[CANCER RESEARCH 58. 4543-4547, October 15, 1998] Advances in Brief Ambient Particulate Matter Causes Activation of the c-jun Kinase/Stress-activated Protein Kinase Cascade and DNA Synthesis in Lung Epithelial Cells1 Cynthia Timblin,2 Kelly BeruBe, Andrew Churg, Kevin Driscoll, Terry Gordon, David Hemenway, Eric Walsh, Andrew B. Cummins, Pamela Vacek, and Brooke Mossman Departments of Pathology ¡C.T., E. W., A. B. C., B. M.], Civil and Mechanical Engineering ¡D.//./, and Binstalistics (P. V.l. University of Venn/ml, Burlington. Vermont 05405: School of Molecular and Medical Biosciences. Universitv of Cardiff, Cardiff CFl 3US. United Kingdom ¡K.B.¡.Department of Laboratory Medicine. University of British Columbia, Vancouver. British Columbia, V6T 2B5 Canada [A. C.\; Miami Valley Laboratories, Procter & Gamble, Inc., Cincinnati. Ohio 45239 IK. D.¡; und institute of Environmental Medicine, New York University Medical Center. Long Meadow Road, Tuxedo, New York 10987 ¡T.G.\ Abstract Numerous epidemiológica! studies have demonstrated a positive asso ciation between ambient air pollution and adverse health effects including respiratory morbidity, asthma, and lung cancer. It has been suggested in some experimental studies that airborne particulate matter (PM) can produce inflammatory effects, but nothing is known about the possible proliferative and carcinogenic effects of these particles on cells of the lung. We show here that exposure of pulmonary epithelial cells, a cell type affected in acute lung injury, asthma, and lung carcinomas, to nontoxic concentrations of PM in vitro results in increases in c-jun kinase activity, levels of phosphorylated c.liin immunoreactive protein, and transcrip- tional activation of activator protein-1-dependent gene expression. These changes are accompanied by elevations in numbers of cells incorporating 5'-bromodeoxyuridine, a marker of unscheduled DNA synthesis and/or cell proliferation. Data here are the first to demonstrate that interaction of ambient PM with target cells of the lung initiates a cell signaling cascade related causally to aberrant cell proliferation and carcinogenesis. Introduction Epidemiológica!evidence indicates that exposure to PM,1a component of urban air pollution, is linked to increased acute and chronic respiratory morbidity (1), possible elevations in lung cancer risk (2), and episodes of asthma (3), chronic bronchitis (4), and pneumonia (5) in predisposed indi viduals. These observations have engendered intense scrutiny by regulatory agencies and health assessment panels. In experimental animals, PM evokes an inflammatory response. However, nothing is known of the critical mo lecular and pathogenic effects of PM on lung epithelium. Ambient PM is generated primarily during the combustion of fossil fuels and is composed of organic or elemental carbon aggregates containing various metals, acid salts, organic pollutants, and biological contaminants such as endotoxin. Samples of PM may differ somewhat depending upon the site and method of collection. However, an association between high airborne concentrations of PM and increases in adverse respiratory effects are ob served regardless of geographic site or time of year. Recent attention has Received 7/16/98: accepted 8/24/98. The costs of publication of this article were defrayed in pan by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported by a Special Emphasis Research Career Award from National Institute for Occupational Safety and Health (to C. T.), Grants ES0649 and 09213 from National Institute of Environmental Health Sciences (to B. T. M.). Grant HL-39469 from the National Heart. Lung, and Blood Institute (to B. T. M.), Grants MT6097. MT805I, and M77820 from the Medical Research Council of Canada (to A. C.). Health Effects Institute Contract 95-8, Grant G7061234 from the Environmental Protection Agency, and Grant ES0256 from the National Institute of Environmental Health Sciences. 2 To whom requests for reprints should be addressed, at the University of Vermont, Department of Pathology. Burlington, VT 05405. Phone: (802) 656-0382: Fax: (802) 656- 8892; E-mail: ctimblin@zoo.uvm.edu. •¿ ' The abbreviations used are: PM. paniculate matter; AP-I. activator protein-1; TEM, transmission electron microscopy; EPXMA. electron probe X-ray microanalysis: RLE, rat lung epithelial: NBS. newborn calf serum; 0-gal. /3-galactosidase: BrdUrd, 5'bromode- oxyuridine; JNK. c-jun NH-»-terminalkinase; ERK. extracellular signal-related kinase. focused on fine PM2iS,i.e., particles of an aerodynamic diameter <2.5 /urn, because the United States Environmental Protection Agency has introduced new standards for PM, 5 particles in addition to retaining the present stand ards for larger particles of PMK) (< 10-/xm diameter). One premise for these new policies is that fine, and especially ultrafine, particles of submicrometer diameters may be more pathogenic and/or present in high numbers in PM samples (although they comprise a minor component of the total particulate mass). Ultrafine particles also have a greater surface area per unit weight and may be retained more efficiently than larger particles in the peripheral lung. A critical cell type encountering inhaled particles after inhalation and affected in a number of respiratory diseases is the epithelial cell of the airways and alveoli. Previous work in our laboratory has demonstrated that epithelial cells in various compartments of the lung exhibit increased c-jun expression, DNA synthesis, and hyperplasia after inhalation or in vitro exposures to pathogenic minerals such as asbestos (6-8). The proto-oncogene, c-jun, is a member of the im mediate-early response, multigene family transiently expressed in response to a variety of external stresses, c-jun and c-fos members encode protein subunits (Jun/Jun homodimers or Jun/Fos het- erodimers) that comprise the AP-1 transcription factor involved in the transition of the G, phase and entry into the S phase of the cell cycle (9). In epithelial and mesothelial cells exposed to the fibrogenic and carcinogenic mineral fiber, asbestos, increased c-jun mRNA levels are accompanied by elevated cJun protein and increased AP-1 to DNA binding activity (6, 10). These changes are not observed after expo sure of cells to nonpathogenic particles. Moreover, overexpression of c-jun in trachéal epithelial cells causes increased cell proliferation and morphological transformation (11), supporting a causal role of cJun in mitogenesis and carcinogenesis. Links between cJun expression and the development of lung cancers have also been suggested from studies evaluating immunoreactive cJun protein in hyperplasias and metaplasias from human lungs (12). In this study, we examined the hypothesis that PM initiates a cell signaling cascade causing increased expression of c-jun and transactivation of AP-1-dependent genes in epithelial cells of the lung. We speculated that these early molecular changes would lead to increased cell proliferation, a critical event in the pathogenesis of a number of respiratory disorders and diseases. Materials and Methods Preparations and Characterization of PM, Asbestos, and TiO2 for in Vitro Studies. PM,,, samples were collected on filters from the Burlington, VT monitoring station using a Wedding collection apparatus. To prepare a PM2 5 fraction, PM-containing filters were placed in sterile centrifuge tubes, immersed in endotoxin-free double-distilled H2O, and sonicated to release particles. Particle samples representative of PM, 5 were obtained by filtration through a 2.5-/im diameter pore size, polycarbonate filter. Samples were concentrated by lyophilization and stored at -80°C until use. A total of 5 VT PM preparations were used in experiments in duplicate. Fine and ultrafine TiO2 samples were a generous gift from Dr. Gunter Oberdörster from the 4543 Research. on November 10, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from