2008 Progress Report: Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects

EPA Grant Number: R832413C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R832413
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Southern California Particle Center
Center Director: Froines, John R.
Title: Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects
Investigators: Nel, Andre E. , Harkema, Jack , Kleinman, Michael T. , Lusis, Aldons
Institution: University of California - Los Angeles , Michigan State University , University of California - Irvine
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, 2007 through September 30,2008
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air

Objective:

The primary objective is to elucidate the mechanism(s) of PM-induced asthma and atherosclerosis exacerbation in vitro and in vivo.  This is accomplished by animal studies in the mobile trailer in downtown Los Angeles as well as in vitro studies in representative tissue culture cells.  The principal hypothesis is that a major PM injury mechanism is the induction of ROS production and oxidative stress that promotes respiratory and cardiovascular inflammation as the major pathology feature underlying asthma and atherosclerosis (Nel 2005; Xia et al 2006a, 2006b; Nel et al 2006). We propose that oxidative stress is a hierarchical event in which the induction of antioxidant defense pathways in tier 1 is in dynamic equilibrium and defends against the pro-inflammatory (tier 2) pro-apoptotic (tier 3) effects of higher levels of oxidative stress (Nel et al 2006).

Approach:

In Aim 1, we will use normal and genetically susceptible murine models to study the role of oxidative stress in PM-induced exacerbation of asthma and atherosclerosis. We will use low grade OVA sensitization to study the effects of fine and ultrafine particles (UFP) on allergic airway inflammation, oxidative stress, IgE production, mucus hypersecretion, and airway hyperreactivity (AHR) in a BALB/c model. We will use Nrf2 knockout mice, with a weakened antioxidant response, to determine whether this will enhance airway inflammation. A third component of this Aim will be to use atherosclerosis-prone apoE knockout mice to assess dose-dependent atherogenesis and oxidative modification of LDL and HDL during CAPS exposure. In Aim 2, we will use in vitro toxicology approaches to assess the effects of various PM sources, with unique and varying chemical composition, on the induction of oxidative stress and inflammatory responses in tissue culture macrophages, epithelial and endothelial cells. This study will use coarse, fine and UFP, collected at different sites and during different seasons (Project 1), to determine their effects on: (i) phase II enzyme expression by Western blotting and real-time PCR (Tier 1); (ii) cytokine and chemokine expression as determined by ELISA assays and protein arrays (Tier 2); (iii) perturbation of mitochondrial function and induction of apoptosis as determined by flow cytometry and functional studies on isolated mitochondria (Tier 3). These biological responses will be compared to the chemical composition of the particles (Project 1), their activity in the chemical reactivity assays (Project 3), and their ability to promote asthma and atherosclerosis in animal models. In Aim 3, we will use serum samples, collected from indoor-exposed elderly human subjects with ischemic heart disease (Project 4), to determine how oxidative modification of HDL affects its anti-inflammatory and atheroprotective effects. We will assess how the increase in oxidized phospholipids in LDL affects its pro-inflammatory effects in an endothelial co-culture assay. We will determine whether oxidative modification of HDL-associated paraoxonase activity modifies its anti-inflammatory effects in this assay.

Progress Summary:

ApoE knockout mice exposed to CAPs on the freeway shows increased atherogenic potential of ultrafines (Araujo et al 2008)
Epidemiological studies unveil that exposure to ambient PM increases cardiovascular morbidity and mortality. Both epidemiological and animal-based studies suggest that if the exacerbation of atherosclerosis plays an important role in these outcomes. We hypothesized that PM synergizes with known pro-atherogenic stimuli and mediators in their ability to elicit oxidative stress and promote atherosclerosis, and that most of the pro-inflammatory potential resides in the ultrafine particles (aerodynamic diameter <0.1 μm, UFP) that are highly enriched for redox cycling PM chemicals.
 
