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2007 Progress Report: San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
EPA Grant Number: R832414Center: San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Center Director: Wexler, Anthony S.
Title: San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Investigators: Wexler, Anthony S. , Pinkerton, Kent E.
Institution: University of California - Davis
EPA Project Officer: Stacey Katz/Gail Robarge,
Project Period: October 1, 2005 through September 30, 2010
Project Period Covered by this Report: October 1, 2006 through September 30, 2007
Project Amount: $7,999,767
RFA: Particulate Matter Research Centers (2004)
Research Category: Particulate Matter
Description:
Objective:1.0 RESEARCH PERFORMED AND RESULTS
This is a general progress report for the SAHERC. The progress for the specific research projects conducted by the Center is reported separately.
1.1 OBJECTIVES
This research investigates the mechanistic links between ambient particles and health effects. This objective includes two goals: (1) Understanding the metabolic response of tissue and organs when they are exposed to particulate pollutants, and (2) Understanding the characteristics of the particulate pollutants and their gaseous co-pollutants that elicit these responses. The five projects investigate the metabolic response to pollutant exposure in pulmonary and cardiovascular tissues, whole animal effects of exposure, transport of particles from the airways to other tissues, and the effects of particles and gases on lung development in juveniles.
Both field and laboratory studies are being performed. The field studies take place in the San Joaquin Valley of California, the second worst area violating the National Ambient Air Quality Standards for particulate matter in the U.S. The laboratory studies at UC Davis examine the effects of particles from ambient sources and laboratory-generated particles with carefully controlled properties that model ambient particles or particles from dominant sources.
Air-quality–related health indicators for the SJV reflect the severity of the region’s air quality problems; for example, asthma and cardiovascular disease rates are some of the highest in the country. The San Joaquin Valley routinely experiences some of the highest fine airborne particulate matter concentrations in the U.S., with fine and ultrafine particle concentrations that rival those found in Los Angeles.
EPA
PM Centers Committee
Working
Executive Committee
Director
Co-Director
Administrator
Science Advisory Committee
Quality Management Committee
Projects
Pulmonary Metabolism
Cardiovascular Metabolism
SJV Inhalation Exposure
Transport and Fate of Particles
Architecture Development
Cores
San Joaquin Valley
Animal Exposure
Particle Generation & Characterization
Bioanalytical
Imaging
1.2 PROGRESS SUMMARY
All studies in the SAHERC are being undertaken within the primary theme of ‘ambient particulate matter and resulting health effects in the San Joaquin Valley’. Below is a chart outlining the center’s five projects. All five research projects are complementary and focus on the role of particulates in pulmonary and cardiovascular health effects. The research progress for these studies is summarized below in a paragraph for each project.
Project 1--Pulmonary Metabolic Response
Principal Investigator: Michelle Fanucchi
Co-Investigators: Charles Plopper, Alan Buckpitt
During this period of the grant, we focused on finding a suitable carrier in which to instill 1-nitronaphthalene into the lungs of rats. We found that the best fluid that allows for the solubilization of 1-nitronapthalene with the least amount of irritation is Pluronic F-127.
Our intraperitoneal administration of 1-nitronaphthalene to rats resulted in vacuolization and exfoliation of airway epithelial cells. Intratracheal instillation of 1-nitronaphthalene resulted in increased epithelial thickness and an increase in vacuolated cells present. We also evaluated the cytotoxicity and clearance (24-hr post-insufflation) from a single intra-tracheal insufflation of 2.5 mg submicron carbon black or flame generated soot particles (see particle generation core report) adsorbed with 0 or 5% 1-nitronaphthalene in adult rats. Carbon black particles not coated with 1-nitronaphthalene did not cause any appreciable cellular injury to airway epithelium at any airway level and the majority of the particles were cleared from the airways at 24 hours. However, particles coated with 1-nitronaphthalene caused focal areas of exfoliation and cellular injuries in all airways evaluated [proximal, mid-level and distal airways] and were not completely cleared from the airways. Flame generated soot not coated with 1-nitronaphthalene did cause cytotoxic injury throughout the airways and this cytotoxicity was increased with adsorption of 1-nitronaphthalene in all airways evaluated [proximal, mid-level and distal airways].
