Ozone Induces Oxidative Stress in Rat Alveolar Type II and Type I-Like Cells ⁎ Jieru Wang, Shuanglin Wang, Rizwan Manzer, Glen Mcconville, Robert J
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Free Radical Biology & Medicine 40 (2006) 1914–1928 www.elsevier.com/locate/freeradbiomed Original Contribution Ozone induces oxidative stress in rat alveolar type II and type I-like cells ⁎ Jieru Wang, Shuanglin Wang, Rizwan Manzer, Glen McConville, Robert J. Mason Department of Medicine, National Jewish and Medical Research Center, 1400 Jackson Street, Denver, CO 80206, USA Received 21 July 2005; revised 10 January 2006; accepted 16 January 2006 Available online 9 February 2006 Abstract Ozone is a highly reactive gas present in urban air, which penetrates deep into the lung and causes lung injury. The alveolar epithelial cells are among the first cell barriers encountered by ozone. To define the molecular basis of the cellular response to ozone, primary cultures of rat alveolar type II and type I-like cells were exposed to 100 ppb ozone or air for 1 h. The mRNA from both phenotypes was collected at 4 and 24 h after exposure for gene expression profiling. Ozone produced extensive alterations in gene expression involved in stress and inflammatory responses, transcription factors, antioxidant defenses, extracellular matrix, fluid transport, and enzymes of lipid metabolism and cell differentiation. Real-time reverse transcription–polymerase chain reaction and Western blot analysis verified changes in mRNA and protein levels of selected genes. Besides the increased stress response, ozone exposure downregulated genes of cellular differentiation. The changes were more prominent at 4 h in the type I-like phenotype and at 24 h in the type II phenotype. The type I-like cells were more sensitive to ozone than type II cells. The genome-wide changes observed provide insight into signal pathways activated by ozone and how cellular protection mechanisms are initiated. © 2006 Elsevier Inc. All rights reserved. Keywords: Alveolar epithelium; Microarray analyses; Oxidative stress; Differentiation; Free radical Introduction cytotoxicity, DNA damage, and injury through acute and chronic oxidative stress, which ultimately produces necrosis, Ozone is a highly reactive oxidant gas, and a significant sloughing, and increased epithelial permeability [5]. However, component of contemporary ambient air pollution. Many the underlying process and pathways involved in regulation of observational epidemiological studies have indicated significant epithelial injury and repair in response to ozone are not well associations between ambient ozone concentration and a wide understood. Moreover, many previous studies evaluated high range of adverse respiratory health outcomes [1,2]. Ozone has concentrations of ozone (greater than 200 ppb) or were carried been shown to produce lung injury in several animal models as out on cancer cell lines, which may not be representative of well as in normal human subjects and increases airway differentiated epithelial cells [2,6–8]. None have compared responsiveness [2–4]. Ozone is relatively insoluble and targets alveolar type I and type II cell phenotypes. the distal airway epithelium and proximal alveolar units. The present study was designed to explore the cellular However, little is known about the cellular responses to ozone, response of primary cultures of alveolar type II cells and type I- especially those of lung epithelial cells. like cells to a low concentration of ozone (100 ppb for 60 min). The alveolar epithelium is one of the primary targeted sites of Specifically, we sought to define the stress response to ozone ozone toxicity [2]. Inhalation of ozone induces epithelial and to identify sensitive molecular targets in the two alveolar epithelial phenotypes. To our knowledge, this is the first study to Abbreviations: DMEM, Dulbecco's modified essential medium; EHS, focus on genome-wide changes induced by a low concentration Engelbreth-Holm-Swarm; FBS, fetal bovine serum; RS, rat serum; MHC, major of ozone on differentiated alveolar epithelial cells. The genome- histocompatibility complex; MMPs, matrix metalloproteinases; RT-PCR, wide changes observed should help to define how the epithelial reverse transcription-polymerase chain reaction; GSH, glutathione; GRX, glutaredoxin. cells respond to ozone, and, therefore, provide insight into signal ⁎ Corresponding author. Fax: +1 303 398 1806. pathways activated by ozone and how cellular protection E-mail address: [email protected] (R.J. Mason). mechanisms are initiated. 