We conducted two experimental protocols (Araujo et al 2008). In the first (chow protocol), 6-week-old male C57BL/6J apoE null mice were placed on a chow diet and exposed to CAPs over a 40-day period, while in the second study (high fat diet protocol), 2-month-old male apoE null mice were fed a HFD over a 56-day period. Food and water were administered ad libitum.  Mice destined for CAPs exposure were transported to the mobile research laboratory in downtown of Los Angeles, close (~300 m) to the I-110 freeway.  The animals were housed in a Hazelton Chamber ventilated with air from which 99.9% of the incident particles were removed using a HEPA filter (chow protocol) or in top-filter cages (HFD protocol).  There were three exposure groups (17-18 mice/group), namely filtered air (FA), particles < 2.5 μm (FP) and particles < 0.18 μm (UFP).  Whole body exposures were performed simultaneously for five hours per day (exposure session), three days per week, for a combined total of 75 and 125 hours in the chow-fed and HFD-fed protocols, respectively. Animals were euthanized 24-48 hours after completion of the last CAPs exposure, and aortas and various organs harvested.
 
Chow-fed apoE null mice exposed to concentrated ultrafines developed significantly (p<0.05) larger early aortic atherosclerotic lesions (33011 +/- 3741, n=15) than animals exposed to PM2.5 (26361 +/- 2275, n= 16), filtered air (21362 +/- 2864, n= 14) or left non-exposed (17261 +/- 1659, n= 17) (Araujo et al 2008). Exposure to ultrafine particles resulted in an inhibition of the anti-inflammatory capacity of plasma high density lipoproteins and increased systemic oxidative stress markers as evidenced by a significant: (i) increase in hepatic malondialdehyde levels, (ii) upregulation of Nrf2-related phase-2 response genes (e.g., catalase, superoxide dismutase) when compared to FA or NE mice (Nel et al 2005). While HFD-fed apoE null mice did not exhibit differences in their aortic atherosclerosis, they still show evidence of increased systemic oxidative stress as evidenced by significant upregulation of Nrf2 and Nrf2-regulated genes.
 
Genome-wide analysis screening in endothelial cells revealed synergistic cellular stress responses during exposure to organic DEP extracts and oxidized LDL components (Gong et al 2007).
We used human microvascular endothelial cells (HMEC) to test the hypothesis that pollutant particles synergize with known pro-atherogenic stimuli and mediators in their ability to elicit oxidative stress and promote atherosclerosis (Gong et al 2007). We studied the combined effects of a model air pollutant, diesel exhaust particles (DEP), and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on genome-wide gene expression. HMEC were treated in triplicate wells with an organic DEP extract (5 mg/ml), ox-PAPC (10, 20 and 40 mg/ml) or combination of both compounds for 4 hours. Gene expression profiles were assessed by Illumina microarray technology. We found that ox-PAPC as well as DEP regulated a large number of genes (up-hand down-regulated) in a dose-dependent fashion (Gong et al 2007). More importantly, a marked degree of co-regulation was present where the combined action of DEP and ox-PAPC resulted in a different effect than DEP and ox-PAPC alone. All together, 1555 genes were significantly upregulated (> 1.5 fold, p < 0.05) by the three DEP and ox-PAPC combinatory treatments. Notably, some genes were uniquely regulated by ox-PAPC and not by DEP, and vice versa, some genes were regulated by DEP but not by ox-PAPC.
 