Project 2 -- Endothelial Cell Responses to PM -- in vitro and in vivo
Principal Investigator: Dennis Wilson
Co-Investigator: John Rutledge
We obtained our first SJV derived CAPs and performed experiments evaluating overall gene responses and potential signaling mechanisms induced by direct exposure of human endothelial cell cultures (HAEC) to CAPs. Our initial studies evaluated our original hypothesis that general endothelial cell activation through the TGFβ family of signaling molecules would occur. While previous studies in our laboratory have documented the activation of this family of receptors by a variety of stimuli, treatment with CAPs collected from an urban setting in the SJV failed to stimulate the translocation of the common transcription factor (SMAD4) associated with this family of growth regulation factors. Organic chemical analysis demonstrated that these particles contained multiple PAH compounds associated with automotive fuel combustion. RNA isolated from HAECs exposed for 3 hr to 10 ug/ml CAP was analyzed using high density oligonucleotide arrays. CAP exposure resulted in the up-regulation of 30 and the down regulation of 15 genes. Compared with similar experiments from another project examining responses to lipolysis products derived from human blood lipids, this was a modest response.
We collected heart and lung tissues from mice exposed in the HRV protocol in project 3 during the February 2007 Fresno exposure project and are presently being analyzed by RT-PCR for altered expression of the same panel of homologous genes as found in the HAEC array studies. Another group of mice from the same experiment had tissues from all visceral organs harvested and embedded for future immunohistochemical analysis. These will be immunostained for vascular proinflammatory molecules including E-selectin and ICAM-1. Additional immunostains will evaluate expression of CYP 1a1 as a possible correlate of the increased expression of this gene in HAEC cultures treated with collected CAPs from the fall exposure at the same site. Current activity in this project is directed towards optimizing the immunostaining protocols using lungs from rats treated with monocrotaline, an endothelial activating and proinflammatory pulmonary toxin.
Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
Principal Investigator: Kent Pinkerton,
Co-Investigators: Mike Kleeman, Ann Bonham and John Rutledge
To date we have completed two field measurement and exposure studies in the San Joaquin Valley at an urban site located in Fresno, CA (500 East Shaw Avenue). The first field study was conducted during the late summer season (September 5-16, 2006), and the second study at the identical site during the winter season (February 13-24, 2007). In each study male C57/BL6 mice were exposed to concentrated ambient particles (CAPs) for 6 hours/day, 5 days/week for two weeks. Fine/ultrafine ambient particles were collected and concentrated onsite using a Versatile Aerosol Concentrator Enhancement System (VACES). CAPs samples collected during each exposure were analyzed for chemical composition.
Organic carbon, elemental carbon, and water soluble ions (sulfate, nitrate, etc) make up the majority of the particle size distribution. The Fresno sampling site was located in relatively close proximity to busy surface streets and highways in Fresno. It is expected that the majority of these particles are derived from tailpipe exhaust emissions and / or road dust sources. Elemental analysis of these samples using ICPMS has been completed and the data is being reviewed for QA/QC.
Project 4 -- Transport Mechanisms and Systemic Fate of Inspired Ultrafine Particles
Principal Investigator: Dennis Wilson
Co-Investigators: Angelique Louie, Michelle Fanucchi, Ian Kennedy, Charles Plopper, Alan Buckpitt
Polystyrene nanoparticles have been modified to carry radioactive tracers and these were introduced to rats by insufflation. Two types of radiotracers were used: In-111 for biodistribution studies using gamma counting of harvested organs, and Cu-64 for Positron Emission Tomography (PET) imaging of the model ultrafine particles. Overall trends indicate that there is elevated accumulation of particles in the heart in the first 4 hours after exposure and that this elevation disappears by 24 hours. Larger particles (one micron) had less movement out of the lung than smaller particles (78nm). Both In-111 and Cu-64 studies were performed for pharmacokinetics and preliminary blood collection studies indicate that lung deposition results in early appearance of particles in the blood that disappears over time. With esophageal deposition the opposite effect is seen, particles increase in the blood over time.
We examined particle-plasma membrane interactions in endothelial cell culture to test our hypothesis that ultrafine particles can be internalized and transported across endothelial cell membranes using a caveolar transport mechanism. Human aortic endothelial cells (HAECs) were exposed to iron oxide particles (10 µg/ml solution) for 10, 30, 60 min and 4 hrs. At 30 min, iron oxide aggregates interacted with plasma membrane structures and were visible within vesicles close to caveolae. By 4 hrs the electron dense particles were visible as aggregates in membrane bound vesicles stratified throughout the cells and on the basilar side of the endothelial cells. Intercellular junctions remained intact and paracellular transport was not evident.
