title: 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.
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
authors.forename: Steven
authors.initials: S
authors.lastname: Wink
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
authors.forename: Steven
authors.initials: S
authors.lastname: Hiemstra
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
authors.forename: Bram
authors.initials: B
authors.lastname: Herpers
authors.affiliation: Division of Toxicology, Leiden Academic Centre for Drug Research, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands. b.water@lacdr.leidenuniv.nl.
authors.forename: Bob
authors.initials: B
authors.lastname: van de Water
mesh_terms: D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology*; D022202:Chromosomes, Artificial, Bacterial; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects; D006801:Humans; D057054:Molecular Imaging / Q000379:methods*; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D013312:Stress, Physiological / Q000187:drug effects / Q000235:genetics; D018675:Toxicity Tests / Q000379:methods*; D056811:Unfolded Protein Response / Q000187:drug effects / Q000235:genetics
chemical_list: D049452:Green Fluorescent Proteins
keywords: Adaptive stress signaling; DILI; High-content imaging