We used weighted gene co-expression network analysis (WGCNA) to identify 12 modules of densely interconnected genes that were given unique color codes (Gong et al 2007). We found three modules (Brown, Green and Yellow) that were most highly enriched in genes that were differentially regulated by the stimuli. Interestingly, all these three modules exhibited patterns of additive/synergistic interaction where the combined action of DEP and ox-PAPC resulted in a greater effect than each compound alone. We developed a novel synergistic index that allows us to differentiate in between additive effects and synergistic effects. Conceptually, we defined synergy as a response to DEP plus ox-PAPC that was greater than the effects induced by either compound alone and greater than the summation of those individual effects. Interestingly, the brown, green and yellow modules concentrated 83% of the synergistically expressed genes identified in the gene network. These three modules were also enriched in synergistically coregulated genes and pathways relevant to vascular inflammation. We validated our gene expression data by quantitative PCR (qPCR) in the same set of samples analyzed by microarray analysis and in a set of samples from an independent experiment. Representative genes from various pathways were selected including ARE-regulated genes [e.g. HO-1, selenoprotein S (SELS)], inflammatory response genes [e.g. Interleukin 8 (IL-8), chemokine (C-X-C motif) ligand 1 (CXCL1)], immune response genes [e.g. Interleukin 11 (IL-11)], UPR genes [e.g. ATF 4, heat shock 70kDa protein 8 (HSPA8), X-box binding protein 1  (XBP1)], oxygen and reactive oxygen species metabolism genes [e.g. dual specificity phosphatase 1 (DUSP1), PDZ and LIM domain 1 (PDLIM1)]. qPCR could confirm 91 % of the synergistic effects that were revealed by microarray technology (Gong et al 2007). All considered, the synergistic response profile represents a combination of hierarchical oxidative stress as well as protein unfolding response genes.
 
We validated this synergy on selected genes in vivo by demonstrating that liver gene expression of hypercholesterolemic mice (HFD protocol from section 1) exposed to ambient ultrafine particles exhibited significant upregulation of the module genes (Gong et al 2007). Indeed, liver tissue was assayed for mRNA expression of HO-1, as well as two key UPR transcription factors, XBP1 and ATF4. UFP-exposed animals exhibited a significant up-regulation (p < 0.05) of all three genes in comparison with FP, FA and NE mice. These results indicate that the synergistic effects predicted by our in vitro studies have important in vivo outcomes, in which pro-oxidative PM chemicals may gain access to the systemic circulation from the lung and may then be able to synergize with circulating ox-LDL. 
 
Progress on studies looking at the effect of the pro-oxidative potential of ambient particles on their adjuvant effect on allergic inflammation in a murine asthma model.
 
Although studies have suggested that ambient PM can act as an adjuvant to promote sensitization to common environmental allergens, there is a paucity of direct evidence showing this effect on allergic sensitization in vivo. We have developed a mouse model, which allows us to demonstrate the adjuvant effect of ambient ultrafine particles (UFP) on ovalbumin (OVA)-induced allergic sensitization in vivo. Intranasal sensitization of Balb/C mice with a low dose of endotoxin-free OVA (10 mg) in the presence of ambient PM followed by OVA (1%) aerosole challenge resulted in a significant increase of allergic inflammation compared to the saline control or OVA alone. At a dose of 0.5 mg/mouse, UFP significantly enhanced OVA-induced eosinophil infiltration, airway inflammation, and serum OVA-specific IgE and IgG1 production. UFP also increased the production of a number of pro-inflammatory cytokines in the lung.  Cytometric bead array anaysis of BAL showed that while the animals in OVA/UFP group had significantly enhanced production of TNFa, IL-5, IL-6, IL-13, KC, MCP-1 and MIP-1a, those in other groups (OVA alone, LPS plus OVA, or F/UF plus OVA) were unaffected. The increase of IL-5 and IL-13 by OVA/UFP is important indicator suggesting that ambient UFP be capable of skewing immune response towards Th2 immunity.
 
Using different controls, we were able to show that neither endotoxin nor ultrafine carbon black particles had any enhancing OVA sensitization. Moreover, side-by-side comparison of UFP and FP indicate that this adjuvant effect is specific to the UFP since FP failed to enhance the effect of OVA. This approach could allows us to compare the contribution of particle size and accompanying differences in the pro-oxidative potential of PM2.5 and UFP in an in vivo model, similar to what we have previously demonstrated in tissue culture cells (Li et al, Environ. Health Perspect.  111:455-460 ; 2003). To provide further evidence that the adjuvant effect of UFP correlates to its redox –active organic chemical contents, we characterized the ambient PM for their chemical composition, redox potential, and the ability to induce intracellular oxidative stress. Ambient UFP consistently had higher organic carbon content compared to the fine PM.  DTT assay, which measures the redox activity of ambient PM based on the interaction between quinones and DTT, showed that ambient UFP had much stronger oxidant potential than the fine particles. Moreover, ambient UFP also had a greater ability to induce antioxidant enzyme HO-1, a sensitive marker of oxidative stress.  Taken together, these data suggest that the degree of adjuvancy is related to the OC content and redox potential of PM. 
 
