Airway epithelial cell exposure to distinct e-cigarette liquid flavorings reveals toxicity thresholds and activation of CFTR by the chocolate flavoring 2,5-dimethypyrazine

Respir Res. 2016 May 17;17(1):57. doi: 10.1186/s12931-016-0369-9.


Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: 2,5-dimethylpyrazine; Airway epithelium; CFTR; Electronic cigarettes; Odorant receptor; xCELLigence RTCA.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Bronchi / drug effects*
  • Bronchi / metabolism
  • Bronchi / pathology
  • Cell Line
  • Cell Survival / drug effects
  • Chocolate*
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cystic Fibrosis Transmembrane Conductance Regulator / drug effects
  • Cystic Fibrosis Transmembrane Conductance Regulator / metabolism
  • Dose-Response Relationship, Drug
  • Electric Conductivity
  • Electronic Nicotine Delivery Systems*
  • Epithelial Cells / drug effects*
  • Epithelial Cells / metabolism
  • Epithelial Cells / pathology
  • Flavoring Agents / toxicity*
  • High-Throughput Screening Assays
  • Humans
  • Mice, Inbred C57BL
  • Permeability
  • Pyrazines / toxicity*
  • Signal Transduction / drug effects
  • Time Factors
  • Vaping / adverse effects*


  • Flavoring Agents
  • Pyrazines
  • Cystic Fibrosis Transmembrane Conductance Regulator
  • Cyclic AMP-Dependent Protein Kinases
  • 2,5-dimethylpyrazine