Abstract
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.
Highlights
Despite major efforts to understand and predict drug-induced liver injury (DILI), unpredicted liver failure upon drug use remains an important adverse drug reaction both in the clinic and during drug development (Raschi and de Ponti 2015).Steven Wink, Steven W
Cellular adaptive responses were activated by non-DILI and severe-DILI compounds alike, dynamic behavior and lower benchmark concentrations (BMCs) of pathway activation were sufficiently distinct between these compound classes
We have established a panel of fluorescent protein reporter liver cell lines based on transgenomics GFP tagging, that capture each of these four pathways using Srxn1, C/EBP homologous protein (CHOP), p21 and intercellular adhesion molecule 1 (ICAM1) as quantitative biomarkers (Wink et al 2017)
Summary
Despite major efforts to understand and predict drug-induced liver injury (DILI), unpredicted liver failure upon drug use remains an important adverse drug reaction both in the clinic and during drug development (Raschi and de Ponti 2015).Steven Wink, Steven W. Gene expression analysis has contributed significantly to our understanding of DILI (Laifenfeld et al 2014; Raschi and De Ponti 2017). This has led to the identification of specific signaling pathways that are activated during DILI and are possibly predictive for chemical-induced liver injury. Key among these are classic stress responses activated to maintain cellular homeostasis, including the oxidative stress response, the endoplasmic reticulum (ER) stress response, the DNA damage response (Laifenfeld et al 2014) and the TNF signaling pathway (Chen et al 2015). We have established a panel of fluorescent protein reporter liver cell lines based on transgenomics GFP tagging, that capture each of these four pathways using Srxn, CHOP, p21 and ICAM1 as quantitative biomarkers (Wink et al 2017)
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