman, MS, Diana Ceballos, MS

Objectives: Two hypothesis are being tested in this project: 1) PM will induce glutamate cysteine ligase [GCLC] (the rate limiting enzyme for glutathione synthesis) and such induction will depend upon antioxidant regulatory elements present in the 5’ promoter region of these genes; and 2) Mice which are deficient in glutathione levels (nullizygous for the glutamate cysteine ligase modifier subunit gene) will display enhanced sensitivity to diesel exhaust-induced oxidative stress in the lungs.

Progress Summary/Accomplishments:
We have assessed the effect of NIST PM 1648 and 1649 on GCLC and GCLM expression in mouse RAW macrophage cells. Both types of PM caused an induction of GCLM to a much greater degree than GCLC. PM 1648 was more toxic to RAW cells than PM 1649.

GCLM nullizygous and wild type mice were exposed to diesel exhaust (DE) at 0 (air), 100 and 400 μg/m3 for 6 hours, and sacrificed 18 – 22 hours later. Parameters measured included BAL differential cell counts, GCL subunit protein expression, total lung glutathione levels, and b-galactosidase activity (as a surrogate for GCLM induction in the GCLM nullizygous mice). DE caused a modest increase in GCLC protein levels, but no change in GCLM protein. Higher levels of DE (~600 μg/m3) caused an increase in b-gal activity.

Assessment of the effects of PM 1648 and 1649 on GCL promoter – luciferase reporter gene constructs are underway. These experiments are designed to identify transcriptional factors responsible for the induction of GCLM and GCLC by these PM.

Assessment of b-gal activity, inflammatory cytokines, F2-isoprostanes and 3-nitrotyrosine levels in lung homogenates from GCLM nullizygous and wild type mice exposed to DE are planned. In addition, DNA microarrays for analysis of global gene expression in the lungs of GCLM null and wild type mice exposed to diesel exhaust are underway. Lung tissue RNA has been extracted, assessed for quality and quantitated. Hybridization to microarrays will occur within the month, and analysis of the expression data will be completed by the end of August.

The mice exposures were delayed due to purchase of specially designed exposure cages. This research group will continue to analyze data that can be used to satisfy the objective of inter-species comparisons of effects of diesel exhaust.


Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other subproject views: All 8 publications 1 publications in selected types All 1 journal articles
Other center views: All 209 publications 113 publications in selected types All 109 journal articles
Type Citation Sub Project Document Sources
Journal Article Corey LM, Baker C, Luchtel DL. Heart-rate variability in the apolipoprotein E knockout transgenic mouse following exposure to Seattle particulate matter. Journal of Toxicology and Environmental Health-Part A-Current Issues 2006;69(10):953-965. R827355 (Final)
R827355C005 (2004)
R827355C005 (Final)
  • Abstract from PubMed
  • Abstract: Taylor and Francis-Abstract
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  • Supplemental Keywords:

    Health, Scientific Discipline, Air, particulate matter, Toxicology, air toxics, Environmental Chemistry, Health Risk Assessment, Epidemiology, Risk Assessments, Biochemistry, Atmospheric Sciences, ambient aerosol, ambient air quality, health effects, particulates, air pollutants, cardiopulmonary responses, human health effects, morbidity, exposure and effects, exposure, hazardous air pollutants, animal model, air pollution, particle exposure, human exposure, inhalation, atmospheric aerosols, ambient particle health effects, mortality studies, mortality, aerosols, atmospheric chemistry, cardiovascular disease, exposure assessment, human health risk

    Progress and Final Reports:

    Original Abstract
  • 1999 Progress Report
  • 2000 Progress Report
  • 2001 Progress Report
  • 2002 Progress Report
  • 2003 Progress Report
  • 2004 Progress Report

  • Main Center Abstract and Reports:

    R827355    Airborne PM - Northwest Research Center for Particulate Air Pollution and Health

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827355C001 Epidemiologic Study of Particulate Matter and Cardiopulmonary Mortality
    R827355C002 Health Effects
    R827355C003 Personal PM Exposure Assessment
    R827355C004 Characterization of Fine Particulate Matter
    R827355C005 Mechanisms of Toxicity of Particulate Matter Using Transgenic Mouse Strains
    R827355C006 Toxicology Project -- Controlled Exposure Facility
    R827355C007 Health Effects Research Core
    R827355C008 Exposure Core
    R827355C009 Statistics and Data Core
    R827355C010 Biomarker Core
    R827355C011 Oxidation Stress Makers