Grantee Research Project Results
Final Report: Pulmonary Toxicity of Particulate Matter and Ozone
EPA Grant Number: R825268Title: Pulmonary Toxicity of Particulate Matter and Ozone
Investigators:
Institution: New York University Medical Center
EPA Project Officer: Chung, Serena
Project Period: November 18, 1996 through November 17, 1998
Project Amount: $172,446
RFA: Air Quality (1996) RFA Text | Recipients Lists
Research Category: Air Quality and Air Toxics , Air
Objective:
Increases in morbidity and mortality associated with elevated levels of air pollution suggest that exposure to particulate matter (PM) below the present National Ambient Air Quality Standard (NAAQS) may adversely affect human health. Yet, few toxicologic studies have demonstrated plausible biological mechanisms by which PM could elicit adverse effects. Because ozone is an important copollutant of PM, it will be useful to demonstrate if there is any interaction between PM and ozone when animals are exposed to their mixtures. We hypothesize that if ambient PM enhances a clearly defined ozone response, this interaction will provide strong evidence supporting the biological plausibility of ambient PM-induced effects. The goals of this project are to: (1) establish a concentration-response relationship and the time-course of the pulmonary response of rats exposed to concentrated ambient PM with or without the addition of ozone; and (2) determine the cellular response of pulmonary cells to size-selected ambient PM with or without the addition of ozone.Summary/Accomplishments (Outputs/Outcomes):
During the research period, we have developed a centrifugal ambient particle concentrator for use in an inhalation study (jointly sponsored by the Health Effects Institute [HEI] and the National Institute for Environmental Health Sciences [NIEHS]). However, the task took longer than we anticipated, and we did not perform the original goal (i.e., coexposure of concentrated ambient PM with ozone) before the funding ended. Instead, we concentrated on investigating the mechanisms of ozone- and PM-induced lung injury using in vitro exposure techniques. We found that ozone is capable of inducing the production of inflammatory cytokine interleukin-8 (IL-8) by airway epithelial cells through increasing the DNA-binding activity of nuclear factors. Airway epithelial cells also were responsive to PM exposure by releasing IL-8 and granulocyte-macrophage colony-stimulating factors (GM-CSF) in a size-dependent manner, with the ultrafine/fine fraction (PM < 0.16 m) producing the greatest effect. Furthermore, PM from an upwind urban area was the primary contributor to the cellular response, while locally generated coarse particles had less of an effect.The following section summarizes the results of these studies:
- Development of a centrifugal particle concentrator for use in inhalation toxicology (Collaboration with HEI, Terry Gordon, PI). Epidemiologic studies have provided strong evidence that episodic exposure to ambient PM is associated with increases in morbidity and mortality. These adverse effects have been demonstrated at concentrations far below the NAAQS and, thus, the biological plausibility of these effects has been questioned. For the purpose of exposing test animals to relevant and reproducible exposure concentrations of ambient PM, we have developed a simple and inexpensive concentrator system that can concentrate ambient particles tenfold. A high-volume blower is used to deliver ambient air to the inlet manifold of a centrifugal concentrator, and the entrained particles travel along a concentric annulus formed by a stationary solid outer cylinder and a porous inner cylinder rotating at high speed (up top 12,500 rpm). Suction applied at one end of the porous shaft causes the dispersion medium (air) to pass through the porous cylinder and into the shaft. Because the rotational velocity of airborne particles is comparable to that of the rotating cylinder near its surface, the particles move radially outward due to the centrifugal force, in addition to their motion laterally along the cylinder, and inward due to the suction of air into the rotating porous cylinder. The particles reach their highest concentration near the outlet manifold, when they enter the exposure chamber under positive pressure (~0.4 cm H2O). Except for coarse particle loss due to impaction and diffusional loss of ultrafine particles in the concentrator, the increase in particle concentration is the ratio of the flow rates for the inlet air and the air delivered to the exposure chamber. We used the centrifugal concentrator to deliver concentrated ambient urban PM to a nose-only exposure chamber and examined the concentrating effect across ambient particle sizes.
- Induction of IL-8 and transcription factors regulating its expression in airway epithelial cells exposed to ozone. Ozone, one of the most reactive oxidant gases to which humans are routinely exposed, induces inflammation in the lower airways. The airway epithelium is one of the first targets that inhaled ozone will encounter, but its role in airway inflammation is not well understood. Expression of inducible genes involved in the inflammatory response, such as IL-8, is controlled by transcription factors. Expression of the IL-8 gene is regulated by the transcription factors NF-kB, NF-IL6, and possibly AP-1. Type II-like epithelial cells (A549) were grown on a collagen-coated membrane and exposed in vitro to 0.1 ppm ozone or air. Exposure to ozone induced DNA-binding activity of NF-kB, NF-IL6, and AP-1. IL-8 mRNA and IL-8 protein levels also were increased after ozone exposure. These results link ozone-induced DNA-binding activity of transcription factors and the production of IL-8 by epithelial cells, thus demonstrating a potential cellular cascade resulting in the recruitment of inflammatory cells into the airway lumen.