To confirm the caveolar transport process, we treated similar cultures with FITC conjugated silica particles (10 µg/ml) for 30 min and 4 h. Caveolin-1 was predominantly centralized at 30 min with FITC-labeled silica particles isolated to the outer plasma membrane with little co-localization. At 4 hours Caveolin-1 was widely disseminated through the cell and there was a strong co-localization between caveolin-1 and the FITC-labeled particles visible in the cells. Our results suggest that ultra fine particles can be internalized and transported across endothelial cell membranes, potentially using a caveolar transport mechanism similar to the observed transport of other macromolecules and drugs.
Project 5 -- Architecture Development and Particle Deposition
Investigator(s): Anthony Wexler, Charles Plopper and Michelle Fanucchi
The lung casts from seven male Sprague Dawley rats were imaged using a commercially available micro CT scanner, MicroCAT II in high resolution mode with a 0.5 mm aluminum filter. The image was reconstructed using the Feldkamp reconstruction algorithm as a 768 x 768 x 1000 array with corresponding voxel size of 0.053 mm x 0.053 mm x 0.053mm. Higher resolution data (pixel size is 0.026 mm) from a single rat was also analyzed to confirm that the resolution mode used is fine enough to distinguish the smallest airways. We analyzed the airway architecture of seven normal rats using an algorithm tp model airway architecture that we developed previously. The modeling algorithm was validated by error analysis and a statistical comparison of our airway model results with measurements from previous studies of rat lung anatomy. The statistical results from our modeling study and Raabe’s measurements were very similar except that the small airways (diameter smaller than 1mm) in our analysis were much more symmetric than Raabe’s. Most notably the current study showed that the mean value and standard deviation of the geometric parameters are insufficient to characterize airway architecture in the lung. For example, we found that the twist angle, i.e., the angle between successive bifurcations, is far from normally distributed . Thus, the typical distribution of the values of airway geometry must be taken into account to quantitatively describe pulmonary architecture. Using SAS we are performing statistical analysis including intersubject variance.
Particle Generation Core
We developed a flame method for the synthesis of soot particles with variable loading of PAHs and other volatile and semi-volatile hydrocarbons. Soot is generated using a flat premixed flame (McKenna) burner. Ethylene is used as the fuel. The ratio of elemental carbon (EC) to organic carbon (OC) can be controlled by either varying the ratio of fuel to air, or by varying the position of the collection probe. This capability will help us in our efforts to understand how EC/OC correlates with toxicity of ultrafine PM. In addition, we have developed a system for doping flame-generated soot particles with 1-Nitronaphthalene (1-NN), in support of Project 1. The soot aerosol is then coated with 1-NN by promoting heterogeneous condensation of 1-NN vapor. Acetylene was used as the fuel in our preliminary work. However, ethylene will be used in future work since acetylene soot was found to be contaminated with metals during acetylene manufacture.
2.0 DIFFICULTIES ENCOUNTERED AND REMEDIAL ACTIONS
2.1 Artifacts in Flame-Generated Soot. In Project 1, the current studies used sub-micron flame-generated soot doped with 1-nitronaphthalene provided by the Particle Generation, Modification and Characterization Core. We recently learned that the acetylene flame-generated soot may have metallic contaminants from the acetylene manufacture, so we will evaluate the cytotoxicity of ethylene flame generated soot.
2.2 Radioactive Labeling of Particles. In Project 4, one challenge was coupling radioactive labels to the particles. We originally proposed to couple derivatized chelators to the particles through amine functional groups at the surface of the particles and then insert the radioactive cations. This uses standard conjugation chemistry that we have used extensively in other systems. However, nanoparticles proved to be recalcitrant to coupling by this method and the degree of labeling was low (~6%). We explored a number of synthetic protocols and finally have been able to increase the degree of labeling to 45%. It is unclear why nanoparticles were so resistant to labeling by the original method. We are currently exploring the mechanism for this resistance as we have since observed it for many other types of nanoparticles, regardless of composition, that are amine-functionalized.
