2003 Progress Report: Pro-inflammatory and the Pro-oxidative Effects of Diesel Exhaust Particulate in Vivo and in Vitro

EPA Grant Number: R827352C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R827352
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Southern California Particle Center and Supersite
Center Director: Froines, John R.
Title: Pro-inflammatory and the Pro-oxidative Effects of Diesel Exhaust Particulate in Vivo and in Vitro
Investigators: Nel, Andre E.
Current Investigators: Nel, Andre E. , Cho, Arthur K. , Froines, John R. , Li, Ning , Sioutas, Constantinos
Institution: University of California - Los Angeles
Current Institution: University of California - Los Angeles , University of Southern California
EPA Project Officer: Chung, Serena
Project Period: June 1, 1999 through May 31, 2005 (Extended to May 31, 2006)
Project Period Covered by this Report: June 1, 2002 through May 31, 2003
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air


The objectives of this research project are to:

  1. Establish a murine model for ovalbumin (OVA)-induced allergic inflammation and use that model to study the adjuvant effects of diesel exhaust particles (DEP) on allergic inflammation. This model seeks to address the hypotheses that particulates induce proinflammatory effects in the lung by inducing oxidative stress and that particle composition determines the level of oxidative stress and the extent of the inflammatory changes.
  2. Establish an in vitro model of toxicity in tissue culture macrophages and epithelial cells in which the effects of particle composition can be studied in terms of oxidative stress effects, cytokine production, and induction of cytotoxicity (apoptosis). This includes the study of concentrated ambient particles, which are collected in a liquid impinger (BioSampler).

Our studies are premised on the presence of redox-cycling chemicals that may be responsible for the biological and adverse health effects of particulate matter (PM). Our principal hypothesis is that these chemical compounds engage in redox-cycling reactions in the lung, where the generation of reactive oxygen species (ROS) and/or covalent modification of critical cellular molecules induce oxidative stress. We propose that the generation of oxidative stress in epithelial cells and macrophages leads to a hierarchical response that is protective in nature, but may yield to injurious effects if the level of oxidative stress increases. We propose that the proinflammatory effects are responsible for the adjuvant effects of DEP on allergic inflammation and asthma.

This is one of the project progress reports for the SCPCS. The progress for the other research projects conducted by the Center is reported separately (see reports for R827352, R827352C001 through R827352C007, and R827352C009 through R827352C021).

Progress Summary:

Objective 1

We have been experimenting with murine asthma models to elucidate the effects of DEP on allergic airway inflammation and airway hyperreactivity (AHR). In Year 3 of the project, we showed that it is possible to use aerosolized DEP in an OVA inhalation-sensitization model to elicit adjuvant effects in terms of OVA-specific IgE and IgG1 antibody responses. This model, however, did not yield an increase in bronchoalveolar lavage (BAL) eosinophils or an increase in AHR as determined by whole body plethysmography (WBP). We concluded that combined DEP/allergen inhalation challenge is insufficient for achieving the sensitization threshold that is required to induce widespread allergic inflammation and AHR. Numerous attempts to develop a short-term animal model in which DEP exert adjuvant as well as AHR effects have failed. This includes the use of intraperitoneal (ip) OVA sensitization as well as varying the time point and dose of DEP administration. We did, however, notice that ip OVA administration in the absence of alum can induce a state of mild allergen sensitization in which DEP exert effects on AHR. Because PM exposure can induce an acute asthma exacerbation, we set out to develop protocols that can be used to study DEP effects on AHR independent of the adjuvant effects of these particles.


The following three protocols were developed (two require OVA sensitization and the third is OVA independent):

  1. In the mild sensitization protocol, Balb/c mice received ip OVA without alum and then were challenged with aerosolized OVA ± DEP.
  2. In the postchallenge model, DEP was delivered after OVA challenge to animals sensitized by ip OVA plus alum.
  3. Nebulized DEP also were delivered to IL-5 overexpressing mice that exhibit constitutive airway inflammation. Animals were subjected to WBP and then sacrificed for performance of BAL, histology, and serology.

