2009 Progress Report: Biological Assessment of the Toxicity of PM and PM ComponentsEPA 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 , Garcia, Joe , Irizarry, Rafael , Moreno, Liliana , 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: August 1, 2008 through July 31,2009
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
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.
- 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.
- Phase II: 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 emphasis on in vitro approaches.
- Particulate matter (PM) collected from Ft. McHenry tunnel, Baltimore enhanced oxidative stress and endothelial barrier dysfunction as measured by changes reactive oxygen species generation and transendothelial electrical resistance (TER).
- PM increased myosin light chain (MLC) phosphorylation mediated by non-muscle myosin light chain kinase (nmMLCK), which was ROS dependent.
- Down-regulation of nmMLCK expression with siRNA or inhibition with ML-7 attenuated PM-induced MLC phosphorylation and degradation of tight junction proteins, ZO-1/ZO-2 in human lung endothelial cells.
- These results suggest a novel role for nmMLCK and ZO-1/ZO-2 tight junction proteins in PM-induced endothelial barrier dysfunction and pulmonary edema. A manuscript describing this study is ready for submission to Circulation Research.
- Phase II: In vivo Effects of PM exposure in a Murine Model of Asthma
Recently published results by our group (Wang et al., Environmental Health Perspectives 116: 1500-1508, 2008) showed a dose-dependent effect of Baltimore PM instillation (0.01 – 30mg/kg body weight, n=3) on airway hyperresponsiveness (AHR), BAL protein, and BAL inflammatory leukocytes infiltration in both asthmatic and naïve animals. Based on these findings, an optimal dose range (1, 3, and 10 mg/kg body weight) was selected to conduct the following comparative study of fine-PM collected from Phoenix (PHX) and Sacramento (SAC).
- Instillation of fine-PM from PHX and SAC, in a dos-dependent fashion, altered pulmonary inflammatory phenotypes, including AHR, BAL white blood cell count (total cell counts, neutrophils and eosinophils) and BAL protein content as compared to control mice. Standard urban-PM (1648a from NIST) also exhibited a significant induction of similar pulmonary inflammatory phenotype in the murine asthmatic model. Interestingly, fine-PM from PHX, compared to SAC, induced a significantly higher level of AHR, and infiltration of white blood cell count, including neutrophils and eosinophils into the alveolar space; however, no difference in BAL protein (protein leak) between PHX and SAC PM was observed.
- Regulation of lung inflammatory gene expression by fine PM from PHX and SAC was assessed by quantitative RT-PCR. Relative mRNA levels of ten genes (Tff2, Cxcl2, Ear11, Clca3, Rgs9, Cfb, Muc5b, Timp1, Gsn, and Tlr2) were quantified and compared. Both PHX and SAC PM dysregulated expression of Tff2, Cxcl2, Ear11, Clca3, Rgs9, Cfb, Muc5b, Timp1, and Tlr2. However, fine-PM from PHX, compared to SAC, exhibited significantly higher regulation on Cxcl2, Cfb, Rgs9, Ear11, and Tlr2, and weaker regulation of Timp1 than SAC PM.
Overview and Summary: These results suggest a differential effect of fine-PM from PHX and SAC on regulation of gene expression in a murine model of Asthma.
2. Cardiomyopathy Model
A mechanistic link between human exposure to PM pollution and increased cardiovascular morbidity and mortality observed in people with congestive heart failure is poorly defined. Therefore, we have developed a murine model of cardiomyopathy (expressing a cardiac-specific, dominant negative CREB mutant transcription factor, CREB A133) to investigate PM-mediated cardiac arrhythmias/respiratory dysynchrony.
- 20 Week old CREB, but not CD-1 control, mice exposed to bulk-PM (20 mg/kg body weight) for 36 h exhibited mildly increased PVCs at baseline with a marked increase post-exposure.
- Analyses of the left ventricles of control- and PM-treated mice utilizing microarray, OntoExpress and Ingenuity Pathway revealed that PM dysregulated 171 genes in CD-1 and 41 genes in CREB mice compared to controls involving critical pathways like complement system, beta-catenin signaling and glycosaminoglycans degradation.
- Bulk-PM challenge of CREB A133, but not CD-1, mice displayed respiratory Dysynchrony with waxing and waning patterns of respiration under basal conditions which was pronounced during hypoxia. Such a breathing pattern is often observed in patients with severe congestive heart failure; thus, a prognostic sign for sudden cardiac death and heart failure progression.
- Mouse carotid body gene expression was dysregulated by PM as determined by quantitative RT-PCR. These included signaling genes (Cd44, Cxcl2, Rhoc, and Edg3), inflammatory, oxidative/stress genes (Tgfbr1, Tnfsf9, IL6, Tlr2, Nfe212, Hmox1, Sod1, Sod2, and Cfb), and carotid body function genes (Scn8a, Kcnmb2, Gdnf, Scnn1b, Kcnc1, Kcnd1, Kcnd2, Sstk38l, Slc8a1, and PKCc). A strong role of ion channel dysregulation by PM-induced oxidative stress, which facilitated cardiac arrhythmia, was observed. These data have been incorporated into our newly to be submitted manuscript to Nature Medicine, 2009.
- Comparison of left ventricular tissue samples obtained from Baltimore PM exposed CREB A133 mice and PBS-challenged CD-1 mice (SAM software) identified 1927 probe sets. Within this probe set, only 60 probe sets displayed significant differential expression (p < 0.05) between PM- and PBS-treated CREB A133 mice whereas PM alone minimally impacted cardiac gene expression. The magnitude of gene dysregulation in this subset of genes, however, was greater in PM-challenged CREB A133 mice than in PBS-exposed CREB A 133 mice.
