Menthol flavoring in e-cigarette condensate causes pulmonary dysfunction and cytotoxicity in precision cut lung slices

Am J Physiol Lung Cell Mol Physiol. 2023 Mar 1;324(3):L345-L357. doi: 10.1152/ajplung.00222.2022. Epub 2023 Jan 24.


E-cigarette consumption is under scrutiny by regulatory authorities due to concerns about product toxicity, lack of manufacturing standards, and increasing reports of e-cigarette- or vaping-associated acute lung injury. In vitro studies have demonstrated cytotoxicity, mitochondrial dysfunction, and oxidative stress induced by unflavored e-cigarette aerosols and flavoring additives. However, e-cigarette effects on the complex lung parenchyma remain unclear. Herein, the impact of e-cigarette condensates with or without menthol flavoring on functional, structural, and cellular responses was investigated using mouse precision cut lung slices (PCLS). PCLS were exposed to e-cigarette condensates prepared from aerosolized vehicle, nicotine, nicotine + menthol, and menthol e-fluids at doses from 50 to 500 mM. Doses were normalized to the glycerin content of vehicle. Video-microscopy of PCLS revealed impaired contractile responsiveness of airways to methacholine and dampened ciliary beating following exposure to menthol-containing condensates at concentrations greater than 300 mM. Following 500 mM menthol-containing condensate exposure, epithelial exfoliation in intrabronchial airways was identified in histological sections of PCLS. Measurement of lactate dehydrogenase release, mitochondrial water-soluble-tetrazolium salt-1 conversion, and glutathione content supported earlier findings of nicotine or nicotine + menthol e-cigarette-induced dose-dependent cytotoxicity and oxidative stress responses. Evaluation of PCLS metabolic activity revealed dose-related impairment of mitochondrial oxidative phosphorylation and glycolysis after exposure to menthol-containing condensates. Taken together, these data demonstrate prominent menthol-induced pulmonary toxicity and impairment of essential physiological functions in the lung, which warrants concerns about e-cigarette consumer safety and emphasizes the need for further investigations of molecular mechanisms of toxicity and menthol effects in an experimental model of disease.

Keywords: PCLS; e-cigarettes; lung toxicity; menthol; nicotine; precision cut lung slices.

Publication types

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

MeSH terms

  • Animals
  • Electronic Nicotine Delivery Systems*
  • Flavoring Agents / toxicity
  • Lung
  • Menthol / toxicity
  • Mice
  • Nicotine* / toxicity
  • Respiratory Aerosols and Droplets


  • Nicotine
  • Menthol
  • Flavoring Agents

Associated data

  • figshare/10.6084/m9.figshare.20323689.v2