![[logo] US EPA](https://webarchive.library.unt.edu/eot2008/20081106010035im_/http://www.epa.gov/epafiles/images/logo_epaseal.gif){kind=logo}
Final Report: Mechanisms of Ozone Toxicity to the Lung
EPA Grant Number: R825702C014Subproject: this is subproject number 014 , established and managed by the Center Director under grant R825702
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: Environmental Lung Disease Center (National Jewish Medical and Research Center)
Center Director: Mason, Robert
Title: Mechanisms of Ozone Toxicity to the Lung
Investigators: White, Carl W. , Murphy, Robert C.
Institution: National Jewish Medical and Research Center
EPA Project Officer: Glenn, Barbara
Project Period: February 16, 1998 through February 28, 2003 (Extended to February 28, 2004)
RFA: Environmental Lung Disease Center (National Jewish Medical and Research Center) (1998)
Research Category: Targeted Research
Description:
Objective:The objectives of this research project were to: (1) determine effect of ozone on mitochondrial function and energetics (ATP) in lung epithelial cells (A549 cell line and primary epithelial cells); (2) determine structure of novel phospholipids produced during exposure of purified lipids, isolated plasma membranes, and intact cells to ozone; and (3) evaluate effects of novel phospholipids, proteins, and/or protein/lipid products on epithelial cell viability, respiration, and mitochondrial membrane potential.
Summary/Accomplishments (Outputs/Outcomes):Determine Effect of Ozone on Mitochondrial Function and Energetics (ATP) in Lung Epithelial Cells (A549 Cell Line and Primary Epithelial Cells)
In this aim, we made the novel discovery that although there were not changes in total intracellular ATP during acute exposures to “physiologic” levels of ozone, instead there was release of ATP acutely (7-30 within onset of exposure or less) from epithelial cells from all levels of the airways. This release of ATP was not caused by nonspecific damage but instead appeared to be a result of a vesicular transport process that was inhibitable by brefeldin A. This extracellular ATP acted on cellular ATP receptors to assure cellular energy metabolism (glucose uptake) and survival during sustained ozone exposure. Stimulation of cell ATP receptors with ATP, UTP, or nonhydrolyzable ATP analogs all allowed cell survival in ozone, preventing both apoptotic (mitochondrially mediated) and necrotic cell death. Antagonists of P2 and P2Y classes of ATP receptors with suramin and Cibacron blue (reactive blue 2), respectively, prevented cell survival in ozone. Signaling pathways caused by Akt and ERK phosphorylation were activated by these ATP receptors and contributed to cell survival. These results are published (Ahmad, et al., 2005). The work led to a National Institutes of Health grant proposal that received a very high merit score (2.7 percentile) and was funded for a 5-year award. Further studies are in progress to evaluate the function of this pathway in epithelial cells from patients with cystic fibrosis (CF). So far, CF cells appear to be more susceptible by killing caused by ozone, even at low levels that are very relevant to contemporary air pollution.
Determine Structure of Novel Phospholipids Produced During Exposure of Purified Lipids, Isolated Plasma Membranes, and Intact Cells to Ozone
In this aim, the reaction products of calf lung surfactant extract with ozone was identified as 1-palmitoyl-2-(9'-oxo-nonanoyl)-glycerophosphocholine (16:0a/9-al-GPCho; aka “9-al”). This oxidized phospholipid was found to be biologically active in inducing macrophage toxicity with an ED50 of 6 micromolar. Concentrations in the range of 100-200 nM caused apoptosis in lung epithelial-like A549 cells as indicated by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, nuclear shrinkage, and caspase 3 activation and loss of viability indicated by declines in mitochondrial dehydrogenase activities. IL-8 release was stimulated in the range of 50-100 nM 9-al. Physiologic levels of ozone (0-250 ppb) resulted in dose-response increases in 9-al formation during periods of surfactant exposure from 2-48 hours. This work is published (Uhlson, et al., 2002). Although Dr. Murphy did not remain active in this project after 2003, his work has led to further studies in this field, including additional grant support and publications.
Evaluate Effects of Novel Phospholipids, Proteins, and/or Protein/Lipid Products on Epithelial Cell Viability, Respiration, and Mitochondrial Membrane Potential
As indicated above, 9-al was evaluated and found to have potent cytotoxic effects, as well as the capacity to induce IL-8 release at considerably lower concentrations. Both viability and mitochondrial dehydrogenase activity, as an indicator of function, were evaluated. With respect to protection from ozone, the extracellular ATP-dependent protective pathway was identified and characterized as described above. Assessment of mitochondrial preservation was obtained because mitochondrial-dependent cell death pathways (e.g., apoptosis) were inhibited by extracellular ATP through receptor-mediated mechanisms. Overall viability of respiratory epithelial-like cells also was increased significantly through this mechanism.
