title: Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine.
abstract: The potential for adverse respiratory effects following exposure to electronic (e-) cigarette liquid (e-liquid) flavorings remains largely unexplored. Given the multitude of flavor permutations on the market, identification of those flavor constituents that negatively impact the respiratory tract is a daunting task. In this study we examined the impact of common e-liquid flavoring chemicals on the airway epithelium, the cellular monolayer that provides the first line of defense against inhaled particulates, pathogens, and toxicants.
  
  
  
  We used the xCELLigence real-time cell analyzer (RTCA) as a primary high-capacity screening tool to assess cytotoxicity thresholds and physiological effects of common e-liquid flavoring chemicals on immortalized human bronchial epithelial cells (16HBE14o-). The RTCA was used secondarily to assess the capability of 16HBE14o- cells to respond to cellular signaling agonists following a 24 h exposure to select flavoring chemicals. Finally, we conducted biophysical measurements of well-differentiated primary mouse tracheal epithelial (MTE) cells with an Ussing chamber to measure the effects of e-cigarette flavoring constituents on barrier function and ion conductance.
  
  
  
  In our high-capacity screens five of the seven flavoring chemicals displayed changes in cellular impedance consistent with cell death at concentrations found in e-liquid. Vanillin and the chocolate flavoring 2,5-dimethylpyrazine caused alterations in cellular physiology indicative of a cellular signaling event. At subcytotoxic levels, 24 h exposure to 2,5-dimethylpyrazine compromised the ability of airway epithelial cells to respond to signaling agonists important in salt and water balance at the airway surface. Biophysical measurements of 2,5-dimethylpyrazine on primary MTE cells revealed alterations in ion conductance consistent with an efflux at the apical airway surface that was accompanied by a transient loss in transepithelial resistance. Mechanistic studies confirmed that the increases in ion conductance evoked by 2,5-dimethylpyrazine were largely attributed to a protein kinase A-dependent (PKA) activation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel.
  
  
  
  Data from our high-capacity screening assays demonstrates that individual e-cigarette liquid flavoring chemicals vary in their cytotoxicity profiles and that some constituents evoke a cellular physiological response on their own independent of cell death. The activation of CFTR by 2,5-dimethylpyrazine may have detrimental consequences for airway surface liquid homeostasis in individuals that use e-cigarettes habitually.
authors.affiliation: Asthma and Airway Disease Research Center, Arizona Health Sciences Center, 1501 N. Campbell Avenue, Tucson, AZ, 85724-5030, USA. caras@email.arizona.edu.|Bio5 Collaborative Research Institute, Arizona Health Sciences Center, Tucson, AZ, USA. caras@email.arizona.edu.
authors.forename: Cara L
authors.initials: CL
authors.lastname: Sherwood
authors.affiliation: Asthma and Airway Disease Research Center, Arizona Health Sciences Center, 1501 N. Campbell Avenue, Tucson, AZ, 85724-5030, USA.|Bio5 Collaborative Research Institute, Arizona Health Sciences Center, Tucson, AZ, USA.|Department of Physiology, Arizona Health Sciences Center, Tucson, AZ, USA.
authors.forename: Scott
authors.initials: S
authors.lastname: Boitano
mesh_terms: D000818:Animals; D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D000069956:Chocolate; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D066300:Electronic Nicotine Delivery Systems; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005421:Flavoring Agents / Q000633:toxicity*; D057166:High-Throughput Screening Assays; D006801:Humans; D008810:Mice, Inbred C57BL; D010539:Permeability; D011719:Pyrazines / Q000633:toxicity*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D000072137:Vaping / Q000009:adverse effects*
chemical_list: D005421:Flavoring Agents; D011719:Pyrazines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases; C075524:2,5-dimethylpyrazine
keywords: 2,5-dimethylpyrazine; Airway epithelium; CFTR; Electronic cigarettes; Odorant receptor; xCELLigence RTCA