Morphometric analysis of the lung showed that the major changes in the lungs of OVA/UFP-treated mice consisted of marked mucous cell metaplasia in the surface epithelium lining the conducting airways (large- and small-diameter bronchioles) and an associated mixed inflammatory cell influx consisting mainly of eosinophils, lymphocytes and plasma cells in the interstitial tissues surrounding these airways.  Airway lesions were most severe in the main axial airways, but were also present to a slightly lesser degree in the small-diameter, terminal bronchioles of the mice exposed to both OVA and UFP. Along the axial airways, the volume densities of intraepithelial mucosubstances in the proximal and distal generations (5 and 11) were approximately 22 and 24 times greater, respectively, than those measured at the same airway generations in saline-instilled control mice.  Mice exposed only to OVA had epithelial and inflammatory alterations that were similar to those in the OVA/UFP mice, but the severity of these changes in the large-diameter, pre-terminal and small-diameter, terminal bronchioles were must less severe.  As a comparison, the morphometrically determined volume densities of mucosubstances in the airway epithelium lining the proximal axial airways (generation 5) of OVA/UFP mice were approximately twice as much as those in mice exposed only to OVA. In the distal axial airway (generation 11), OVA/UFP mice had almost five times more intraepithelial mucosubstances compared to those in OVA-alone mice.
 
In addition to the lower airway, we have investigated changes in the nasal mucosa. Nasal histopathology and morphometry showed that mice exposed to both OVA and UFP had airway epithelial and inflammatory changes consistent with an acute allergic rhinitis. The principal morphologic alterations were mucous cell metaplasia/hyperplasia of airway epithelium accompanied by inflammatory cell influx including eosinophils and mononuclear cells (lymphocytes and plasma cells) in the underlying lamina propria of the nasal mucosa. These changes were restricted to intranasal regions lined by transitional or respiratory epithelium.  No alterations were present in regions lined by olfactory epithelium. There was a markedly greater amount of mucosubstances in the nasal transitional epithelium lining the maxilloturbinates compared to the mice in the control or OVA-alone groups.  These data suggest that UFP exert its adjuvant effect immediately upon contact with the allergen in the nasal turbinate and this may explain the association between air pollution and allergic rhinitis. 
           
Taken together, these results suggest that intranasal OVA delivery caused minimal allergic airway, epithelial and inflammatory responses in the lungs and nose of mice without concomitant intranasal UFP instillation. Thus, intranasal exposure to UFP exerts an adjuvant effect that could not be obtained with intranasal fine PM.  The results are currently being written up for publication and will serve as the launching pad for carrying out in vivo inhalation exposure studies that will attempt to show that CAPs, UFP in particular, can lead to similar sensitization.
 
Exposure to pro-oxidative DEP chemicals perturb the antigen-presenting function of dendritic cells, which may explain the adjuvant effect of PM in asthma (Chan et al 2006; Gilmour et al 2006; Phalen et al 2006).
Dendritic cells (DCs) play a key role in antigen presentation in the immune system. There is growing evidence that the redox equilibrium of DCs influence their ability to induce T-cell activation and to regulate the polarity of immune response. Ambient PM acts as an adjuvant that promotes sensitization to common environmental allergens. Systematic dissection of the molecular pathways of PM-induced adjuvancy is of great general interest and it is also a key priority in the research of asthma and allergy (Gilmour et al 2006). We are studying the hypothesis that altered cellular redox equilibrium by PM and adsorbed redox cycling organic chemicals leads to the perturbation of DC function and favors Th2 skewing of the immune response. In year 2 we investigated how DEP chemical-induced oxidative stress interferes with DC function including maturation, antigen uptake, antigen presentation, expression of costimulatory molecule, cytokine/chemokine production, and T-cell activation (Chan et al 2006). DCs were prepared from mouse bone marrow cells. Exposure of DCs to organic DEP extract (DEPext) resulted in a dose-dependent glutathione depletion and the induction of antioxidant enzyme, heme oxygenase-1 (HO-1) (Chan et al 2006).
 
