2008 Progress Report: Project 1 -- Pulmonary Metabolic Response

EPA Grant Number: R832414C001
Subproject: this is subproject number 001 , established and managed by the Center Director under grant R832414
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Center Director: Wexler, Anthony S.
Title: Project 1 -- Pulmonary Metabolic Response
Investigators: Winkle, Laura Van , Buckpitt, Alan , Fanucchi, Michelle V.
Current Investigators: Fanucchi, Michelle V. , Buckpitt, Alan , Plopper, Charles , Winkle, Laura Van
Institution: University of California - Davis
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2011)
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


To determine whether the increased pulmonary vulnerability to polycyclic aromatic hydrocarbons (PAHs) in neonates is exacerbated when the PAH is adsorbed to particulate matter. By compromising detoxification mechanisms, particles of mixed composition, such as carbon and a PAH, carbon and a transition metal, or carbon with both a transitional metal and a PAH, will result in more injury than particles composed of only one component.


We will apply transdisciplinary approaches to test these hypotheses, including histological, biochemical, and microarray analyses. Using defined synthetic particles consisting of graphitic carbon, a PAH, and a transitional metal, we will compare responses in intact airways at different sites of susceptibility (branch points and airway wall) in postnatal and adult rats to responses in human airway epithelial cell lines. As the Architecture Development Project defines the sites of deposition, we will adjust the sites that we are evaluating in the lung accordingly.

Progress Summary:

1. Insufflation with 3 different carbon particles +/- 1-NN: 1-Nitronaphthalene (1-NN) is a compound that is not very volatile, making it difficult to create a consistent atmosphere for dose-response studies. In addition, 1-nitronapththalene is also water-insoluble, making it difficult to administer it by instillation. However, significant amounts of 1-NN are associated with particulate air pollution. During this period of the grant, we focused on completing the analysis of the insufflation studies using a dry powder insufflator (PennCentury Model DP-4 Dry Powder Insufflator) which we used to resuspend soot particles with minimal impact on their original size and distribution. Rats were exposed to 2.5 mg of particles and necropsied at 24hrs after exposure. We compared carbon black, acetylene soot, ethylene soot and 1-NN (40 ug/mg) coated ethylene soot and 1NN coated acetylene soot. Sample were taken for high resolution histopathology of particle effects and of the particles themselves as well as cytokine analysis in the airway tissue. We found that equivalent doses of “clean” carbon black and flame generated soots illicit different cytokine responses and that the addition of 1-NN induced changes in epithelial cytokine profiles are unique to each carbon particle. Further the source and coating of the particle had an influence on cytotoxicity. “Clean” carbon black is cleared from the airways by 24 hours and shows little evidence of cytotoxicity. “Clean” flame generated soots do not clear from the airways by 24 hours and airway epithelium shows evidence of cytotoxicity (very thin epithelium, change in epithelial cell composition). Ethylene generated soot translocates into the subepithelial interstitium. 1-Nitronaphthalene-doped carbon black doesn’t clear completely from airways and results in changes in epithelial cell composition (mucous cells). Further, 1-Nitronaphthalene-doped flame generated soots do not clear from the airways and both result in extensive epithelial sloughing and cytotoxicity. One of the more interesting findings of these studies is the possibility of a cytokine “signature” for the different pollutants (Figure 1).
Figure 1:  Comparison of the cytokine profile within the airway tissue for 4 different particles in the airways of adult rats.
Paper in preparation: Acute Airway Epithelial Injury from Three Different Carbon Particles With and Without Adsorbed 1-Nitronapthalene (Fanucchi lead author)
2. Analysis of soluble metabolites and protein bound I-NN adducts in neonatal and adult rats. 1-nitronaphthalene cytotoxicity in the conducting airways of postnatal mice and rats cannot solely be attributed to site-specific bioactivation of the PAH (neonates have less) or baseline levels of GSH (neonates have more). Yet neonates are more susceptible- one hypothesis is that adduction of a critical protein for cellular function occurs in neonatal cells but not in adults. To address this hypothesis we incubated microdissected airways from adult and postnatal rats with 2 hrs with14C-1NN, 100 mM 1.1 E6 DPM/nmole. Then the samples were examined for total covalent binding to proteins, the types of proteins covalently adducted (analyzed by 2Dgel electrophoresis) and the types of metabolites formed (separated by HPLC and to be analyzed using MS). We made significant progress on this aim and initial analysis indicates that the some of the proteins adducted in the neonatal animals (susceptible) are different that in the adult animals (less susceptible).
Papers in preparation:
Age-Specific Pulmonary Metabolism of 1-Nitronaphthalene (Fanucchi lead author)
Alterations in Extracellular Nasal and Pulmonary Glutathione Pools in Adult and Postnatal Rat Following 1-Nitronapthalene Exposure (Fanucchi lead author)
Formation of 1-NN adducts in airways of neonatal and adult rats (Van Winkle, Buckpitt lead authors)
3. Acute exposure to diffusion flame PM in neonatal and adult rats in vivo and in vitro
The goal with these studies is to develop a reproducible exposure system for low PAH and high PAH particles which we can then subsequently modify to generate “custom” particles containing various PAHs of interest. We have completed (in collaboration with Project 5) assembly and testing of a diffusion flame generated soot chamber exposure system (low PAH) and have exposed both neonatal and adult rats to diffusion flame soot (130-170 ug/m3) acutely for 6 hours. We examined timepoints at 2 and 24 hours after exposure so that we could gauge temporal responses. Endpoints included peripheral blood CBC and cytokines, Cell counts in lavage and lavage cytokines, immunohistochemistry of key antioxidant genes, RT-PCR/microarray and histopathology. With the exception of the array, all the endpoints have received preliminary analysis. We did not detect airway cytotoxicity in the airways of exposed rats. Although diffusion flame particles do NOT cause frank cellular toxicity that is visible morphologically, they do cause a release of cytokines and stimulate neutrophil responses that differ by age and compartment with the adults having more of a peripheral blood increase at 24 hours and the neonates having an increase in lavage at 2 hrs. Further, there is up regulation of key antioxidant proteins: HO-1 and gamma GCS.
Figure 2: Up regulation of the key enzyme in glutathione synthesis, gamma gluamyl cysteine synthetase is apparent in the PM exposed groups only (dark staining).
The in vitro aspects of these studies have been shifte to incorporate field particulates as well as laboratory generated particulates in microdissected airway explaints. We are using airway explants because they are metabolically competent and many cell lines are not. Endpoints include cytotoxicity and cytokine release.

