2004 Progress 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 , Feigl, Eric , Kavanagh, Terrance J , Larson, Timothy V. , Leaman, Susan , McConnachie, Lisa , Rosenfeld, Michael
Current 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 Period Covered by this Report: June 1, 2004 through May 30, 2005
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:

This project involves two parts. The objective of the first part of the project is to test the hypothesis that particulate matter (PM) deposited in the lung causes release of inflammatory cytokines from the lung that target the cardiovascular system. Transgenic mouse strains with specific cardiovascular genetic alterations are used to address the research need to identify potential health conditions that enhance susceptibility to adverse PM health effects. The nature of such health conditions provides insight into the biological mechanisms by which PM mediates acute and chronic health effects.

The objective of the second part of this project is to test the following two hypotheses: (1) PM will induce glutamate cysteine ligase (the rate limiting enzyme for glutathione synthesis) and such induction will depend on antioxidant regulatory elements present in the 5´ promoter region of these genes; and (2) mice that 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:

In work that will be reported at this year’s American Thoracic Society (ATS) meeting in San Diego, we used microarray technology to examine global genomic changes in the apolipoprotein E knockout (apoE-/-) transgenic mouse. Mice were aged to 11–12 months (n = 5/group) and 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 pooled in each group to minimize 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.

To date, we have results analyzed for the 72 hour post-exposure time point only. 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. We are continuing the analysis of animals from the 6 and 24 hour post-exposure time points.

Another part of this study analyzed changes in cytokine levels. These results also will be reported at the ATS meeting. Serum was collected in 1 mL serum separator tubes and centrifuged at 1000 rpm for 10 minutes. 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).

We found 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. We are continuing with analyses of cytokine levels in lavage fluid and further results are imminent.

So far, we conclude 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.

In the second part of the project, we have assessed the effect of NIST PM 1648 and 1649 on glutamate cysteine ligase catalytic (GCLc) and modifier (GCLm) subunit 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 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). Diesel exhaust caused a modest increase in GCLc protein levels, but no change in GCLm protein. Higher levels of diesel exhaust (~600 μg/m3) caused an increase in b-gal activity.

Future Activities:

What the above studies lack and what we hope to accomplish soon is to repeat the above dose-response study using the normal background strain on which the apoE-/- transgenic is based, the C57/BL6 strain, also using mice aged to 11-12 months. This will allow us to compare normal aged animals (the C57/BL6 strain) versus health compromised aged mice (the apoE-/- transgenic).

We also will assess the effects of PM 1648 and 1649 on GCL promoter¾luciferase reporter gene constructs are underway. These experiments are designed to identify transcriptional factors responsible for the induction of GCLm and GCLm by 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 diesel exhaust 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.


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:

    ambient particles, fine particles, combustion, health, exposure, biostatistics, susceptibility, human susceptibility, sensitive populations, air toxics, genetic susceptibility, indoor air, indoor air quality, indoor environment, tropospheric ozone, California, CA, polyaromatic hydrocarbons, PAHs, hydrocarbons, acute cardiovascular effects, aerosols, air pollutants, air pollution, air quality, airborne pollutants, airway disease, airway inflammation, allergen, ambient aerosol, ambient aerosol particles, ambient air, ambient air quality, ambient particle health effects, animal model, assessment of exposure, asthma, atmospheric aerosols, atmospheric chemistry, biological markers, biological response, cardiopulmonary response, cardiovascular disease, children, children’s vulnerability, combustion, combustion contaminants, combustion emissions, epidemiology, exposure, exposure and effects, exposure assessment, harmful environmental agents, hazardous air pollutants, health effects, health risks, human exposure, human health effects, human health risk, incineration, inhalation, lead, morbidity, mortality, mortality studies, particle exposure, particle transport, particulates, particulate matter, risk assessment,, Health, Scientific Discipline, Air, particulate matter, Toxicology, air toxics, Environmental Chemistry, Health Risk Assessment, Epidemiology, Risk Assessments, Biochemistry, Atmospheric Sciences, health effects, ambient aerosol, particulates, ambient air quality, air pollutants, morbidity, cardiopulmonary responses, human health effects, exposure and effects, animal model, hazardous air pollutants, exposure, air pollution, particle exposure, human exposure, atmospheric aerosols, ambient particle health effects, mortality studies, inhalation, mortality, particle transport, cardiovascular disease, human health risk, aerosols, atmospheric chemistry

    Relevant Websites:

    http://depts.washington.edu/pmcenter/ Exit

    Progress and Final Reports:

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