Although DEP chemicals per se did not change the expression of DC surface molecules (I-Ad, CD54, and CD86), DEPext was able to suppress LPS-induced expression of these molecules in a dose-dependent fashion. Similarly, while DEPext alone failed to exert an effect on IL-12p40 and IL-12p70 production by DCs, it suppressed the LPS-induced production of this cytokine (Chan et al 2006). The inhibitory effects of DEPext on LPS-induced CD86 expression and IL-12 production could be neutralized by thiol antioxidant, N-acetyl cysteine, indicating the involvement of oxidative stress.
 
Using CD4+ T cells from a T-cell receptor transgenic mouse strain (DO11.10) that recognizes OVA323-339 in the context of the BALB/c MHC class II (I-Ad), we demonstrate that simultaneous exposure of DCs to DEPext and LPS induced a significant decrease in IFN-g production compared with that in the cells treated with LPS only. Furthermore, exposure of DCs to DEPext alone, before antigen pulsing, significantly increased IL-10, a Th2 cytokine, production in the co-cultured T cells. In addition to inhibiting LPS effect (TLR4), organic DEP chemicals also suppressed CD86 expression induced by TLR2, TLR3, and TLR9 agonists suggesting that TLRs may be targets of DEP chemicals (Chan et al 2006). Using BMDCs from Nrf2-deficient mice, we show that Nrf2 is required for the suppression of LPS effects by DEP chemicals.  We demonstrate that DEPext inhibits LPS effects on DC by interfering with NFkB signaling pathway. The findings from the our second-year studies indicate that organic DEP chemicals indeed alter the redox equilibrium in DCs and that oxidative stress does interfere with several DC functions leading to the suppression of Th1 response (Chan et al 2006).  Our studies also indicate that Nrf2-mediated phase II response and NF-kB signaling pathway play keys roles in modulating DC function under conditions of PM-induced oxidative stress. 
 
The adjuvant effect at the level of DC could play out in select sites of mucosal immunity in the lung.  Utilizing tracheobronchial particle dose considerations, we were able to demonstrate that it is theoretically possible to achieve the in vitro levels of oxidative stress that are described in the aforementioned paragraph at so-called hotspots of deposition in the lung (Phalen et al 2006).  These deposition sites that are located at point of airway bifurcation could be the mucosal areas where the allergen as well as the inhaled particles come together to drive adjuvant effects in the immune system.
 
Proteome analysis of the oxidative stress responses in a bronchial epithelial cell line BAL Fluid (Jung et al 2007).
We have previously demonstrated that organic redox cycling diesel exhaust particle (DEP) chemicals induce a tiered oxidative stress response in a macrophage cell line as determined by proteome analysis (Xiao at al; J Biol Chem, 278:50781-50790; 2003). In collaboration with Dr. Joseph Loo from the Keck Proteomics facility at UCLA, a 2D-difference gel electrophoresis (DIGE) technology was used to demonstrate that organic DEP extracts induce a pro-inflammatory response in a BEAS-2B human bronchial epithelial cell line (Jung et al 2007).  The DIGE method uncovered induction of an unfolding protein response (UPR) that is characterized by increased IL-6 and IL-8 production in parallel with increased expression of Hsp70, HSF-1, and ATF4 (Jung et al 2007).  The UPR pathway is activated under stress conditions in order to govern the protein processing and/or folding in the ER.  These results corroborate the demonstration of a protein unfolding response by the gene clustering analysis that was performed in endothelial cells as well as the gene response profile in the livers of apoE mice exposed to concentrated air pollution particles as reported above.
 
