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

Steven Wink,Bob Van De Water,Steven Hiemstra,Bram Herpers

doi:10.1007/s00204-016-1781-0

Steven Wink, Bob Van De Water + Show 2 more

Open Access

https://doi.org/10.1007/s00204-016-1781-0

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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.

Highlights

In the past decades, hepatic toxicity has contributed disproportionately to drug withdrawals (Stevens and Baker 2009)
We established a panel of fluorescent protein reporter HepG2 cell lines using bacterial artificial chromosome (BAC) cloning technology to follow the dynamics of several adaptive stress response pathways essential in chemical-induced cytotoxicity
We focused on target genes that are central in the regulation of three key adaptive stress response programs; for each pathway, we successfully established reporters for the sensory machinery, downstream transcription factor and one of the transcription factors downstream targets

Summary

Introduction

Hepatic toxicity has contributed disproportionately to drug withdrawals (Stevens and Baker 2009). Transcriptomics has contributed much to our mechanistic understanding and has helped to initiate and populate the adverse outcome pathway (AOP) framework (Ankley et al 2010; Vinken 2013). AOPs are described as a sequential chain of causally linked events at different levels of biological organization that together culminate in the adverse health outcome. While some AOPs have so far been established, a important step is to translate AOP-related mechanistic understanding in advanced, preferably quantitative, high throughput assays that reflect pathways essential in target organ toxicity. Our vision is to establish an imaging-based platform that can quantitatively assess the activation of individual key events relevant to AOPs. Our initial focus is on adaptive stress response pathways, which are typically part of AOPs and related to adverse drug reactions

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