Expected Results:

Humans are exposed to multiple compounds early in life, yet most toxicological studies focus on the effects in adults. In addition, decisions regarding acceptable levels of environmental contaminants are based on adult data, which may not translate to children, the most susceptible portion of the population. Our work has already demonstrated that exposure to bioactivated pollutants produces much higher pulmonary toxicity in neonates than in adults. These studies will further define the role of particles in neonatal pulmonary susceptibility to environmental pollutants. Understanding the changes in lung cells following particle exposure at the gene and protein expression level will provide a basis for the development of biomarkers for assessing exposure and toxicity in young children.

Future Activities:

The need to address all the aims in the time allotted means that Project 1 needs to choose an exposure paradigm and stick with it to facilitate comparisons between the biological responses to different particle compositions. For the in vivo exposures to laboratory generated particles, we will continue whole body chamber exposures. Therefore, Project one will emphasize acute exposure of a single day duration with additional timepoints to take some advantage of the most recent SAC recommendation regarding injury/proliferation responses. Following the SAC panel’s recommendation we will monitor cell proliferation, but we will use PCNA labeling instead of BRdU so that we don’t have to inject a nucleotide reagent (the BrdU) which may compromise our RT-PCR endpoints. We have considerable experience with this sort of staining and feel that this of other particles, many of which contain more factors that may contribute to hyperreponsiveness, is unlikely to be informative until we have a good handle on the dose response. Planned exposures in the next year include a repeat of the diffusion flame exposures to add endpoints and the completion of the high PAH particle exposures using the pre-mixed flame apparatus which is now operational. Then we will start testing the effect of the addition of ozone into the exposure paradigm.
For the in vitro studies, we will take the panels recommendation to put the particles in the agarose of the airway explants. We will continue to monitor PAH content and to determine total mass of all our particles but will also use surface area as a normalizing parameter. Project 1 will test the particles from the field studies but will also test extracted fractions from the same particles to measure soluble PAHs from the particles and their effects on cells in culture.

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

Other subproject views: All 43 publications 10 publications in selected types All 8 journal articles
Other center views: All 128 publications 71 publications in selected types All 64 journal articles
Type Citation Sub Project Document Sources
Journal Article Day KC, Plopper CG, Fanucchi MV. Age-specific pulmonary cytochrome P-450 3A1 expression in postnatal and adult rats. American Journal of Physiology-Lung Cellular and Molecular Physiology 2006;291(1):L75-L83. R832414 (2009)
R832414C001 (2007)
R832414C001 (2008)
R832414C001 (Final)
  • Abstract from PubMed
  • Full-text: Full Text AJP
  • Abstract: AJP
  • Other: Full Text AJP PDF
  • Journal Article Kennedy IM. The health effects of combustion-generated aerosols. Proceedings of the Combustion Institute 2007;31(2):2757-2770. R832414 (2009)
    R832414C001 (2008)
    R832414C001 (Final)
  • Abstract: Science Direct
  • Supplemental Keywords:

    ambient air, health effects, vulnerability, susceptibility, sensitive population, ozone, exposure, human health, metabolism, sensitive populations, infants, children, metals, oxidants, agriculture, transportation,, RFA, Health, Scientific Discipline, Air, particulate matter, Environmental Chemistry, Health Risk Assessment, Epidemiology, Risk Assessments, ambient aerosol, lung injury, air toxics, toxicology, long term exposure, lung disease, airway disease, airborne particulate matter, particle exposure, endothelial function, pariculate matter, human exposure, ambient particle health effects, ultrafine particulate matter, epidemiological studies, PM, human health risk

    Progress and Final Reports:

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

  • Main Center Abstract and Reports:

    R832414    San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R832414C001 Project 1 -- Pulmonary Metabolic Response
    R832414C002 Endothelial Cell Responses to PM—In Vitro and In Vivo
    R832414C003 Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
    R832414C004 Project 4 -- Transport and Fate Particles
    R832414C005 Project 5 -- Architecture Development and Particle Deposition