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

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.Electronic supplementary materialThe online version of this article (10.1007/s00204-018-2178-z) contains supplementary material, which is available to authorized users.

High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability

“…Next, we systematically determined the GFP reporter activation in individual spheroids for all DILI compounds in the six reporter cell lines. An increase of GFP reporter activity was observed for various DILI compounds, also those for which responses in 2D HepG2 have so far not been observed (Wink et al 2018). Some variability in responses between spheroids was evident for different reporters, indicating the importance to include multiple spheroids in the analysis (Fig.…”

“…To further study the applicability of the 3D BAC-GFP reporter cell lines in drug safety assessment screening, a systemic analysis of in total 33 drugs was performed with known DILI liabilities: 20 drugs categorized as most-DILIconcern drugs, 7 drugs with less-DILI concern, and 6 drugs with no-DILI-concern drugs were selected (Supplemental Table 6). These compounds were selected based on limited sensitivity in our 2D HepG2 reporter study (Wink et al 2018). The evaluation was conducted with six GFP reporter cell lines: Srxn1-GFP, Nqo1-GFP, p21-GFP, Btg2-GFP, BIP-GFP, and Chop-GFP.…”

“…Using bacterial artificial chromosome recombineering technology (Poser et al 2008), we tagged individual components of three different stress response pathways with green fluorescent protein (GFP): oxidative stress, unfolded protein response and DNA damage (Wink et al 2016). Using high-content live cell confocal microscopy, we can quantitatively evaluate the dynamics of the activation of individual stress response pathway components at the individual cell level (Wink et al 2016(Wink et al , 2018.…”

“…We have used the HepG2 cell line to establish the BAC-GFP reporter platform. These reporters show excellent performance in detecting mode-of-action of stress response activation (Wink et al 2018). A caveat of HepG2 cells cultured in a conventional 2D monolayer is the high proliferative capacity and a dedifferentiated phenotype (LeCluyse et al 2012).…”

Screening of Cellular Stress Responses Induced by Ambient Aerosol Ultrafine Particle Fraction PM0.5 in A549 Cells

“…Therefore, they constitute an ideal first-line set of parameters suitable for identification of in vitro toxicity. Determination of stress markers in the cells exposed to xenobiotics such as drugs, environmental contaminants, or their mixtures is a novel approach in in vitro toxicity profiling [34][35][36].…”