Significance of Research
The concept that lipid oxidation or ozonation products could incite pulmonary inflammation and/or damage is not entirely new, but our findings did provide support for existing concepts in this area. In addition, our novel approach where whole surfactant was first reacted with ozone and then fractionated and tested for cytotoxic effects did lead to the identification of two major types of toxic lipid products: 9-al and cholesterol oxidation products. The latter has continued to be an important research direction for the laboratory of Dr. Murphy.
The potential importance of the extracellular ATP-dependent protection pathway against ozone toxicity was unsuspected previously. This area is important because this is being identified as a new pathway that potentially is important in many different biologic actions in the lung, some of which could contribute to pulmonary diseases like asthma, CF, and chronic obstructive pulmonary disease. These would include cytotoxicity and cell death or survival, mucus production, mucociliary clearance, neurotransmission, and surfactant release, among others. In addition, because defects in these signaling pathways are beginning to be discovered in some disease states like CF, their relevance may be even greater than was anticipated. Although some of these effects may be beneficial to survival of airway epithelial cells, the activation of certain pathways also could have implications for airway remodeling and/or fibrosis in populations with recurrent ozone exposures.
Importance to the Scientific Community
In addition to the considerations listed immediately above, it should be noted that ATP analogs also are being developed as therapeutic agents. Because changes in survival of airway epithelium may be mediated by ozone in susceptible individuals, ATP analogs could have different roles in treatment of individuals that reside in high pollution areas.
Importance to the U.S. Environmental Protection Agency
In addition to the considerations discussed in the sections above, these research findings may have implications for guidelines and restrictions upon ambient ozone levels, especially with respect to certain high-risk populations. These findings also suggest that further research into health effects in those high-risk populations in high ozone areas may be indicated.
Journal Articles on this Report: 3 Displayed | Download in RIS Format
| Other subproject views: | All 3 publications | 3 publications in selected types | All 3 journal articles |
| Other center views: | All 69 publications | 39 publications in selected types | All 39 journal articles |
| Type | Citation | ||
|---|---|---|---|
|
|
Ahmad S, Ahmad A, McConville G, Schneider BK, Allen CB, Manzer R, Mason RJ, White CW. Lung epithelial cells release ATP during ozone exposure: signaling for cell survival. Free Radical Biology and Medicine 2005;39(2):213-226. |
R825702C014 (Final) |
not available |
|
|
Pulfer MK, Taube C, Gelfand E, Murphy RC. Ozone exposure in vivo and formation of biologically active oxysterols in the lung. Journal of Pharmacology and Experimental Therapeutics 2005;312(1):256-264. |
R825702 (Final) R825702C014 (Final) |
not available |
|
|
Uhlson C, Harrison K, Allen CB, Ahmad S, White CW, Murphy RC. Oxidized phospholipids derived from ozone-treated lung surfactant extract reduce macrophage and epithelial cell viability. Chemical Research in Toxicology 2002;15(7):896-906. |
R825702 (Final) R825702C014 (Final) |
not available |
air toxics, acute lung injury, air pollutants, airway disease, animal studies, environmental toxicant, exposure, genetic susceptibility, health effects, human exposure, human health risk, lung disease, lung epithelial cells, occupational disease, occupational exposure,
,
Economic, Social, & Behavioral Science Research Program, Scientific Discipline, Health, RFA, Biology, Risk Assessments, Disease & Cumulative Effects, Environmental Statistics, Health Risk Assessment, Epidemiology, biomarkers, exposure assessment, database management, human health risk, statistical models, biostatistics, data quality assurance, statistical methods, data analysis, lung disease
Relevant Websites:
http://www.nationaljewish.org/patient-info/progs/med/environmental/index.aspx 
Progress and Final Reports:
Original Abstract
Main Center Abstract and Reports:
R825702 Environmental Lung Disease Center (National Jewish Medical and Research Center)
Subprojects under this Center:
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R825702C001 SP-A and SP-D in Environmental Lung Disease
R825702C003 Adaptation to Nitrogen Dioxide: Role of Altered Glycolytic Pathway Enzyme Expression and NF-κB-Dependent Cellular Defenses Against Apoptosis
R825702C005 Inhalation of Particulate Matter Alters the Allergic Airway Response to Inhaled Allergen
R825702C006 Particle-Induced Lung Inflammation and Extracellular EC-SOD
R825702C007 Indoor-Outdoor Relationships of Airborne Particle Count and Endotoxin Concentrations
R825702C008 The Role of Mitochondrial DNA Mutations in Oxidant-Mediated Lung Injury
R825702C009 Immunopathogenesis of Hypersensitivity Pneumonitis in the Mouse
R825702C010 Activation of Natural T Lymphocytes by Diesel Exhaust Particulates Leads to Their Production of Interleukin-4 and TH2 Lymphocyte Differentiation to Allergen
R825702C011 Latex Antigen Levels During Powdered and Powderless Glove Use
R825702C012 Adjuvant Effects of Ozone in a Model of Allergen-Induced Airway Inflammation and Hyperresponsiveness
R825702C013 Acute Exposure to Particulate Air Pollution in Childhood Asthma
R825702C014 Mechanisms of Ozone Toxicity to the Lung