The identification of global protein expression changes in BAL fluid (BALF) and lung tissue from ovalbumin (OVA) sensitized mice could provide new insights into the complex molecular mechanisms involved in asthma.  We are using two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify significantly increased protein expression in a murine asthma model and potential protein targets of N-acetylcysteine (NAC) in allergic airway inflammation.  Six proteins were found to be significantly increased in BALF from OVA-challenged Balb/c mice compared to a control group: chitinase 3-like protein 3 (Ym1), chitinase 3-like protein 4 (Ym2), acidic mammalian chitinase (AMCase), pulmonary surfactant-associated protein D (SP-D), resistin-like alpha (FIZZ1), and haptoglobin a-subunit.  A total of 11 protein spots on 2D-gels were significantly increased in lung tissue from the murine asthma model, including Ym1, Ym2, FIZZ1, and other lung remodeling related proteins.  Western blotting confirmed increased Ym1/Ym2, SP-D, and FIZZ1 expression measured from BAL fluid and lung tissue from OVA-challenged mice.  Administration of NAC intraperitoneally before OVA inhalation inhibited Ym1/Ym2, SP-D, and FIZZ1 expression from BALF and lung tissue. Proteins Ym1/Ym2, FIZZ1 and SP-D identified in this study could be associated with the pathogenesis of asthma and suggest a link between oxidative stress-induced inflammation and asthma.  A manuscript reporting these findings upon completion of the immunohistochemical analysis is being prepared.
 
Conclusions: All considered, our data indicate that oxidative stress indeed plays a major role in driving inflammatory responses in the respiratory and cardiovascular systems.  We have developed study methods and tools that are capable of following these oxidative stress effects under abiotic and biotic conditions, with the capability of linking that to specific PM chemical components such as the OC fraction.  We are capable of using predictive in vitro models to link the oxidant injury to pathological processes in vivo, in particular atherosclerosis and asthma.  These data are being integrated in the center with studies source and aerosols studies as well as ongoing research in humans in order to develop a comprehensive understanding of the role of oxidant injury in PM-induced adverse health effects.
 

Expected Results:

We expect that due to the presence of redox cycling chemicals, ambient PM induce a series of pro-oxidative and pro-inflammatory effects which enhance asthma and atherosclerosis. We expect that these effects will be related to particle dose, size, source, composition, and season, and will be exaggerated in individuals and animals with a weakened antioxidant defense. This study will yield important biomarkers that will be linked to specific toxicological components that could be monitored to prevent adverse health effects.

Future Activities:

