High-content imaging-based BAC-GFP toxicity pathway reporters to assess chemical adversity liabilities

Abstract

Adaptive cellular stress responses are paramount in the healthy control of cell and tissue homeostasis and generally activated during toxicity in a chemical-specific manner. Here, we established a platform containing a panel of distinct adaptive stress response reporter cell lines based on BAC-transgenomics GFP tagging in HepG2 cells. Our current panel of eleven BAC-GFP HepG2 reporters together contains (1) upstream sensors, (2) downstream transcription factors and (3) their respective target genes, representing the oxidative stress response pathway (Keap1/Nrf2/Srxn1), the unfolded protein response in the endoplasmic reticulum (Xbp1/Atf4/BiP/Chop) and the DNA damage response (53bp1/p53/p21). Using automated confocal imaging and quantitative single-cell image analysis, we established that all reporters allowed the time-resolved, sensitive and mode-of-action-specific activation of the individual BAC-GFP reporter cell lines as defined by a panel of pathway-specific training compounds. Implementing the temporal pathway activity information increased the discrimination of training compounds. For a set of >30 hepatotoxicants, the induction of Srxn1, BiP, Chop and p21 BAC-GFP reporters correlated strongly with the transcriptional responses observed in cryopreserved primary human hepatocytes. Together, our data indicate that a phenotypic adaptive stress response profiling platform will allow a high throughput and time-resolved classification of chemical-induced stress responses, thus assisting in the future mechanism-based safety assessment of chemicals.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1781-0) contains supplementary material, which is available to authorized users.

Unraveling cellular pathways contributing to drug-induced liver injury by dynamical modeling

“…As a first approach, the activation of all stress pathways upon exposure to a specific drug can be measured in vitro to determine which stress pathway models to use for this drug. This can for example be done in cell lines with built-in fluorescent reporters for the sensor, transcription factor, and downstream targets during live microscopy imaging [11]. For example, Wink et al showed that known DILI drugs are frequently stress pathway specific, but some induce multiple stress pathways at a molecular level.…”

“…This computational approach potentially offers a mechanism-based tool towards predictive toxicology to understand and eventually to reduce the occurrence of DILI. For illustration purposes, we include a 2D representation of cells in a single microscopy image reproduced from [11] under the terms of the Creative Commons Attribution License.…”

“…Currently available methods include the use of reporter cell lines, in which the stress or cell death pathways can be monitored over time. In contrast to endpoint assays, live confocal microscopy gives the opportunity to measure single-cell behavior over time [11].…”

“…These key events can occur on different biological levels (subcellular, cellular, tissue, organ, individual, and population level) ( Figure 1, bottom with a microscope image reproduced from [11]). Adverse outcomes in DILI include clinical observations such as liver fibrosis, liver steatosis, and cholestatis.…”

Highlight report: ‘Big data in the 3R’s: outlook and recommendations’, a roundtable summary

“…Through fluorescent protein genetic engineering approaches such as BAC-GFP transgenomics and CRISPR/Cas9 technology, the behaviour of relevant intracellular molecular markers derived from gene expression data can be followed in individual cells over time. With the advantage of high-throughput microscopy technology we can now monitor the dynamics of pathway activation at single cell level in hundred parallel treatment conditions with hundreds of cell per conditions (Wink et al 2017). Parallel perturbation conditions allow the fast screening of temporal concentration responses of thousands of compounds or the application of RNA interference screening to identify relevant signalling molecules that determine the pathway activation in connection to the adverse outcome.…”