Grantee Research Project Results
2003 Progress Report: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
EPA Grant Number: R827354C005Subproject: this is subproject number 005 , established and managed by the Center Director under grant R827354
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Rochester PM Center
Center Director: Oberdörster, Günter
Title: Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
Investigators: Finkelstein, Jacob N. , O'Reilly, Michael , Phipps, Richard
Current Investigators: Finkelstein, Jacob N.
Institution: University of Rochester
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, 2003 through May 31, 2004
RFA: Airborne Particulate Matter (PM) Centers (1999) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Particulate Matter , Air
Objective:
Our goals are to define mechanisms that follow particle cell contact and test the specific hypothesis that many of the subsequent physiologic effects are the consequences of cellular oxidative stress. We also examine host and environmental factors, including age, the influence of co-exposure to gaseous oxidants or prior priming or activation by pre-exposure to other inflammatory stimuli.
A key component of the proposed studies is our plan to examine these particle cell interactions in individual cell populations to begin to assess the role of interactions among epithelial, inflammatory and vascular endothelial cells in the systemic response to ultrafine particles (UFP). The proposed in vitroexperiments are intended to provide a link between the whole animal (core 4) and controlled clinical (human) exposures (core 3), described in the other subproject reports of this Particulate Matter (PM) Center.
The experiments proposed within this project are designed to address specific mechanistic hypotheses regarding the interactions between inhaled UFP and pulmonary cell populations. We have used a number of cell lines and primary cells derived from rats and mice to test the overall PM Center hypothesis that the unique physicochemical characteristics of UFP in comparison to accumulation mode particles of similar composition contribute to the observed increases in morbidity and mortality in susceptible populations exposed environmentally.
This is one of the projects of the Rochester PM Center. The progress for the other projects is reported separately (see reports for R827354 C001 through R827352C004).
Progress Summary:
Recent work has continued the refinement of in vitro models of particle cell interactions with the goal to define mechanisms of cellular activation, the effects of age or prior activation on cytokine gene activation, and the differential responses of epithelial cells, macrophages, and vascular endothelial cells to particles of different size and composition.
Particle Effects on Vascular Endothelium
During the current year, in an effort to better bridge the experiments that are being carried out in core 3 and core 4, we have developed vascular endothelial models that could be useful in assessing particle-induced changes in endothelial gene expression. To more accurately reflect the complex nature of endothelial interactions with particles, we have used two complementary culture models. Many of our experiments utilize a standard monolayer culture of primary vascular endothelial cells. A second model, a bilayer epithelial/endothelial co-culture system, permits study of cell-cell interactions mediated by particles.
Monolayer cultures of human umbilical vein endothelial cells (HUVEC) were established and optimized with regard to media, serum, and other culture conditions. Production of IL-6 and PGE2 when endothelial cell were cultured in the presence of LPS or TNF for 24 hours was used to establish the basic parameters. Our major objective with these cultures was to establish appropriate dose and time parameters of incubation with particles so that we could begin testing with UFP of various compositions. Based on our experiments with cultured epithelial cells we began our studies using laboratory-generated particles containing 25 percent Fe. Particles were added to the cells at concentrations ranging from 0.47 to 19.0 μg/cm2, and media was collected at 6 and 24 hours. Particle induced cytotoxicity was measured by LDH release. Addition of particles in the presence of a priming dose of LPS stimulated the release (production) of both IL-6 and PGE2 at both 6 and 24 hours. The PGE2 response appears to be more sensitive as it is observed at doses as low as 0.47 μg/cm2 (which converts to a total mass dose of ~1.5 μg of particle).
In this system we also assessed response of these cells to laboratory-generated ultrafine carbon, similar to the material used in the human clinical studies, TiO2, and a laboratory-generated MnO2. In contrast to our experiments with epithelial cells, the endothelial cell cultures were moderately responsive to the carbon alone. After 24 hours of incubation, PGE2 production was increased 2-3 fold. In contrast C/Fe particles increased PGE2 by 5-6 fold. Overall comparison of particle, dose, and time parameters suggest that PGE2 is the most reliable marker of endothelial activation. It was also noted that particle composition was a major response factor, with TiO2 being most active and MnO2 being most directly cytotoxic. The endothelium appears to respond to lower particle mass burdens than does the epithelium. This may account for the enhanced sensitivity of the vascular endothelium in vivo.
Using a bilayer culture model, we have continued to characterize cytokine production in response to various stimuli, including particle and LPS. We also determined if coculturing these cells with A549 pulmonary epithelial cells would alter their ability to be stimulated by LPS or by particles. Both cell types appear to be responsive to particles and LPS with apparently different concentration dependence.
Effects of Age on Macrophage Response to Particles
We continue to maintain our main focus in the area of understanding how age affects the interaction of particles. Most significant, in the context of our investigation of age effects and the ability of particles to induce effects at low dose, was the fact that in the aged animals co-administration of particles and LPS lead to synergistic effects at the lowest dose of particles of 0.47 μg/cm2.
Our current results show both an age-dependent change in cytokine production as well as a response at low, environmentally relevant doses. We have continued our studies on dose effects by refining our indicator cell line to be useful in detecting particle-induced effects at a wide range of doses.
