2010 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. , Plopper, Charles
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: July 1, 2009 through June 30,2010
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 will result in more injury than particles composed of only one component.

Progress Summary:

Progress this year was primarily on two fronts: 1) acute animal exposures and 2) data preparation and submission for publication based on work previously reported. Collaborations continued and flourished. Highlights of the accomplishments in these areas are listed below. A section for each specific aim also is included.

1. Acute Animal Exposures

We have completed to date 5 exposure scenarios at 3 timepoints (2 hrs, 24 hrs, and 48 hrs post exposure) in neonatal and adult rats:

  1. Normal adult and neonatal rats (7D)
  2. Neonatal and Adult rats exposed to low PAH PM (diffusion flame, DFP210)
  3. Neonatal and Adult rats exposed to high PAH PM (premixed flame, PFP70)
  4. Neonatal and Adult rats exposed to high PAH PM (PFP70) + O3, and
  5. Neonatal and adult rats exposed to a larger particle size high PAH PM (PFP210).

We added scenario 5 in the last year to determine whether the substantial effects we see in the PFP exposed animals are due to either particle composition or to particle size. To completely answer this question, we also will need to be able to generate a chamber atmosphere that has a low PAH particle of small size (DFP70).

2. Data Preparation and Submission for Publication

In the past year we published 2 book chapters and 6 abstracts, and we have 3 papers either submitted or in press.The new methods we developed to obtain site specific RNA without amplification from the lung using laser capture microdissection has resulted in a manuscript on the method that is in its second revision to the Journal of Histochem Cytochem. These methods have been used, or are under consideration for use, by other SAHERC funded labs including (Pinkerton, Proj 3; Wilson, Proj 4).

The diffusion flame studies (DFP210), including the RT profiler array results and the chamber characterization results from our laboratory generated exposures in neonatal and adult rats, were submitted to the journal Inhalation Toxicology and have been provisionally accepted pending minor revision. This manuscript involved substantial cross center collaboration (Fannuchi and Van Winkle, Proj 1; Wexler, Kleeman and Kennedy, Proj 5).

Proj 1 contributed information on cell proliferation in lung tissue in response to pre-mixed flame exposures (PFP70) in neonatal and adult rats that added to the interpretation of the lung cast results described in a recently accepted publication in Journal of Applied Physiology led by Dr. Wexler (Wexler and Schelegle, Proj 5; Van Winkle, Proj 1).

We assisted Dr. Fern Tablin with sample collection of PM exposed mice and she, in turn, assisted us with the inflammation analysis of peripheral blood in our rats and taught us new methods (Tablin Project 4, Van Winkle Project 1). A paper that describes the inflammatory response in peripheral blood and lung tissue for diffusion flame (DFP210) exposed neonatal and adult rats is in the late stages of preparation.

The specific aims are listed below with progress on each described.

Specific Aim 1 PM alone vs PM with PAH

4 papers late stages this aim;

  1. Effects of PAH 1-NN and prtein binding by 1-NN (Van Winkle and Buckpitt)
  2. In vivo exposures insufflation of Carbon Black, acetylene and ethylene soot (Fanucchi)
  3. Age-Specific Pulmonary Metabolism of 1-Nitronaphthalene (Fanucchi and Plopper)
  4. Alterations in Extracellular Nasal and Pulmonary Glutathione Pools in Adult and Postnatal Rat Following 1-Nitronapthalene Exposure (Fanucchi and Buckpitt)

Specific Aim 2 PM with PAH vs PM with low PAH

Data collection and most of the analysis is complete for this aim. All that remains is publication of the results. We expect 4 papers from this aim. The first paper for this aim has been provisionally accepted for publication. The paper describes the effects of a moderate exposure level of diffusion flame soot (DFP210) exposures on the pulmonary metabolic response (Van Winkle, Fanucchi). Summary of data: Diffusion flame PM is cytotoxic, causing vacuolated ciliated cells in both neonates and adults. LDH in BALF is only increaed in neonates and the distribution of permeable cells to a larger number of airways is more apparent in neonates. Interestingly there is no overlap in significantly changed genes between neonatal and adult rates exposed to the same particle. This indicates that the pattern of responsiveness in neonatal animals cannot be predicated based on adult data.

A second paper on the inflammatory response is in preparation. The second paper finds that the temporal pattern of inflammatory cell responses (including BALF cell numbers of neutrophils, cytokines in both BALF and peripheral blood, and cells in the lung tissue) varies by compartment (blood vs lung tissue vs BALF) and age. Only neonates have increased neutrophils in BALF, only adults have persistent increases in cytokines in blood (still increasing at 48hr, see Figure at left) but neonates have decreased levels of this cytokine. In general peripheral blood cytokine responses were greater in the animals exposed to the low PAH containing particle, DFP, but we cannot exclude that this may be due to a difference in dose. This is why the planned exposures with a smaller size DFP exposure, similar in size to the PFP exposure, are important. In collaboration with Fern Tablin from Project 4 we have also assessed platelet abundance in both neonatal and adult peripheral blood and find that it is not affected by DFP exposure.

