2008 Progress Report: Biological Assessment of the Toxicity of PM and PM Components

EPA Grant Number: R832417C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R832417
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Johns Hopkins Particulate Matter Research Center
Center Director: Samet, Jonathan M.
Title: Biological Assessment of the Toxicity of PM and PM Components
Investigators: Spannhake, Ernst , DeCastro, Rey , Garcia, Joe , Irizarry, Rafael , Natarajan, Viswanathan
Current Investigators: Spannhake, Ernst , DeCastro, Rey , Garcia, Joe , Irizarry, Rafael , Natarajan, Viswanathan , Vinasco, Liliana Moreno , Wang, Ting
Institution: The Johns Hopkins University , University of Chicago
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010
Project Period Covered by this Report: October 1, 2007 through September 30,2008
Project Amount: Refer to main center abstract for funding details.
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

Exposure to particulate matter (PM) is currently associated with development of various respiratory diseases such as lung cancer, COPD, and asthma. Hallmarks of asthma include airflow obstruction, bronchial hyper-responsiveness, and airway remodeling. Particulate matter less than 2.5 µm in diameter (PM2.5)  is derived mainly from industrial heating as well as the combustion of vehicle fuels and is  considered to have clinical relevance since it deposits in the respiratory bronchioles of the lungs.  PM2.5 has been associated with premature mortality. Recent studies suggest an association between acute exposure to PM and daily mortality and morbidity, which was strongest for respiratory- and cardiovascular-related hospital admissions and cause of death in susceptible individuals. The specific objectives to be completed across the three phases of this Project are: 1.To characterize secretion of inflammatory cytokines/chemokines in human bronchial epithelial cells induced by PM; 2. To characterize airway inflammation in murine models of lung inflammation induced by bioavailable PMs; 3. To evaluate the role of ROS in PM-induced in vitro and in vivo airway inflammation and toxicity; 4. To link in vitro and in vivo gene expression patterns induced by PM with morbidity and mortality rates of the city where the sample was collected; 5. To link fluctuations in ambient bioavailable PM levels with relevant biomarkers (cytokines, epithelial/endothelial activation, peripheral blood mononuclear cell gene expression, exhaled breath condensates) in a panel of PM exposed human subjects; 6. To characterize signaling mechanisms of PM-induced secretion of inflammatory cytokines/chemokines and ROS burden in human bronchial epithelial cells.

Progress Summary:

  1. Project #3: Biological Assessment of Toxicity of PM and PM Components

    Rationale. Despite numerous epidemiologic studies pointing to diverse adverse health effects of exposure to urban airborne particulate matter (PM), the physical and chemical characteristics of PM that contribute to cardiopulmonary toxicity and dysfunction remain poorly understood.  Further, relatively little is known regarding the molecular mechanism(s) of PM-induced airway inflammation and cardiovascular dysfunction, processes considered to play a critical role in cardiopulmonary morbidity and mortality. Elaboration of reactive oxygen species (ROS) and secretion of pro-inflammatory cytokines from airway epithelium exposed to urban PM may be involved not only in airway inflammation, but also in PM-mediated toxicity to cardiac tissue, distant from the lung. Project #3 studies are encompassed within 3 phases. In vitro and in vivo Phase I studies have been initiated to establish the models that will be used in carrying out bioassays with specimens collected in the various cities throughout the United States.  In Phase I, in developing the models, emphasis has been placed on  PM collected by cyclone-generated (single stage) extraction method for bulk PM collection from the roof of the School of Public Health (April-June 2005), yielding a PM sample in the size range of 0.1 to 10 microns (provided by Project #2 investigators: Drs. Patrick Breysse and Alison Geyh). Phase I studies have included both PM-induced changes in lung and cardiac tissue gene expression using the Baltimore PM.  Several manuscripts are in preparation. Similar studies using PM derived from specific US cities will be carried out subsequently under Phase II (murine asthma) and Phase III (murine dilated cardiomyopathy). As noted in the prior review of Project #3 studies, future studies will not be emphasizing in vitro approaches, which were evaluated in Phase I.