2.3 Insufflation Technique for Radioactive Particles. The second technical challenge in Project 4 was training personnel to perform the insufflation technique on rats. The insufflation technique uses a spritzer device that delivers a fine mist solution to the lungs of the animals when performed properly. Consistently delivering material to the lung required many hours of training. Training was initially performed using fluorescent material and whole animal optical imaging to prevent risk of exposure to radioactivity while personnel were learning to perform the insufflation. While more consistent lung deposition was achieved over time, we were still unable to reliably avoid deposition to the esophagus. Therefore, in the future we are moving to an endotracheal catheter method of deposition which we have found gives much more reliable deposition exclusively to the lungs.
3.0 ABSENCE OR CHANGES OF KEY PERSONNEL
3.1 Key personnel. There are no absences or changes in key personnel involved.
3.2 Goals and hypotheses. There were no revisions in goals or hypotheses.
4.0 EXPENDITURES TO DATE
4.1 PERCENTAGE OF THE PROPOSED WORK COMPLETED
About twenty months of research work has been completed to date, since the grant award was made to UC Davis in October 2005. Expenditures in this second year have been for staffing, equipment purchase, supplies, communications, monitoring site set up, grad student fees, and salaries/benefits. Spending was less than projected in the first year, with unspent funds moved forward to cover research costs in this project. At fiscal year end (July 2006 – June 2007), our expenditures for twenty months were about 45% less than originally budgeted based upon a monthly spending plan.
4.2 EXPLANATION OF ANY HIGHER THAN ESTIMATED COSTS
We did not have any higher than estimated costs.
5.0 QUALITY ASSURANCE REQUIREMENTS
The quality assurance requirements of the following are being met: (1) 40 C.F.R 30.54; (2) G-1 STAR, Guidance on Satisfying EPA Quality System Requirements for STAR Grants (for individual projects), and (3) the PM agreement through preparation and implementation of a Quality Assurance plan. The QAP for this project was approved by the Director of Quality Assurance at ORD, EPA on July 14, 2006. Quality Management Plans for each project were prepared and submitted in 2007.
6.0 PLANNED ACTIVITIES FOR THE NEXT REPORTING PERIOD
Project 1 -- Pulmonary Metabolic Response
The administration of 1-nitronaphthalene by aerosol/intra-tracheal instillation has been less than optimal for postnatal animals due to their small size and their intolerance of sedation. Future studies will be performed by utilizing whole animal inhalation of laboratory generated particles and use 1-nitronaphthalene to probe the susceptibility of animals exposed to particles. Using this approach will allow us to generate more data in a reasonable amount of time. It will also facilitate the evaluation of multiple particles (soot + PAH, soot + metal, soot + PAH + metal). In addition, it will allow us to evaluate the susceptibility of postnatal animals to particulate matter.
Project 2 -- Endothelial Cell Responses to PM -- in vitro and in vivo
In the next project year we plan the following experiments:
- Compare gene responses of endothelium to CAPs with that in airway epithelial cells
- Compare specificity of AhR responses with ROS/Nrf-Keap
- Complete evaluation of mouse exposure to PM collected in Winter with histopathology, immunohistochemistry, and gene expression analysis
- Compare gene responses to PM from urban sources to agricultural origin PM
Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
For the next reporting period, our plan is to (1) continue exposures in the summer season (i.e., August 2007), (2) conduct CAPs experiments in our designated rural site of the San Joaquin Valley (Westside, CA), (3) develop pulmonary and cardiovascular measures of PM effects for week 1 and week 2 of rural summer exposure as well as for the combined 2 weeks of exposure and 4) complete the analysis of respiratory, HRV and stress test impacts for murine studies completed in Fresno during the late summer (September 2006) and winter (February 2007) seasons as well as particle concentration and composition.
Project 4 -- Transport Mechanisms and Systemic Fate of Inspired Ultrafine Particles
In ongoing work we will explore the utility of a mouse model using catheter deposition techniques, to consistently deliver desired volumes to precise locations. In addition the smaller size of the mice makes it more feasible to increase the number of animals imaged per study. We will also be investigating modification to the liquid spritzing protocol that may improve the deposition profiles with that technique, as well as testing a new device designed by a UCD undergraduate team that automates the “spritzing” process and delivers a fixed volume at a velocity every time. We will continue to collect both In-111 distribution data and Cu-64 imaging data. The question of whether the In or Cu could escape from the probes will be answered through stability assays. However, given the very high stability of macrocyclic compounds we do not believe that there can be release of the ions during the course of these imaging and biodistribution studies.