Mechanistic Studies of Particle Cell Interactions
A proposed mechanism for particle-induced alterations in gene expression is through generation of reactive oxygen species leading to intracellular oxidative stress. This oxidative stress in turn activates redox sensitive transcription factors such as NFkB leading to increased gene expression. To test this mechanism we have begun to evaluate transcription factor activation in epithelial and macrophage cells. Interestingly, experiments with NFkB inhibitors that act by inhibition of proteosome activation were found to increase particle-induced gene expression rather than inhibit it. Comparing the effect of these inhibitors on particle-induced gene expression and a soluble activator (TNF) showed that TNF activation was blocked while particle effects were enhanced. This suggests a different mechanism of activation for these two stimuli. Continued investigation into these differences and the possible cellular specificity of these effects are the goals for the coming year.
A second aspect of these studies is to directly alter the oxidative stress component of activation. Supplemental antioxidants have been used in many studies to approach this question. In our recent studies we have compared the effect of the addition of N-acetylcysteine, a sulfhydryl reagent, with BHA, a lipophilic antioxidant. In both epithelial cells and endothelial cells, BHA is a more potent inhibitor of the particle response. This suggests lipid peroxidation as a mechanism of particle cell interaction. Studies measuring lipid peroxidation products in these cells are planned for the coming year.
Future Activities:
In the coming year we plan to continue to characterize the difference in response to stimuli, alone and in combination, as a function of age. We also expect to extend these studies from macrophages to parenchymal cells, fibroblasts and epithelial cells, as well. We will also investigate other markers of response. Measurement of prostaglandin production, and COX-2 activation will be evaluated with respect to its usefulness as a marker. Studies have shown COX-2 to be important in the induction of the inflammatory response and systemic responses.
In support of the in vivo projects, we will evaluate in vitro effects of particles of differing composition. We will continue to examine the cytokine response to UFP containing elemental carbon and iron and organic carbon compounds and begin studies of concentrated real world particles.
Journal Articles:
No journal articles submitted with this report: View all 9 publications for this subprojectSupplemental Keywords:
pollution prevention, urban air pollution, atmosphere, metals, air, health, waste, atmospheric sciences, biochemistry, children’s health, environmental chemistry, epidemiology, genetics, virology, molecular biology, health risk assessment, risk assessments, incineration, combustion, combustion engines, air toxics, tropospheric ozone, PM2.5, particulates, ultrafine particles, particulate matter, particle exposure, particle size, aerosol, aerosols, ambient air, ambient air monitoring, ambient air quality, animal model, atmospheric, cardiopulmonary, cardiopulmonary responses, cardiovascular disease, cardiovascular vulnerability, coronary artery disease, cytokine production, fine particles, human exposure, human health, human health effects, environmental health effects, inhalation toxicology, lead, lung, lung inflammation, metals, morbidity, mortality, pathophysiological mechanisms, pulmonary, pulmonary disease, stratospheric ozone, sensitive populations, susceptible populations,, RFA, Scientific Discipline, Health, Air, Toxicology, particulate matter, Environmental Chemistry, Health Risk Assessment, air toxics, Risk Assessments, Biochemistry, Atmospheric Sciences, Molecular Biology/Genetics, ambient air quality, cytokine production, particle size, particulates, sensitive populations, biostatistics, atmospheric, health effects, risk assessment, altered gene expression, cardiopulmonary responses, fine particles, human health effects, morbidity, ambient air monitoring, lung, cardiovascular vulnerability, pulmonary disease, susceptible populations, animal model, ambient air, environmental health effects, particle exposure, ambient monitoring, particulate exposure, lung inflamation, pulmonary, coronary artery disease, inhalation toxicology, urban air pollution, PM, mortality, urban environment, aerosol, cardiopulmonary, human health, aerosols, cardiovascular disease, ultrafine particles, pathophysiological mechanisms, metals, cell kinetic modelsRelevant Websites:
http://www2.envmed.rochester.edu/envmed/PMC/ Exit
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R827354 Rochester PM Center Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R827354C001 Characterization of the Chemical Composition of Atmospheric Ultrafine Particles
R827354C002 Inflammatory Responses and Cardiovascular Risk Factors in Susceptible Populations
R827354C003 Clinical Studies of Ultrafine Particle Exposure in Susceptible Human Subjects
R827354C004 Animal Models: Dosimetry, and Pulmonary and Cardiovascular Events
R827354C005 Ultrafine Particle Cell Interactions: Molecular Mechanisms Leading to Altered Gene Expression
R827354C006 Development of an Electrodynamic Quadrupole Aerosol Concentrator
R827354C007 Kinetics of Clearance and Relocation of Insoluble Ultrafine Iridium Particles From the Rat Lung Epithelium to Extrapulmonary Organs and Tissues (Pilot Project)
R827354C008 Ultrafine Oil Aerosol Generation for Inhalation Studies
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
- Final Report
- 2004 Progress Report
- 2002 Progress Report
- 2001 Progress Report
- 2000 Progress Report
- 1999 Progress Report
- Original Abstract
7 journal articles for this subproject
Main Center: R827354
106 publications for this center
91 journal articles for this center