  2. Phase I: In vitro Toxicity Assessment of Baltimore PM. Overview and summary: These studies have utilized human bronchial epithelium and human  lung endothelium with evaluation of cytokine secretion (GM-CSF, IL-6, IL-8, and IL-1β), generation of ROS, such as H2O2 and superoxide, and signaling mechanisms regulating cytokine/ROS production, cytotoxicity, and vascular/epithelial permeability. These in vitro effects of Baltimore PM on lung cell function evidence a “pro-inflammatory lung cell phenotype” with increases in epithelial and endothelial permeability in PM fraction-specific pathways. PM also induces elaboration of ROS, effect which is partially reversed by the anti-oxidant N-acetyl-L-cysteine (NAC). Reflective of the prior SAC review of Project #3, future studies will focus on in vivo animal models with less emphazise on in vitro approaches.  

    Effects of Baltimore PM on human bronchial epithelium: Regulation of COX-2 Expression and IL-6 Release by Particulate Matter  in Airway Epithelial Cells to hydroxyethidium by reaction with superoxide.  Exposure of HBEpCs to PM stimulated phosphorylation

    • Effect of Baltimore PM on human lung endothelium: We determined that PM decreases trans-endothelial electrical resistance (TER), a reflection of loss of vascular integrity in dose-dependent and time-dependent fashion. Water–soluble PM supernatants, in contrast, enhance endothelial cell barrier function whereas the water–insoluble PM pellet produces endothelial cell barrier dysfunction. The presence of NAC partially reverses the PM effect on permeability and barrier dysfunction and induces stress fiber formation, a finding consistent with increased permeability. This particulate endothelial cell biology study of PM toxicity elucidated that PM disrupts EC barrier via an ROS-p38 MAPK-HSP27 signaling pathway. These results partially explained acute inflammatory pulmonary injury induced by PM via the induction of vascular leakage of protein into BAL.
  3. Phase I: In vivo Effects of PM exposure in a Murine Model of Asthma

    Overview and summary: We have developed and characterized an experimental model of  murine asthma induced by ovalbumin (OVA) in the asthma-susceptible AJ mouse strain in order to evaluate PM effects.

    Briefly, 10-12 week old AJ mice received OVA (0.4 mg/kg i.p, day 0) followed by an intratracheal OVA challenge (30 mg/kg, day 14). Then PM (20 mg/kg) was administered through an intratracheal aspiration three days after OVA challenge. After 1 day, 4 days or 7 days post PM exposure, airway hyperresponsiveness (AHR) was determined via acetylcholine (1 mg/kg) intravenous injection through the inferior vena cava and animals were sacrificed for BAL extraction and tissue harvesting. As noted below, these studies highlight the interaction between PM and lung inflammatory responses in the sensitized mouse--interactions which result in enhancement of airway hyperresponsiveness. In this study, we have used a high dose of PM; however, instillation of lower doses of PM (0.01 to 1.0 mg/kg body weight) is planned in all the future studies with fine particles from different locations (Project 2). These lower doses are more comparable to exposure of humans to ambient PM.  Key findings are highlighted below:  
    • Baltimore PM induces AHR. Reactivity of the airways was determined by the response to endogenous bronchoconstrictors, such as acetylcholine. Airway pressure change stimulated by exogenous infused acetylcholine was measured to represent airway responses. OVA challenge increased AHR. PM induced significant increases in AHR in both control AJ mice and asthmatic OVA challenged mice.
    •  Baltimore PM induces protein leakage into airway. BAL protein level increase indicates vascular leakage and is a key parameter of inflammatory lung injury. PM, not OVA, increased protein levels in BAL an indication of disruption of epithelial/endothelial barriers.
    • Baltimore PM induces inflammatory leukocyte infiltration into the airways. PM induced inflammatory leukocyte infiltration into the alveolar and airway in both PBS control AJ mice and OVA challenged asthma mice. OVA challenge induced eosinophil and macrophage increases in BAL. Baltimore PM induced eosinophil and neutrophil infiltration in BAL.
    • Baltimore PM induces Th1 and Th2 type cytokines in BAL. OVA challenge induced TH2 cytokine IL-4 and IL-5 secretion into BAL. PM induced not only TH2 cytokine IL-4 and IL-5 in asthma mice, and TH1 cytokine IL-6, IFN-g and TNF-a in BAL.
    • Baltimore PM induces mucus production in murine airways. PM and OVA challenge induced mucus-producing goblet cell generation in mice (pink PAS stained epithelial cells). PM and OVA challenge synergistically induced positive-stained goblet cells at day 4 post PM challenge.
    • Baltimore PM induces gene transcription signaling in murine asthmatic lung. PM had a strong impact on the global expression of lung genes. 436 genes survived filtering and were identified as significantly dysregulated by PM exposure (375 genes upregulated and 61 gene downregulated). In contrast, OVA-challenge had less impact on lung gene expression than PM at day 4 post exposure. Only 37 genes (21 genes upregulated and 16 gene downregulated) were differentially regulated by OVA sensitization even when less stringent criteria (FDR <5% and fold change >2 fold) were applied. The combination of PM and OVA treatment exhibited synergistic effects on lung gene expression, with a total of 591 genes identified as differentially regulated (492 genes upregulated and 99 genes downregulated). The PM-regulated genes were related to 22 biological processes, including innate immune response, chemotaxis, cell surface receptor linked signal transduction, inflammatory response, defense response, cell cycle, nervous system development, and DNA-dependent regulation of transcription. Similar to GO analysis, cell cycle, inflammatory response (interleukin signaling, interferon signaling) and cell surface receptor (B-cell receptor, T-cell receptor and Toll-like receptor) pathways were among the most distinctly regulated pathways. The majority of these signaling pathways are closely related to asthma development. For example, the genes in the complement system were significantly regulated by PM in both the control (PM group) and the asthma animals (PM & OVA group). These genes are implicated in the development of asthmatic phenotypes. Some asthmatic marker genes such as It1na (SI>1.52), Tff2 (SI>1.60), and Clca3 (SI>1.16) were all upregulated by PM and OVA synergistically.  
  4. Phase I: In vivo Effects of PM exposure in a Murine Model of Cardiomyopathy