Next we will also evaluate transport in dynamic (flow through culture) as opposed to static conditions and to characterize transport in airway epithelial cell cultures. Transport in perfused vessels will also be initiated in year 3.
Project 5 -- Architecture Development and Particle Deposition
We will analyze rat lungs exposed to ozone and/or particles, and compare them to normal lungs. Ozone exposures are currently underway.
Particle Generation Core
The interaction of certain metals with organic species on the surface of particles is believed to lead to the continuous generation of reactive oxygen species (ROS). In order to evaluate this idea, we would like the capacity to synthesize soot particles that are doped with both transition metals, such as Fe or Cu, and organics/PAHs. We are currently evaluating the inverse coflow flame as a potential system for this task. We also aim to dope soot particles with hydroquinone, using the previously described condensation system, with the hopes of producing soot with stable quinone-type radicals that also may generate ROS.
Journal Articles: 10 Displayed | Download in RIS Format
| Other center views: | All 19 publications | 10 publications in selected types | All 10 journal articles |
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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. |
R832414C001 (2007) R832414C001 (2008) |
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Donaldson K, Borm PJA, Oberdorster G, Pinkerton KE, Stone V, Tran CL. Concordance between in vitro and in vivo dosimetry in the proinflammatory effects of low-toxicity, low-solubility particles:the key role of the proximal alveolar region. Inhalation Toxicology 2008;20(1):53-62. |
R832414C003 (2008) |
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Kennedy IM. The health effects of combustion-generated aerosols. Proceedings of the Combustion Institute 2007;31(2):2757-2770. |
R832414C001 (2008) |
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Kleeman MJ, Riddle SG, Jakober CA. Size distribution of particle-phase molecular markers during a severe winter pollution episode. Environmental Science & Technology 2008;42(17):6469-6475. |
R832414C003 (2008) |
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Lee DY, Wexler AS, Fanucchi MV, Plopper CG. Expiration rate drives human airway design. Journal of Theoretical Biology 2008;253(2):381-387. |
R832414C005 (2008) |
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Lee D, Park SS, Ban-Weiss GA, Fanucchi MV, Plopper CG, Wexler AS. Bifurcation model for characterization of pulmonary architecture. Anatomical Record 2008;291(4):379-389. |
R832414C005 (2008) |
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Lee D, Fanucchi MV, Plopper CG, Fung J, Wexler AS. Pulmonary architecture in the conducting regions of six rats. Anatomical Record 2008;291(8):916-926. |
R832414C005 (2008) |
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Riddle SG, Robert MA, Jakober CA, Hannigan MP, Kleeman MJ. Size-resolved source apportionment of airborne particle mass in a roadside environment. Environmental Science & Technology 2008;42(17):6580-6586. |
R832414C003 (2008) |
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Smith KR, Veranth JM, Kodavanti UP, Aust AE, Pinkerton KE. Acute pulmonary and systemic effects of inhaled coal fly ash in rats:comparison to ambient environmental particles. Toxicological Sciences 2006;93(2):390-399. |
R832414C003 (2006) R832414C003 (2007) R832414C003 (2008) |
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Tebockhorst S, Lee DY, Wexler AS, Oldham MJ. Interaction of epithelium with mesenchyme affects global features of lung architecture:a computer model of development. Journal of Applied Physiology 2007;102(1):294-305. |
R832414C005 (2007) R832414C005 (2008) |
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ambient air, ozone, exposure, health effects, human health, metabolism, sensitive populations, infants, children, PAH, metals, oxidants, agriculture, transportation,
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ENVIRONMENTAL MANAGEMENT, Air, Scientific Discipline, Health, RFA, PHYSICAL ASPECTS, Risk Assessment, Risk Assessments, Health Risk Assessment, Physical Processes, Biochemistry, particulate matter, Environmental Chemistry, exposure assessment, cardiovascular disease, chemical characteristics, particulate matter components, cardiopulmonary responses, human health risk, acute cardiovascular effects, human health effects, atmospheric particulate matter, exposure, airborne particulate matter, human exposure, PM
Progress and Final Reports:
2006 Progress Report
Original Abstract
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