We demonstrated that DEP could induce AHR in all three protocols. In the mild OVA sensitization protocol, DEP delivery concomitant with OVA challenge induced increased airway obstruction as well as increased inflammatory changes and mucin production in large and intermediary airways (manuscript in preparation, 2004). A possible explanation for the impact of nebulized DEP on large and intermediary airways is the deposition characteristics of the relatively large nebulized droplets. Animals in this protocol did not exhibit an increase in OVA-specific IgE or TH2 cytokine levels. The second model utilizes constitutive eosinophilic inflammation to elicit an effect on AHR and airway inflammation in IL-5 transgenic mice (manuscript in preparation, 2004). The realization that a lesser, but above threshold level, of airway inflammation is optimal for elucidating DEP effects led us to adapt the classical OVA-sensitization model (OVA + alum administration ip) for DEP-induced AHR. Instead of delivering the DEP at the time of OVA challenge, the nebulized particles were given after the allergen challenge. At this point, there is a decline in OVA-induced allergic inflammation, allowing us to observe the effects of nebulized DEP on AHR and lung morphology. This approach overcomes previous problems with the classical model in which DEP administration concomitant with OVA challenge was not potent enough to elicit effects on AHR and airway inflammation. With these exposure protocols in place, we should be able to dissect chemical components by which DEP induce acute asthma exacerbations.

Objective 2

We have extended the concept that the biological effects of DEP are mediated through the generation of oxidative stress. We provided evidence that a range of biological effects indicate a stratified oxidative stress response to DEP chemicals. In the past year, we have extended these concepts to study concentrated ultrafine (UF) particles collected in the Los Angeles Basin. We previously have shown that these particles induce oxidative stress via a perturbation of mite contra function. For that reason, we began testing these particles for their effects on oxidative stress responses and isolated mitochondrial preparations in vitro.


We demonstrated that ambient UF, but not commercial, nanoparticles exert potent functional effects on isolated mitochondria (manuscript in preparation). In addition, we showed that aliphatic, aromatic, and polar organic compounds, fractionated from DEP, exert differential toxic effects in RAW 264.7 cells. Cellular analyses showed that the quinone-enriched polar fraction was more potent than the polycyclic aromatic hydrocarbon (PAH)-enriched aromatic fraction in O2·¯ generation, decrease of membrane potential (ΔΨm), loss of mitochondrial membrane mass, and induction of apoptosis. When studying these chemical fractions in isolated liver mitochondria, a major effect of the polar fraction was to promote cyclosporin A-sensitive permeability transition pore (PTP) opening, both directly at low doses as well as by inducing ΔΨm depolarization at higher doses if mitochondria were first Ca2+ loaded. The PTP effect was mimicked by redox-cycling DEP quinones. Although the aliphatic fraction failed to perturb mitochondrial function, the aromatic fraction at low dose increased the Ca2+ retention capacity, whereas at high dose it induced mitochondrial swelling and a decrease in ΔΨm. This swelling effect was mostly cyclosporin A insensitive and could be reproduced by a mixture of PAHs present in DEP. The above chemical effects on isolated mitochondria could be reproduced by ambient UF particles, which contain an abundance of the same organic chemical compounds. In contrast, commercial polystyrene UF particles failed to exert a mitochondrial effect, suggesting that DEP and UF effects are mediated by their chemical compounds and not by the particles.

The proinflammatory effects of particulate pollutants, including DEP, are related to their content of redox-cycling chemicals and their ability to generate oxidative stress in the respiratory tract. To prevent this effect and to protect against asthma, there is an antioxidant defense pathway in place, which mediates the expression of phase II enzymes. This induction is dependent on the expression of a genetic antioxidant response element (ARE) in the promoters of these antioxidant and detoxification enzymes. We recently investigated the mechanism by which redox-cycling organic chemicals, prepared from DEP, induce phase II enzyme expression as a protective response. We demonstrate that aromatic and polar DEP fractions, which are enriched in PAHs and quinones, respectively, induce the expression of heme oxygenase-1 (HO-1), glutathione-S-transferase, and other phase II enzymes in macrophages and epithelial cells. We show that HO-1 expression is mediated through the accumulation of the bZIP transcription factor, Nrf2, in the nucleus. Nrf2 accumulation and ARE activation are regulated by the proteasomal degradation of Nrf2; this pathway is sensitive to the effect of prooxidative and electrophilic DEP chemicals. This effect could be reproduced by ambient UF particles and establishes an important antioxidant defense mechanism that may protect against the development of allergic inflammation and asthma in atopic people that are exposed to particulate pollutants.