    Overview and summary: Transgenic mice engineered to express a cardiac-specific dominant/ negative form of transcription factor CREB-(Ser-Ala)133, essential for cardiac muscle function were used as a cardiomyopathy model for studying the cardiac effects of PM. This model induces: progressive ventricular failure, cardiac dilatation, decreased systolic & diastolic pressures, hypertrophy and interstitial fibrosis. Measurements were obtained in 10- and 20-wk-old mice exposed to 1 mg/mouse of PM/mouse with evaluation 72 hours after PM challenge. Experimental groups (CD1-PBS, CD1-PM, CREB-PBS, and CREB-PM) were exposed to PM or PBS by intratracheal instillation at 10 or 20 wks of age.  Continuous electrocardiograms were recorded prior to and for 36 hours following exposure.  Arrhythmia scores were based on the frequency of ventricular premature beats and episodes of ventricular tachycardia.  Cardiac function was assessed by cardiac ultrasound at baseline and following PM exposure.  cDNA microarray analyses were performed on the left ventricles, lung, and left atrial tissues of 20 wk groups.   In conclusion, this study is the first to demonstrate that PM exposure acutely increases ventricular arrhythmias in transgenic mice with severe cardiac dysfunction.  Genomic assessment revealed differential regulation of numerous genes,some of which may be involved in the pathogenesis of PM triggered ventricular arrhythmias.  These results are consistent with epidemiologic studies that suggest that PM is more likely to trigger phenotypic changes in individuals with severe cardiac dysfunction. We currently are pursuing the novel molecular signatures that PM may have on lung,, left ventricle, left atrium, and carotid body tissues. In these studies, exposure of mice to 1mg/kg body weight represents ~10 times greater exposure compared to human exposures; therefore, future studies will be carried out at 0.01 to 1.0 mg/kg body weight fine particle doses.  Key findings are highlighted below:

    • Baltimore PM induces reductions in baseline fractional shortening (FS) in the 20    wk CREB groups when compared to 10 wk CREB groups (18% vs 35%; p=0.04).
    • Baltimore PM induces ventricular arrhythmias in CREB mutant mice with CHF. CD1 mice at any age do not exhibit ventricular arrhythmias: either at baseline or 36 hours post PM exposure. CREB mice at 10 or 14 weeks of age do not demonstrate arrhythmias: either at baseline or following PM/PBS exposure. CREB mice at 20 wks demonstrate ventricular arrhythmias at baseline (arrhythmia score 2.0 vs. 1.8; p=0.77). CREB mice at 20 wks exhibit marked increases in ventricular arrhythmias following PM in conjunction with increased expression of genes involved in cardiac arrhythmias (arrhythmia score 5.5 vs. 2.2; p=0.02). CREB mice exhibit numerous PVC’s (pre-ventricular contractions) at baseline. After PM administration, the same CREB mice demonstrate increased PVC’s and an idioventricular rhythm. This rhythm may be similar to that of a slow ventricular tachycardia.
    • Baltimore PM induces left ventricular differential gene expression of several gene ontologies in CREB mice including inflammation, signal transduction, and ion channel regulation. LV RNA from control and 20 wk CREB-PM groups was utilized in Affymetrix arrays.

