Altered surfactant homeostasis and alveolar epithelial cell stress in amiodarone-induced lung fibrosis

Toxicol Sci. 2014 Nov;142(1):285-97. doi: 10.1093/toxsci/kfu177. Epub 2014 Aug 27.


Amiodarone (AD) is a highly efficient antiarrhythmic drug with potentially serious side effects. Severe pulmonary toxicity is reported in patients receiving AD even at low doses and may cause interstitial pneumonia as well as lung fibrosis. Apoptosis of alveolar epithelial type II cells (AECII) has been suggested to play an important role in this disease. In the current study, we aimed to establish a murine model of AD-induced lung fibrosis and analyze surfactant homeostasis, lysosomal, and endoplasmic reticulum (ER) stress in this model. AD/vehicle was instilled intratracheally into C57BL/6 mice, which were sacrificed on days 7, 14, 21, and 28. Extent of lung fibrosis development was assessed by trichrome staining and hydroxyproline measurement. Cytotoxicity was assessed by lactate dehydrogenase assay. Phospholipids (PLs) were analyzed by mass spectrometry. Surfactant proteins (SP) and markers for apoptosis, lysosomal, and ER stress were studied by Western blotting and immunohistochemistry. AECII morphology was evaluated by electron microscopy. Extensive lung fibrosis and AECII hyperplasia were observed in AD-treated mice already at day 7. Surfactant PL and SP accumulated in AECII over time. In parallel, induction of apoptosis, lysosomal, and ER stress was encountered in AECII of mice lungs and in MLE12 cells treated with AD. In vitro, siRNA-mediated knockdown of cathepsin D did not alter the AD-induced apoptotic response. Our data suggest that mice exposed to intratracheal AD develop severe pulmonary fibrosis, exhibit extensive surfactant alterations and cellular stress, but AD-induced AECII apoptosis is not mediated primarily via cathepsin D.

Keywords: ER stress; alveolar epithelial cell apoptosis; amiodarone; lung fibrosis; lysosomal stress; surfactant.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amiodarone / toxicity*
  • Animals
  • Anti-Arrhythmia Agents / toxicity*
  • Apoptosis / drug effects
  • Cathepsin D / genetics
  • Cell Culture Techniques
  • Cell Line
  • Disease Models, Animal
  • Endoplasmic Reticulum Stress / drug effects
  • Epithelial Cells / drug effects*
  • Epithelial Cells / metabolism
  • Epithelial Cells / ultrastructure
  • Homeostasis / drug effects*
  • Instillation, Drug
  • Mice, Inbred C57BL
  • Microscopy, Electron, Transmission
  • Pulmonary Alveoli / drug effects*
  • Pulmonary Alveoli / metabolism
  • Pulmonary Alveoli / ultrastructure
  • Pulmonary Fibrosis / chemically induced*
  • Pulmonary Fibrosis / metabolism
  • Pulmonary Fibrosis / pathology
  • Pulmonary Surfactants / metabolism*


  • Anti-Arrhythmia Agents
  • Pulmonary Surfactants
  • Cathepsin D
  • Amiodarone