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.01.017 J. Wang et al. / Free Radical Biology & Medicine 40 (2006) 1914–1928 1915 Materials and methods like phenotype the plated type II cells were switched from 5% rat serum to 5% fetal bovine serum (FBS). The 6-well plates were Animals incubated at 37°C on a rocking platform inside a humidified incubator gassed with 10% CO2; the media were replaced every Male Sprague-Dawley rats weighing 150–250 g were 48 h. On Day 3 and Day 5 of culture, 10−8 M dexamethasone (dex) purchased from Harlan (Indianapolis, IN). Animals were fed was added to media for both phenotypes. The ozone exposure was ad libitum and housed in an IAUAC-accredited facility in HEPA- carried out on Day 7 of culture, 6 days under air/liquid conditions. filtered cages at the National Jewish Medical and Research Center, Denver. In each microarray experiment, at least 6 rats 6– In vitro ozone exposure 8 weeks of age were used. Animal care, handling, and expe- rimental procedures were carried out in accordance with a pro- Cells were exposed to ozone in a specially designed com- tocol approved by the Animal Care and Use Committee of puter-regulated exposure facility [11]. Ozone was generated by National Jewish Medical and Research Center, Denver. passing compressed medical grade oxygen through an ozone generator (Model OZ2SS-SS, Ozotech, Yreka, CA). Four spe- Antibodies cifically designed 3.7 L glass chambers were used to expose the cultured cells. One of these specifically plumbed chambers was Rabbit anti-SP-B and SP-C were purchased from Chemicon used as the control chamber receiving humidified and warm air/ (Chemicon International, Inc. Temecula, CA), mouse anti-heme CO2 mixtures, and the other chambers received specified con- oxygenase-1 (HO-1) and heat shock 70-kDa protein (Hsp70) centrations of ozone. All chambers were fitted with a rocking were from Stressgen (SPA-180) (Stressgen, Victoria, Canada) platform to keep the culture plate rocking at the time of exposure. and Santa Cruz (sc-24) (Santa Cruz Biotechnology, Santa Cruz, The medium was completely removed from the apical surface CA), rabbit anti-Fra-1 and goat anti-actin were products from and 1 ml of media was maintained in the basolateral compart- Santa Cruz. Rabbit anti-rat SP-A and SP-D were a gift from Dr. ment for the exposure. In the current study, cells were exposed to Dennis Voelker (National Jewish Medical and Research Center, 100 ppb ozone for 60 min. Ozone concentration in the ozone Denver, CO), mouse anti-T1α was provided by Dr. Mary exposure chamber was analyzed precisely by an ozone analyzer Williams (Boston University, Boston, MA). (Model MD-050-12-f-4, Perma Pure Inc., Toms River, NJ) and was regulated by a computerized system. Type II cell isolation In vivo ozone exposure Alveolar type II cells were isolated from pathogen free adult male Sprague-Dawley rats (Harlan-Sprague-Dawley, Indiana- Rats were randomly assigned to two groups of four and polis, IN) by tissue dissociation with porcine pancreatic elastase placed in individual stainless-steel wire-mesh cages inside a (Roche Molecular Biochemicals, Indianapolis, IN) and partial 135 L exposure chamber and exposed to 2 ppm ozone or air for purification on discontinuous metrizamide gradients as des- 3 h. Chamber ozone concentration was monitored with an cribed previously [9,10]. Advance Pollution Instruments (API) Model 400A (Teledyne Instruments, San Diego, CA). Six hours after exposure, the rats Culture in the apical-access system were euthanized with an intraperitoneal injection of pentobar- bital (Abbott Laboratories, North Chicago, IL). The chest cavity Two and a half million freshly isolated viable type II cells were was opened, and the lungs were removed. plated in 1 ml of DMEM containing 5% rat serum (PelFreez Biologicals, Rogers, AR), 2 mM glutamine, 2.5 μg/ml amphoter- Immunohistochemistry analyses icin B, 100 μg/ml penicillin G, 100 μg/ml streptomycin (GIBCO BRL, Life Technologies Inc., Rockville, MD), and 10 μg/ml The ozone-and air-exposed rat lungs were perfused and fixed in gentamicin (Sigma-Aldrich, St. Louis, MO) on a filter insert acid alcohol overnight at 4°C and embedded in paraffin [12].For (Millicell-CM, 0.4 μm pore, 30 mm diameter, Millipore Corp, immunocytochemical staining, slides were incubated with 3% Bedford, MA) that had been coated with 0.4 ml of a mixture of rat donkey serum for 20 min at room temperature to block the tail collagen and Engelbreth-Holm-Swarm (EHS) tumor matrix unspecific staining and then incubated with mouse monoclonal anti- (Matrigel, Collaborative Biochemedical Products, Bedford, MA). Hsp70 or T1α antibodies overnight at 4°C. Alexa 594 conjugated The coating mixture was prepared at 4°C and allowed to donkey anti-mouse IgG (Molecular Probes. Inc., Eugene, OR) was polymerize at 37°C before the addition of the cells and contained used to detect the monoclonal antibodies. Sections were viewed and approximately 0.8 mg rat tail collagen and 2 mg EHS protein per photographed with a Zeiss Axioskop 2 fluorescent microscope. milliter. Two milliters of the same media was added to the basolateral compartment of the well. After attachment for 24 h, the Measurement of DNA monolayers were rinsed twice with DMEM, and 0.4 ml of medium was added to the apical surface and 2.0 ml to the basolateral DNA from air control and ozone-treated cells were extracted compartment. For the type II phenotype, cells were cultured in at 24 h posttreatment for DNA assay as described previously DMEM containing 5% rat serum and 10 ng/ml KGF.