- In lung tissue, PM-induced greater gene dysregulation in CREB A 133 mice (843 probe sets, fold change > 2, FDR < 1.9%) than in CD-1 mice (589 probe sets, fold change > 2, FDR < 2.7%) suggesting that the congestive heart failure phenotype primes the lung for an exaggerated response to PM.
- A greater number of genes belonging to growth factor signaling, MAPK pathways, cytoskeletal signaling and factors promoting cardiogenesis in vertebrates were down-regulated by PM in CREB A 133 lungs (70%, 575/843 probe sets), whereas the majority of PM-induced dysregulated genes in CD-1 lungs were up-regulated (66%, 384/589 probe sets). Interestingly, none of the pathways enriched with PM-down-regulated genes in CREB A 133 mice were identified in PM-exposed CD-1 mice.
- Carotid bodies from CREB A 133 mice, but not CD-1 mice, responded to Baltimore PM challenge with marked inflammatory and pro-oxidant gene signatures and dysregulation of carotid body genes. These results suggest that carotid bodies from congestive heart failure mice, similar to lung, are primed for an exaggerated response to PM exposure.
- The effect of fine-PM from PHX and SAC on cardiac arrhythmias and respiratory Dysynchrony in CD-1 and CREB transgenic mouse model was examined. Both PHX and SAC fine-PM (10 mg/kg, 24 hr, n=3) manifested a reduction in heart rate variability, respiratory dysynchrony and increased frequency of serious ventricular arrhythmias with no significant difference between the two PM samples. Additional animals (n=5) are planed to compare the effect of different PM samples from various cities (Project 2) for future studies with CREB and CD-1 mice.
Overview and Summary: In summary, our findings in a murine model of dilated cardiomyopathy are consistent with results of population-based studies showing that short-term exposure to PM is a risk factor for acute cardiovascular morbidity and mortality and provide novel mechanistic insights regarding complex PM pathophysiology in susceptible individuals with CHF. Utilizing complementary physiological and genomic approaches with direct measurements of carotid body neural activity, our studies significantly extend earlier in vitro and in vivo studies and now suggest that PM-mediated serious cardiac arrhythmias, decreases in HRV, and respiratory dysynchrony represent multi-organ pathobiology within the CREBA133 CHF phenotype which is driven by heightened sensitivity of carotid body function and augmented peripheral chemoreceptor reflexes.
Studies proposed for Year 4-5:
As noted above, we have completed all planned in vitro experiments under Phase I and proposed in vivo experiments with animal models under Phase II. In Years 4-5, we will concentrate on Phase III studies focused on screening the cardiopulmonary toxicity of the new, characterized PM samples collected from five different locations by Project #2 personnel. Fine PM collected at Phoenix and Sacramento (200 mg) were received from Project 2, and these have been evaluated for toxicogenomics in the model of OVA-induced murine asthma and studies are in progress with CREB model of dilated cardiomyopathy. As per recommendations of the Scientific Advisory Committee, 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 one dose of fine PM (10 mg/kg body weight) for the cardiomyopathy model. 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 using NIST PM for comparison among the samples based on the different geographical distribution and sample size.
The investigators, along with the Center PI and the Quality Assurance Manager of the Center have collaborated to develop the Project Quality Assurance Project Plan for Project 3. The Plan was developed and submitted to the PI and QAM in 2007. The Quality Assurance Manager also conducted a site visit to the University of Chicago in May 2009 to do a separate internal assessment of procedures, review the policy/procedures forms and to give an update on EPA requirements to the team. The QAPP has been implemented, and the investigators have systems in place to review, identify and correct any QA/QC issues. Further, interaction between Project 1 and Project 3 on bioinformatics analyses of data derived from instillation of fine PM to mice of OVA-induced asthma model and CREB model of dilated cardiomyopathy has been initiated. Drs. Barr and Irizarry (Project 1) and Drs. Huang, Wang and Moreno-Vinasco participated in the initial discussion of quality control practices, introduction to NUSE approach and comparison to GCOS. Based on the preliminary suggestions, the bioinformatics group of Project1 and 3 will implement the preferred methods for studying gene sets, processing of microarrays for differential gene expression and gene ontology. Summary of the bioinformatics analyses includes:
Quality Control - Gene Chip operating Software (GCOS); Normalized unscaled errors (NUSE)
Preprocessing – Robust multi-array averaging (RMA); Gene Chip robust multi-array averaging (GCRMA)
Differential Expression – Significance analysis of microarrays (SAM); Linear models for microarrays (LIMMA)
Gene Sets – Gene ontology (GO); Gene set enrichment (GSEA)
Journal Articles on this Report : 3 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|
||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.||
||Wang T, Chiang ET, Moreno-Vinasco L, Lang GD, Pendyala S, Samet JM, Geyh AS, Breysse PN, Chillrud SN, Natarajan V, Garcia JGN. Particulate matter disrupts human lung endothelial barrier integrity via ROS-and p38 MAPK-dependent pathways. American Journal of Respiratory Cell and Molecular Biology 2010;42(4):442-449.||
||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.||
Supplemental Keywords:Differentiated and non-differentiated airway cells; ROS; particulate matter, murine models; cardiopulmonary functions; carotid body; cardiac arrhythmias; respiratory dysynchrony; 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
Progress and Final Reports:Original Abstract
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