Time course of bronchial cell inflammation following exposure to diesel particulate matter using a modified EAVES Brie Hawley a , Dave McKenna a , Anthony Marchese b , John Volckens a,b,⇑ a Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA b Department of Mechanical Engineering, Colorado State University, Fort Collins, CO 80523, USA article info Article history: Received 30 December 2013 Accepted 1 March 2014 Available online 26 March 2014 Keywords: In vitro Bronchial Diesel particulate matter EAVES CYP1A1 IL-8 abstract Electrostatic deposition of particles onto the surface of well-differentiated airway cells is a rapid and effi- cient means to screen for toxicity associated with exposure to fine and ultrafine particulate air pollution. This work describes the development and application of an electrostatic aerosol in vitro exposure system (EAVES) with increased throughput and ease-of-use. The modified EAVES accommodates standard tissue culture plates and uses an alternating electric field to deposit a net neutral charge of aerosol onto air- interface cell cultures. Using this higher-throughput design, we were able to examine the time-course (1, 3, 6, 9, and 24 h post-exposure) of transcript production and cytotoxicity in well-differentiated human bronchial cells exposed to diesel particulate matter at levels of ‘real-world’ significance. Statistically sig- nificant responses were observed at exposure levels (0.4 lg/cm 2 ) much lower than typically reported in vitro using traditional submerged/resuspended techniques. Levels of HO-1, IL-8, CYP1A1, COX-2, and HSP-70 transcripts increased immediately following diesel particulate exposure and persisted for several hours; cytotoxicity was increased at 24 h. The modified EAVES provides a platform for higher throughput, more efficient and representative testing of aerosol toxicity in vitro. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Elevated exposure to ambient air pollution increases one’s risk for chronic lung inflammation & fibrosis (Henderson et al., 1988; Mauderly et al., 1988), allergic immune responses (Sydbom et al., 2001), asthma (Pandya et al., 2002), lung cancer (NIOSH, 1988), and cardiovascular morbidity (Lucking et al., 2008; Peters et al., 2001). Ambient air pollution has recently been identified as the eighth leading cause of death worldwide, outpacing the risks posed by all other environmental hazards (Lim et al., 2012). The U.S. EPA recently reduced the annual standard for fine particulate matter (PM2.5) from 15 to 12 lg/m 3 in an effort to reduce mortality rates, incidents of heart attacks, stroke, and childhood asthma resulting from exposure (Clean Air Act). Despite the increased regulatory action, human exposures to emissions from motor vehicle traffic, coal burning, and other forms of combustion remain prevalent. Human exposure to ultrafine particles is increasing due to increased traffic density (World vehicle, 2012) and to the practice of biomass burning in developing countries (Bruce et al., 2000). The advent of nanotechnology, and nanotoxicology, has also created renewed interest in ultrafine particles (Oberdörster et al., 2005). As a result, a need exists to improve our understanding of the mechanisms associated with ultrafine PM toxicity (Oberdörster et al., 2005; Teeguarden et al., 2007a). Airway cell cultures offer an economical means to study the toxicological effects of exposure to aerosol inhalation hazards. Tra- ditionally, however, models of aerosol exposure/response in vitro have been limited in practical relevance by their dissimilarities to the respiratory epithelium in vivo. The alteration of particle size, surface, and chemistry are also problems when particles are col- lected via filtration, extracted, and resuspended in liquid, and then applied to submerged cell cultures. Furthermore, the calculation of particulate ‘dose’ delivered to cells that are submerged in media is also problematic (Teeguarden et al., 2007a; Lenz et al., 2009; Comouth et al., 2013). Recent advancements, however, have enabled the development of more representative models of aerosol toxicology. Two significant advances are the advent of air–liquid interface (ALI) cultures and direct air-to-cell exposure systems. When cultured at an air–liquid interface, normal human bron- chial epithelial (NHBE) cells progressively differentiate into a mucociliary phenotype that resembles the bronchial epithelium in vivo. Cultured NHBE cells exhibit a pseudo-stratified columnar orientation and show evidence of goblet, basal, and ciliated cell http://dx.doi.org/10.1016/j.tiv.2014.03.001 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author at: Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523, USA. Tel.: +1 (970) 491 6341; fax: +1 (970) 491 2940. E-mail address: John.Volckens@colostate.edu (J. Volckens). Toxicology in Vitro 28 (2014) 829–837 Contents lists available at ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit