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Arch Toxicol. 2019 Aug 27. doi: 10.1007/s00204-019-02552-0. [Epub ahead of print]

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

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Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.
Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
Department of Toxicogenomics, GROW School of Oncology and Development Biology, Maastricht University, Maastricht, The Netherlands.
Department of Pharmacokinetics, Dynamics and Metabolism, Janssen Pharmaceutical Companies of Johnson & Johnson, Beerse, Belgium.
Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.


Adaptive stress response pathways play a key role in the switch between adaptation and adversity, and are important in drug-induced liver injury. Previously, we have established an HepG2 fluorescent protein reporter platform to monitor adaptive stress response activation following drug treatment. HepG2 cells are often used in high-throughput primary toxicity screening, but metabolizing capacity in these cells is low and repeated dose toxicity testing inherently difficult. Here, we applied our bacterial artificial chromosome-based GFP reporter cell lines representing Nrf2 activation (Srxn1-GFP and NQO1-GFP), unfolded protein response (BiP-GFP and Chop-GFP), and DNA damage response (p21-GFP and Btg2-GFP) as long-term differentiated 3D liver-like spheroid cultures. All HepG2 GFP reporter lines differentiated into 3D spheroids similar to wild-type HepG2 cells. We systematically optimized the automated imaging and quantification of GFP reporter activity in individual spheroids using high-throughput confocal microscopy with a reference set of DILI compounds that activate these three stress response pathways at the transcriptional level in primary human hepatocytes. A panel of 33 compounds with established DILI liability was further tested in these six 3D GFP reporters in single 48 h treatment or 6 day daily repeated treatment. Strongest stress response activation was observed after 6-day repeated treatment, with the BiP and Srxn1-GFP reporters being most responsive and identified particular severe-DILI-onset compounds. Compounds that showed no GFP reporter activation in two-dimensional (2D) monolayer demonstrated GFP reporter stress response activation in 3D spheroids. Our data indicate that the application of BAC-GFP HepG2 cellular stress reporters in differentiated 3D spheroids is a promising strategy for mechanism-based identification of compounds with liability for DILI.


BAC-reporter cells; Cellular stress response; Drug-induced liver injury; HepG2 spheroids; High-throughput imaging; Liver transcription factors


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