1. We will continue the collaboration with Dr. Jake Lusis and Jesus Araujo using the genome wide association screening (GWAS) to elucidate gene clusters that can be used for bioassay development that reflect the impact of ambient fine versus UFP exposure in murine lung tissue.  We are currently using lungs that were collected during the performance of atherosclerosis studies with the apoE knockout mice and are the process of analyzing promising gene clusters that reflect the pro-the inflammatory effect of different particle sizes.  These studies will be supplemented by further animal exposures in the mobile laboratory as well as using the lung tissue for immunohistochemistry and proteome analysis. We are looking to establish in vivo biomarkers for oxidative stress that can be used in air pollution research.  We will also use BAL fluid from our OVA adjuvancy model to screen for potential biomarkers of oxidative stress through the use of proteome profiling.
2. We will continue to work on the OVA adjuvancy model including performing studies that will investigate the role of oxidative stress at the level of antigen presenting cells as outlined in our studies using dendritic cells and diesel exhaust particle extracts in year two.  We will determine whether in vivo inhalation exposure the same outcome and whether bronchial epithelial cells contribute to modification of dendritic cell behavior in vitro and in vivo. These studies will be conducted in collaboration with Project 1, Dr Harkema and Dr Kleinman.
3. In collaboration with Project 3, Project 1 and Dr William Hinds, we will attempt to develop online instrumentation that can be used to monitor the oxidant potential of ambient UFP as a metric for their biological potency.  This study is premised on the DTT assay that Dr. Art Cho and Dr. John Froines have previously developed to measure PM oxidant potential based on thiol chemistry.  These studies that will be conducted by a new postdoctoral fellow, Dr. Allen Haddrell, who proposes to design and build instrumentation with the ability to monitor the oxidative stress potential of a given sample of UFP in near real time.  The proposed operation of the device is as follows: Particles are pulled from the air via a vacuum, once in the system the particles are charged via a corona discharge.  The charged particles are then directed into an AC levitation trap where they can be held indefinitely.  The levitated particles are subsequently introduced to a primary thiol in the vapor phase.  The particles oxidize the thiol vapor, generating thiol radicals that are ionized with a deep UV laser.  The thiol ions are ejected from the levitation chamber into an ion trap where they are counted.  The number of thiol ions produced is used to predict the oxidative potential of the particles held within the trap.  Through the use of standardized materials, the data will be converted to report the oxidative stress potential of the suspended particles in a given fraction of air rather than simply reporting the absolute number.
4. In collaboration with Drs Delfino, Araujo, and Lusis, we will begin the work on Aim 3 that proposes to use serum samples collected from indoor exposed elderly human subjects with ischemic heart disease in Project 4 to determine how oxidation of HDL affects the anti-inflammatory and anti-oxidative properties of this lipoprotein fractioin. The utility of this assessment has been demonstratedin the murine atherosclerosis studies that were discussed above (1). Dr Jake Lusis and Dr Jesus Araujo will determine whether oxidation of HDL leads to a decline in the anti-inflammatory protective actibity of human HDL. Dr. Delfino has collected 156 plasma samples that were preserved in a sucrose cryo-preservative solution. These samples correspond to 5-12 repeated measures in 17 individuals belonging to one out of four retirement communities in Dr Delfino's CHAPS study. These samples can be used to assess HDL antioxidant capacity by employing a fluorescent cell-free assay that allows us to evaluate the ability of HDL to inhibit LDL oxidation. Briefly, HDL fractions will be isolated by magnetic bead separation. Once separated, 0.625 μg of HDL samples will be preincubated with 0.25 μg standard LDL in triplicates on 96-well microplates at 37 ºC for 30 minutes. 5 μg DCF will be then incubated at 37 ºC for 1 hour. Reactive oxygen species will be determined by the degree of DCF-fluorescence read in a fluorescence plate reader at an excitation of 485 nm and emission of 530 nm. HDL anti-inflammatory capacity will be expressed in HDL inflammatory index (HII) units, calculated as the ratio of LDL-induced DCF fluorescence in the presence vs. absence of HDL. HII > 1 indicates a pro-inflammatory potential.