- Induction of IL-8 expression by ozone is mediated by protein tyrosine kinase and protein kinase A, but not by protein kinase C. Ozone is one of the most common air pollutants that humans routinely inhale. We have previously shown that in vitro ozone exposure induces the DNA-binding activities of NF-kB and NF-IL6, as well as the expression of IL-8 in respiratory epithelial cells. In this study, we investigated intracellular signaling steps mediating ozone-induced inflammatory mediator release. A549 cells, a type II-like alveolar epithelial cell line, were exposed in vitro to air or 0.1 ppm ozone in the presence of several kinase inhibitors. Exposure to ozone increased IL-8 expression and transcription factor activities in protein tyrosine kinase (PTK) and protein kinase A (PKA), yet protein kinase C (PKC) acted in an independent manner. Furthermore, ozone induced PTK and PKA activities, but failed to induce PKC activity. In addition, our results suggest that ozone-induced PTK and PKA activities were reactive oxygen intermediate (ROI)-dependent and occurred in parallel, because specific inhibitors for PTK and PKA failed to block the other kinase's activity. These results indicate that PTK and PKA kinase activities are early events in the signal transduction cascade mediating the ozone-induced activation of NF-kB and NF-IL6 as well as the release of IL-8.
- Cellular response to size-selected ambient PM: induction of nuclear
factors NF-kB, AP-1, and GM-CSF. A human type II epithelial cell-like cell
line (A549) was grown to a monolayer on Costar Transwells (Costar, Cambridge,
MA) and exposed in vitro for 3 hours to ambient particles (50 percent cut-sizes
of 3.2, 1.0, 0.16, and <0.16 m) collected by MOUDI. Laboratory-generated
carbon particles served as the control. Activation of NF-KB and AP-1 were
assayed using the mobility shift assay. A concentration-dependent increase in
AP-1 binding activity was observed for ambient PM smaller than 1 m, with
ultrafine particles (those below 0.16 m) producing the strongest signal, while
carbon particles had no effect. All particles used in this experiment induced
NF-KB DNA binding, but the highest concentration actually produced weaker
signals. These results indicate that the exposure of airway epithelial cells to
ambient PM can activate cellular signaling pathways in a size-dependent manner.
Human bronchial epithelial cells (HBECs) are the first target for PM and have the capacity to release GM-CSF. Importantly, GM-CSF effects the recruitment and maturation of dendritic cells, the most potent antigen presenting cells. This property may result in localized altered immune responses in the airway. We hypothesized that size-fractionated ambient PM would enhance the airway immune response via the induction of GM-CSF in HBECs. HBECs were derived from endobronchial brushing of normal human volunteers and cultured in a serum-free, hormonally supplemented medium. Size-fractionated ambient PM (<0.18 m, ultrafine; 0.18-1.0 µm; and 1.0-3.6 m) was collected from a model urban environment (New York City) with a micro-orifice uniform deposition impactor. Particles were sonicated off pre-weighed filters, resuspended in media, and used within 3 days. UF PM elicited a dose-dependent increase in GM-CSF (commercial ELISA), which resulted in a sevenfold increase in GM-CSF release (n=3, P<0.05) in cells exposed to 100 g/ml (11 g/cm2). Exposure to larger particles between the sizes of 0.18 and 3.6 m (100 g/ml) resulted in a significant, albeit only twofold, increase in GM-CSF. We previously demonstrated that optimal generation of GM-CSF mRNA and protein by HBECs is mediated by a PKC, extracellular-regulated kinase (ERK) mitogen-activated protein kinase pathway. Because GM-CSF release induced by UF was greater than that induced by TNF-a or IL-1 (36 vs.15 percent of maximal PMA response), we suggest that size-fractionated ambient PM, particularly the UF component, may stimulate a PKC/ERK MAP kinase pathway. These data begin to describe mechanisms by which ambient PM may alter the underlying airway immune response and generate a milieu conducive to the development of allergic asthma.
- Composition of particulate matter as the determinant of cellular
response. We previously reported that exposure of pulmonary epithelial cells
to size-fractionated ambient PM activates cell-signaling pathways leading to
expression of the inflammatory cytokine, IL-8. In this study, we investigated
whether the composition of PM influences the cellular response. In February
1998, 12-hour samples of coarse (2.5-10 m) and fine (< 2.5 m, corresponding
to PM2.5) particles were collected on Teflon filters from four locations in
southwestern Taiwan using dichotomous samplers. The concentrations of anions
(SO42-, NO3-) and 20 relevant elements were determined using ion
chromatography and x-ray fluorescence, respectively. Elemental carbon (analyzed
separately on particles collected on quartz filters) composed 40 percent of the
PM mass, regardless of size fraction. Nitrate contributed between 14 and 17
percent of the mass, while sulfate contributed between 10 and 14 percent of the
PM mass. Aluminum, silica, sulfur, chloride, iron, and zinc were among the major
elements detected. Confluent monolayers of A549 cells were exposed to coarse and
fine particles for 2 hours at concentrations of 25, 50, and 100 g/ml.
