2009 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: October 1, 2008 through September 30,2009
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air


This research project aims 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 major on two fronts, including:  1) methods development, and 2) data acquisition from inhalation studies in adult and neonatal rats. Collaborations continued and flourished. At the end of this discussion, the researchers will present new data on the premixed versus diffusion flame comparisons.

1.  Methods Development - In the past year, the researchers have made major strides in developing as well as applying new methods that enhance the research objectives of this grant. The researchers aquired capabilities in the following methods and assisted other SAHERC projects in applying these methods to their research:

  • Laser capture microdissection of lung tissue for site-specific analysis of gene expression without amplification (Van Winkle, Project 1; Wilson, Project 4). An optimal method for RNA preservation was developed throughout sample preparation (manuscript in preparation).
  • RT-profiler gene array studies, optimized airway microdissection approaches to provide good yield and suitable quality. Results were generated from the diffusion flame studies (Fanucchi, Project 1).
  • Developed a method to image and quantify permeable cells in situ in the lung in paraffin sections (Van Winkle, Project 1). This was necessitated by the decline in confocal capabilities for whole mount tissues as the laboratory ’s confocal microscope is nearing the end of its useful life and a suitable substitute is not immediately available. Short- and long-term workarounds for this situation are in progress.
  • Refined the lysis lavage technique that was developed by Buckpitt and Plopper so that it also can be used to generate samples for total reduced glutathione analysis from the entire airway epithelium of the conducting airways. This approach was applied in diffusion flame-exposed rats and was found to result in a significant time savings over airway microdissection (Van Winkle, Project 1).
  • Flexivent pulmonary function capability for mice was adapted to measure pulmonary function in neonatal rats (Van Winkle, Project 1; Schelegle, Project 5).
  • Flexcell studies of PM in A549 cells exposed to concentrated urban particles in vitro.
  • Chamber exposures to laboratory-generated particles and full characterization of the exposure atmosphere (Van Winkle, Project 1, in collaboration with Wexler and Ian Kennedy, Project 5).
  • The researchers assisted Dr. Fern Tablin with sample collection of PM-exposed mice and she, in turn, assisted the researchers with the inflammation analysis of peripheral blood in rats and taught the researchers new methods (Tablin, Project 4; Van Winkle, Project 1).

2.  Progress in Data Acquisition:

The researchers completed four exposure scenarios at three timepoints (2 hours, 24 hours, and 48 hours post exposure) in neonatal and adult rats. The additional timepoints at 2 and 48 hours were based on advice from the SAC review. It is important to note that this more than doubles the amount of work required per exposure. Exposures completed included:  1) normal adult and neonatal rats (7D), 2) neonatal and adult rats exposed to low PAH PM (diffusion flame), 3) neonatal and adult rats exposed to high PAH PM (premixed flame), and 4) neonatal and adult rats exposed to high PAH PM + O3. To keep the project within the budget originally requested, and in response to recent publications from Pinkertons’group on diffusion flame soot containing iron that duplicate the researchers’ proposed studies, the portions of the specific aims that involved exposures to iron as a transition metal have been dropped. The revised aims are listed below with progress on each described.

Specific Aim 1 - PM alone versus PM with PAH

Four papers related to this aim are in the later stages of development:

Effects of PAH 1-NN and protein binding by 1-NN (Van Winkle and Buckpitt)

In vivo exposures insufflation of carbon black, acetylene, and ethylene soot (Fanucchi)

Age-specific pulmonary metabolism of 1-Nitronaphthalene (Fanucchi and Plopper)

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 versus PM with low PAH

Two papers in draft form for this aim focus on diffusion flame soot (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 increased in neonates and the distribution of permeable cells to a larger number of airways is more apparent in neonates. The temporal pattern of inflammatory cell responses varies by compartment and age-only neonates have increased neutrophils in BALF and only adults have persistent increases in cytokines in blood (still increasing at 48 hours). Differences in gene expression were not detected in neonates but a number of changes were present in adult airways, including metabolism/antioxidant genes and DNA repair genes/cell cycle-associated genes.

PAH containing soot (premixed flame) exposures are complete. Data are still being analyzed. Preliminary results show that premix flame-generated PM had:  1) only modest effects on peripheral blood, 2) transient increase in PMN in adult BALF and a steady increase in neonate out to 48 hours, and 3) pronounced effect on CYP1A1 gene and CYP1B1 protein expression in keeping with the higher level of organics. 

The key question arising from these data is why were there only moderate effects on toxicity (not shown) in a PM exposure that included more PAH than the diffusion flame exposure.  The answer can be found in the chamber analysis profiles listed in the table below:

Premix PM Diffusion PM
[Mass] 20 ug/m3 140 ug/m3
[Particle] 2x10^5 2x10^5
Mean aero diam. 80 nm 200 nm
Tot attached PAH 54 ng/m3 19 ng/m3
Most abund PAH Naphthalene Pyrene
Concentration 15.5 ng/m3 5 ng/m3
Gas Phase 221 ng/m3 225 ng/m3
Most abundant 2-methylbiphen 2-methylbiphen
Concentration 115ng/m3 230ng/m3
Organic C/Tot C 0.52 0.17

The mass and mean aerodynamic diameter were quite a bit lower in the premixed flame exposure even though the particle number was the same. The researchers are working with Project 5 to increase particle size and hope to repeat the exposure at a higher particle size. 

Specific Aim 3 - PM with PAH +/- ozone

Exposures are completed for this aim.  Data are currently under analysis.

Specific Aim 4 - Seasonal urban and environmental particulate matter versus chamber particulate matter

This aim is being addressed using in vitro exposures currently in progress in bronchiolar explants and flexcell alveolar cell cultures.

Future Activities:

The researchers have four goals for the next 6 months:

  1. complete and submit the diffusion flame papers;
  2. finalize the premixed flame data at two particle sizes (this will involve another exposure study);
  3. complete the initial analysis matrix in vitro of cytotoxicity and phase I/II metabolism in the bronchiolar explants, alveolar cell line, and Clara cell line; and
  4. finalize the PM + ozone dataset.

Journal Articles:

No journal articles submitted with this report: View all 43 publications for this subproject

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
  • 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