Journal Articles on this Report : 10 Displayed | Download in RIS Format

Other subproject views: All 34 publications 23 publications in selected types All 23 journal articles
Other center views: All 241 publications 157 publications in selected types All 157 journal articles
Type Citation Sub Project Document Sources
Journal Article Araujo JA, Barajas B, Kleinman M, Wang X, Bennett BJ, Gong KW, Navab M, Harkema J, Sioutas C, Lusis AJ, Nel AE. Ambient particulate pollutants in the ultrafine range promote early atherosclerosis and systemic oxidative stress. Circulation Research 2008;102(5):589-596. R832413 (2008)
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  • Journal Article Ayres JG, Borm P, Cassee FR, Castranova V, Donaldson K, Ghio A, Harrison RM, Hider R, Kelly F, Kooter IM, Marano F, Maynard RL, Mudway I, Nel A, Sioutas C, Smith S, Baeza-Squiban A, Cho A, Duggan S, Froines J. Evaluating the toxicity of airborne particulate matter and nanoparticles by measuring oxidative stress potential—a workshop report and consensus statement. Inhalation Toxicology 2008;20(1):75-99. R832413 (2008)
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  • Journal Article Chan RC-F, Wang M, Li N, Yanagawa Y, Onoe K, Lee JJ, Nel AE. Pro-oxidative diesel exhaust particle chemicals inhibit LPS-induced dendritic cell responses involved in T-helper differentiation. Journal of Allergy and Clinical Immunology 2006;118(2):455-465. R832413 (2008)
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  • Journal Article Chatila TA, Li N, Garcia-Lloret M, Kim H-J, Nel AE. T-cell effector pathways in allergic diseases:transcriptional mechanisms and therapeutic targets. Journal of Allergy and Clinical Immunology 2008;121(4):812-823. R832413 (2007)
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  • Journal Article Gong KW, Zhao W, Li N, Barajas B, Kleinman M, Sioutas C, Horvath S, Lusis AJ, Nel A, Araujo JA. Air-pollutant chemicals and oxidized lipids exhibit genome-wide synergistic effects on endothelial cells. Genome Biology 2007;8(7):R149 (13 pp.). R832413 (2008)
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  • Journal Article Li N, Nel AE. The cellular impacts of diesel exhaust particles:beyond inflammation and death. European Respiratory Journal 2006;27(4):667-668. R832413 (2008)
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  • Journal Article Li N, Xia T, Nel AE. The role of oxidative stress in ambient particulate matter-induced lung diseases and its implications in the toxicity of engineered nanoparticles. Free Radical Biology and Medicine 2008;44(9):1689-1699. R832413 (2007)
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  • Journal Article Xia T, Kovochich M, Brant J, Hotze M, Sempf J, Oberley T, Sioutas C, Yeh JI, Wiesner MR, Nel AE. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Letters 2006;6(8):1794-1807. R832413 (2008)
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    R832413C001 (2008)
    R832413C001 (Final)
    R832413C002 (2006)
    R832413C002 (2008)
    R827352 (Final)
    R827352C002 (Final)
    R827352C014 (Final)
  • Abstract from PubMed
  • Full-text: UCLA-Full Text PDF
    Exit
  • Abstract: ACS-Abstract
    Exit
  • Other: ResearchGate-Abstract & Full Text-PDF
    Exit
  • Journal Article Xia T, Kovochich M, Nel A. The role of reactive oxygen species and oxidative stress in mediating particulate matter injury. Clinics in Occupational and Environmental Medicine 2006;5(4):817-836. R832413 (2008)
    R832413 (Final)
    R832413C002 (2007)
    R832413C002 (2008)
  • Abstract from PubMed
  • Abstract: Dimensions-Abstract
    Exit
  • Journal Article Xia T, Kovochich M, Nel AE. Impairment of mitochondrial function by particulate matter (PM) and their toxic components: implications for PM-induced cardiovascular and lung disease. Frontiers in Bioscience 2007;12(4):1238-1246. R832413 (2008)
    R832413 (Final)
    R832413C002 (2007)
    R832413C002 (2008)
  • Abstract from PubMed
  • Full-text: Frontiers In Bioscience-Full Text HTML
    Exit
  • Abstract: Frontiers In Bioscience-Abstract
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  • Supplemental Keywords:

    Ambient air, health effects, biology, sensitive populations, human health, animal, PAH,, RFA, Health, Scientific Discipline, Air, particulate matter, Toxicology, Health Risk Assessment, Risk Assessments, Biochemistry, Ecology and Ecosystems, atmospheric particulate matter, particulates, human health effects, PM 2.5, animal model, airway disease, airborne particulate matter, cardiovascular vulnerability, air pollution, human exposure, vascular dysfunction, cardiovascular disease, human health risk

    Progress and Final Reports:

    Original Abstract
  • 2006 Progress Report
  • 2007 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • 2011
  • Final Report

  • Main Center Abstract and Reports:

    R832413    Southern California Particle Center

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832413C001 Contribution of Primary and Secondary PM Sources to Exposure & Evaluation of Their Relative Toxicity
    R832413C002 Project 2: The Role of Oxidative Stress in PM-induced Adverse Health Effects
    R832413C003 The Chemical Properties of PM and their Toxicological Implications
    R832413C004 Oxidative Stress Responses to PM Exposure in Elderly Individuals With Coronary Heart Disease
    R832413C005 Ultrafine Particles on and Near Freeways