Immediately after exposure, the intracellular pH (using the fluorescent dye
BCECF), ROI production (using fluorescent dye DCF), and expression of IL-8 mRNA
(using RT-PCR) were measured. For the fine particles, the cellular responses
were correlated with the concentration of sulfur, but not with any metals. It
was determined that PM from an upwind urban area was the primary contributor to
the cellular response, while locally generated coarse particles had less of an
effect.
In this study, we observed that exposure to ozone induced DNA-binding activity of NF-kB, NF-IL6, and AP-1, with subsequent expression of IL-8 mRNA and IL-8 production in human airway epithelial cells. We further concluded that PTK and PKA kinase activities are early events in the signal transduction cascade mediating the ozone-induced activation of NF-kB and NF-IL6 as well as the release of IL-8. Using ambient PM of New York City, we found that ambient PM can activate cellular signaling pathways in a size-dependent manner. Particle exposure can induce DNA-binding activity of nuclear factor NF-kB and AP-1. In addition, the ultrafine/fine fraction of New York City ambient PM induced the greatest change in GM-CSF production in the primary culture of HBECs as compared to other size ranges. The production of GM-CSF is mediated through a PKC/ERK MAP kinase pathway. Using a well-characterized ambient PM collected in Taiwan, we found that particles collected downwind from major metropolitan areas and major highways produced the greater cellular responses as compared to those collected at other sampling sites. The results of principal component analysis showed that sulfur compounds in the fine particles produced by combustion of fossil fuels were associated with the ability of ambient PM in stimulating respiratory cells to produce ROI. Further analysis showed that several metals such as vanadium, cobalt, iron, and copper were the major contributors to the cellular response induced by ambient PM. In addition, increasing sulfur concentrations in PM were associated with decreasing pHi and increasing ROI in respiratory cells. Taken together, these results showed that fine particles of ambient PM contained acids and the acidity of these particles could make transition metals in the ambient PM bioavailable to produce cellular response.
Journal Articles on this Report : 5 Displayed | Download in RIS Format
Other project views: | All 6 publications | 5 publications in selected types | All 5 journal articles |
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Alpert SE, Walenga RW, Jaspers I, Qu QS, Chen LC. Ozone inactivates cyclooxygenase in human tracheal epithelial cells without altering PGHS-2 mRNA or protein. American Journal of Physiology-Lung Cellular and Molecular Physiology 1997;16(5):L879-L887. |
R825268 (Final) |
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Gordon T, Gerber H, Fang CP, Chen LC. A centrifugal particle concentrator for use in inhalation toxicology. Inhalation Toxicology 1999;11(1):71-87. |
R825268 (Final) R826244 (2000) R826244 (Final) |
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Jaspers I, Flescher E, Chen LC. Ozone-induced IL-8 expression and transcription factor binding in respiratory epithelial cells. American Journal of Physiology-Lung Cellular and Molecular Physiology 1997;16(3):L504-L511. |
R825268 (Final) |
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Jaspers I, Flescher E, Chen LC. Respiratory epithelial cells display polarity in their release of the chemokine IL-8 after exposure to ozone. Inflammation Research 1997;46(Suppl 2):S173-S174. |
R825268 (Final) |
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Jaspers I, Chen LC, Flescher E. Induction of interleukin-8 by ozone is mediated by tyrosine kinase and protein kinase A, but not by protein kinase C. Journal of Cellular Physiology 1998;177(2):313-323. |
R825268 (Final) |
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Supplemental Keywords:
air, ambient air, atmosphere, ozone, exposure, health effects, human health, particulates, biology, northeast, New York, NY, EPA Region 2., RFA, Health, Scientific Discipline, Air, Geographic Area, particulate matter, Environmental Chemistry, State, Chemistry, Risk Assessments, Atmospheric Sciences, tropospheric ozone, EPA Region, ambient air quality, copollutant exposures, particle size, particulates, cumulative risk, ecological risk assessment, air toxics, chemical characteristics, human health effects, contaminant transport, exposure, ozone, ambient air, environmental mutagens, particle exposure models, pulmonary toxicity, human exposure, chronic health effects, lung inflammation, inhalation, lung inflamation, Acute health effects, inhaled, sulfuric acid, physicochemical characteristics, Region 2, atmospheric chemistry, New York (NY)Progress and Final Reports:
Original AbstractThe 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.