PAH containing soot (premixed flame) exposures are complete. The draft of the paper that describes the pulmonary metabolic response in response to a higher PAH containing soot in neonatal and adult rats is in the late stages of preparation and the data analysis is complete. One of the key findings from these studies is that either low PAH or high PAH containing PM exposures activate the Nrf2 – Antioxidant Response Element signaling pathway. Following either DFP or PFP exposure, Nrf2 staining was seen specifically localized in the nucleus in both neonate and adult rats. Furthermore, many oxidative stress and antioxidant related genes were significantly changed as evaluated by qPCR array.


Our data show that adult animals are an inappropriate model to understand juvenile responses to PM. Gene and protein levels of metabolic, oxidative stress and antioxidant enzymes are also significantly different between the two ages, thus implying that an age-related difference exists in both the cellular capability and the response from exposure to particulate matter. Further, the temporal pattern and expression of metabolic enzymes differs depending on particle composition and age of the animal. Basal levels of key xenobiotic metabolizing cytochrome P450s are lower in 7 day postnatal rats compared to adults, and their expression pattern is different in the bronchiolar epithelium compared to the surrounding parenchyma. Following high carbon content PFP exposure, CYP1A1 gene and 1B1 protein expression were markedly increased (see Figure below). This phenomenon was not observed in low organic carbon containing DFP exposed animals.

Specific Aim 3 PM with PAH +/- ozone

Exposures completed this Aim. Data under analysis. Data analysis is currently confounded by the lack of an ozone only exposure group. We plan to add an ozone only exposure group in the coming year to assist with data interpretation.

Specific Aim 4 Seasonal urban and environmental particulate matter vs chamber particulate matter

This aim is being addressed using in vitro exposures currently in progress in bronchiolar explants, flexcell alveolar cell cultures +/- mouse liver microsomes to activate the PAH present on the particles. Particles in suspension have also been obtained from Keith Bein of laboratory generated chamber PM and we have begun instillations of these PM in mice. Our plan is to compare the results from the chamber PM vs environmental PM in the next year.

Future Activities:

We have 4 goals for the next 6 months: 
  1. Complete the premixed flame and premixed flame +ozone papers and submit them,
  2. Finish analysis of premixed flame data at 2 particle sizes,
  3. Complete experiments for specific aim 4 including remaining in vitro and instillation studies, and
  4. Complete ozone and DFP70 exposures.

Journal Articles on this Report : 4 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 Greeley MA, Van Winkle LS, Edwards PC, Plopper CG. Airway trefoil factor expression during naphthalene injury and repair. Toxicological Sciences 2010;113(2):453-467. R832414 (Final)
R832414C001 (2010)
R832414C001 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Oxford-Full Text HTML
  • Abstract: Oxford-Abstract
  • Other: Oxford-Full Text PDF
  • Journal Article Lee DY, Wallis C, Wexler AS, Schelegle ES, Van Winkle LS, Plopper CG, Fanucchi MV, Kumfer B, Kennedy IM, Chan JKW. Small particles disrupt postnatal airway development. Journal of Applied Physiology 2010;109(4):1115-1124. R832414 (Final)
    R832414C001 (2010)
    R832414C005 (2010)
    R832414C005 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: JAP-Full Text HTML
  • Abstract: JAP-Abstract
  • Other: JAP-Full Text PDF
  • Journal Article Sutherland KM, Combs TJ, Edwards PC, Van Winkle LS. Site-specific differences in gene expression of secreted proteins in the mouse lung: comparison of methods to show differences by location. Journal of Histochemistry and Cytochemistry 2010;58(12):1107-1119. R832414 (Final)
    R832414C001 (2010)
    R832414C001 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: JHC-Full Text HTML
  • Abstract: JHC-Abstract
  • Other: JHC-Full Text PDF
  • Journal Article Van Winkle LS, Chan JK, Anderson DS, Kumfer BM, Kennedy IM, Wexler AS, Wallis C, Abid AD, Sutherland KM, Fanucchi MV. Age specific responses to acute inhalation of diffusion flame soot particles:cellular injury and the airway antioxidant response. Inhalation Toxicology 2010;22(Suppl 2):70-83. R832414 (Final)
    R832414C001 (2010)
    R832414C001 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Abstract: Taylor and Francis-Abstract
  • Supplemental Keywords:

    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
  • 2008 Progress Report
  • 2009 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