Future Activities:

We will explore a range of organic molecules, including PAHs, their oxy-derivatives, aldehydes and ketones, and metals adsorbed on PM as they exert proinflammatory and tissue damaging effects through covalent binding of organic pollutants to cellular macromolecules, and generation of ROS that results in cardiopulmonary effects. We also aim to explore the notion that PM from vehicles powered by diesel engines is more toxic than PM from gasoline-powered sources based on data from in vitro and source tracer studies by comparing the biological activity of samples collected on freeways and a traffic tunnel using HO-1 expression and regulation by Nrf2, as well as cellular glutathione ratios, as markers for oxidative stress in tissue culture cells.

Journal Articles:

No journal articles submitted with this report: View all 15 publications for this subproject

Supplemental Keywords:

particulate matter, quinones, polycyclic aromatic hydrocarbons, PAHs, aldehydes, ketones, metals, allergic airway disease, human exposure studies, asthma, cardiovascular effects, aerosol sampling, atmospheric aerosol, environmental monitoring, environmental statistics, California, CA, acute exposure, aerosols, air pollution, air quality, air toxics, airway disease, allergen, allergic response, ambient aerosol, assessment of exposure, asthma triggers, atmospheric chemistry, bioaerosols, biological response, childhood respiratory disease, children, dosimetry, environmental hazard exposures, environmental health hazard, environmental triggers, environmentally caused disease, epidemiology, exposure assessment, health effects, home, household, human exposure, human health effects, indoor air quality, inhaled particles, lead, outdoor air, particle concentrator, particle transport, particulate exposure, particulates, sensitive populations, toxicology, toxics,, RFA, Health, Scientific Discipline, Air, Geographic Area, HUMAN HEALTH, particulate matter, Environmental Chemistry, Air Pollutants, State, Risk Assessments, Biochemistry, Health Effects, ambient aerosol, asthma, particulates, human health effects, toxicology, quinones, airway disease, diesel exhaust particulates, allergic airway disease, air pollution, diesel exhaust, PAH, human exposure, toxicity, particulate exposure, California (CA), allergens, aerosols, atmospheric chemistry, human health risk, particle transport, particle concentrator

Relevant Websites:

http://www.scpcs.ucla.edu/exit EPA

Progress and Final Reports:

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

  • Main Center Abstract and Reports:

    R827352    Southern California Particle Center and Supersite

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R827352C001 The Chemical Toxicology of Particulate Matter
    R827352C002 Pro-inflammatory and the Pro-oxidative Effects of Diesel Exhaust Particulate in Vivo and in Vitro
    R827352C003 Measurement of the “Effective” Surface Area of Ultrafine and Accumulation Mode PM (Pilot Project)
    R827352C004 Effect of Exposure to Freeways with Heavy Diesel Traffic and Gasoline Traffic on Asthma Mouse Model
    R827352C005 Effects of Exposure to Fine and Ultrafine Concentrated Ambient Particles near a Heavily Trafficked Freeway in Geriatric Rats (Pilot Project)
    R827352C006 Relationship Between Ultrafine Particle Size Distribution and Distance From Highways
    R827352C007 Exposure to Vehicular Pollutants and Respiratory Health
    R827352C008 Traffic Density and Human Reproductive Health
    R827352C009 The Role of Quinones, Aldehydes, Polycyclic Aromatic Hydrocarbons, and other Atmospheric Transformation Products on Chronic Health Effects in Children
    R827352C010 Novel Method for Measurement of Acrolein in Aerosols
    R827352C011 Off-Line Sampling of Exhaled Nitric Oxide in Respiratory Health Surveys
    R827352C012 Controlled Human Exposure Studies with Concentrated PM
    R827352C013 Particle Size Distributions of Polycyclic Aromatic Hydrocarbons in the LAB
    R827352C014 Physical and Chemical Characteristics of PM in the LAB (Source Receptor Study)
    R827352C015 Exposure Assessment and Airshed Modeling Applications in Support of SCPC and CHS Projects
    R827352C016 Particle Dosimetry
    R827352C017 Conduct Research and Monitoring That Contributes to a Better Understanding of the Measurement, Sources, Size Distribution, Chemical Composition, Physical State, Spatial and Temporal Variability, and Health Effects of Suspended PM in the Los Angeles Basin (LAB)