    Baltimore PM induces carotid body dysfunction in CREB mice After undergoing Dejour’s test, in which murine minute neural ventilation was assessed after a 15 second exposure to hyperoxia, significant carotid body dysfunction was found in CREB-PM mice compared to their PBS counterparts (-59.3 vs. -48.6). Further ex vivo measures of carotid body sensory responses to hypoxia confirmed these results as the function of carotid bodies from CREB/PM mice were significantly altered from the CREB-PBS animals ( 4.3 vs. 2.7).

Future Activities:

As noted above, we have completed all planned in vitro experiments. In Year 3 we will concentrate on Phase II and Phase III studies focused on screening the cardiopulmonary toxicity of the new, characterized PM samples collected by Project #2 personnel. We have received from Alison Geyh bulk fine PM samples from Baltimore, Seattle and Sacramento (200mg). We will continue to use toxicogenomic approaches in the evaluation of PM effects in the model of OVA-induced murine asthma and mice with dilated cardiomyopathy. For these experiments lower doses of particulate matter fine samples (0. 01, 0.1, and 1 mg/kg body weight) collected from various centers will be employed to assess the biological toxicity on asthma and cardiomyopathy models. Our calculations, based on published data, indicate that a dose of 1mg/kg body weight of mouse will be approximately 10 times greater than the reference human exposure. Further, we will be selecting a marker for PM induced inflammation and a marker for asthma / cardiomyopathy for characterization of these new PM fine samples (0.01 to 1.00 mg/kg body weight) as well as for further comparison among the samples based on the different geographical distribution and sample size.


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

Other subproject views: All 12 publications 4 publications in selected types All 4 journal articles
Other center views: All 89 publications 66 publications in selected types All 64 journal articles
Type Citation Sub Project Document Sources
Journal Article Wang T, Moreno-Vinasco L, Huang Y, Lang GD, Linares JD, Goonewardena SN, Grabavoy A, Samet JM, Geyh AS, Breysse PN, Lussier YA, Natarajan V, Garcia JGN. Murine lung responses to ambient particulate matter: genomic analysis and influence on airway hyperresponsiveness. Environmental Health Perspectives 2008;116(11):1500-1508. R832417 (2008)
R832417 (Final)
R832417C003 (2008)
R832417C003 (2009)
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  • Journal Article Zhao Y, Usatyuk PV, Gorshkova IA, He D, Wang T, Moreno-Vinasco L, Geyh AS, Breysse PN, Samet JM, Spannhake EW, Garcia JGN, Natarajan V. Regulation of COX-2 expression and IL-6 release by particulate matter in airway epithelial cells. American Journal of Respiratory Cell and Molecular Biology 2009;40(1):19-30. R832417 (2008)
    R832417 (Final)
    R832417C003 (2008)
    R832417C003 (2009)
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  • Supplemental Keywords:

    Differentiated and non-differentiated airway cells; ROS; particulate matter, murine models; cardiopulmonary functions; cytotoxicity; cytokines,, RFA, Health, Scientific Discipline, Air, particulate matter, Health Risk Assessment, Epidemiology, Risk Assessments, atmospheric particulate matter, atmospheric particles, long term exposure, acute cardiovascular effects, toxicogenomic approaches, airway disease, human exposure, ambient particle health effects, atmospheric aerosol particles, ultrafine particulate matter, PM, toxicologic assessment, aersol particles, cardiovascular disease

    Relevant Websites:

    www.jhsph.edu/particulate_matterexit EPA

    Progress and Final Reports:

    Original Abstract
  • 2006 Progress Report
  • 2007 Progress Report
  • 2009 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R832417    Johns Hopkins Particulate Matter Research Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832417C001 Estimation of the Risks to Human Health of PM and PM Components
    R832417C002 PM Characterization and Exposure Assessment (Project 2)
    R832417C003 Biological Assessment of the Toxicity of PM and PM Components