Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury

Arch Toxicol. 2018 May;92(5):1797-1814. doi: 10.1007/s00204-018-2178-z. Epub 2018 Mar 3.


Drug-induced liver injury remains a concern during drug treatment and development. There is an urgent need for improved mechanistic understanding and prediction of DILI liabilities using in vitro approaches. We have established and characterized a panel of liver cell models containing mechanism-based fluorescent protein toxicity pathway reporters to quantitatively assess the dynamics of cellular stress response pathway activation at the single cell level using automated live cell imaging. We have systematically evaluated the application of four key adaptive stress pathway reporters for the prediction of DILI liability: SRXN1-GFP (oxidative stress), CHOP-GFP (ER stress/UPR response), p21 (p53-mediated DNA damage-related response) and ICAM1 (NF-κB-mediated inflammatory signaling). 118 FDA-labeled drugs in five human exposure relevant concentrations were evaluated for reporter activation using live cell confocal imaging. Quantitative data analysis revealed activation of single or multiple reporters by most drugs in a concentration and time dependent manner. Hierarchical clustering of time course dynamics and refined single cell analysis allowed the allusion of key events in DILI liability. Concentration response modeling was performed to calculate benchmark concentrations (BMCs). Extracted temporal dynamic parameters and BMCs were used to assess the predictive power of sub-lethal adaptive stress pathway activation. Although cellular adaptive responses were activated by non-DILI and severe-DILI compounds alike, dynamic behavior and lower BMCs of pathway activation were sufficiently distinct between these compound classes. The high-level detailed temporal- and concentration-dependent evaluation of the dynamics of adaptive stress pathway activation adds to the overall understanding and prediction of drug-induced liver liabilities.

Keywords: Adaptive stress pathways; DILI; DILI prediction; High content imaging; Live cell imaging.

MeSH terms

  • Chemical and Drug Induced Liver Injury / etiology*
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • Dose-Response Relationship, Drug
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Hep G2 Cells
  • Humans
  • Intercellular Adhesion Molecule-1 / genetics
  • Intercellular Adhesion Molecule-1 / metabolism
  • Models, Biological*
  • Oxidoreductases Acting on Sulfur Group Donors / genetics
  • Oxidoreductases Acting on Sulfur Group Donors / metabolism
  • Single-Cell Analysis
  • Stress, Physiological / drug effects
  • Stress, Physiological / physiology*
  • Support Vector Machine
  • Transcription Factor CHOP / genetics
  • Transcription Factor CHOP / metabolism


  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • DDIT3 protein, human
  • ICAM1 protein, human
  • Intercellular Adhesion Molecule-1
  • Transcription Factor CHOP
  • Green Fluorescent Proteins
  • Oxidoreductases Acting on Sulfur Group Donors
  • SRXN1 protein, human