The extended ToxTracker assay discriminates between induction of DNA damage, oxidative stress and protein misfolding

Hendriks, G.; Derr, R.S.; Misovic, B.; Morolli, B.; Calleja, F.M.G.R.; Vrieling, H.

doi:doi:10.1093/toxsci/kfv323

Chemical exposure of cells may damage biomolecules, cellular structures and organelles thereby jeopardising cellular homeostasis. A multitude of defence mechanisms have evolved that can recognise specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two GFP reporters specific for DNA damage and oxidative stress, respectively. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signalling pathways. This panel of six GFP reporters is able...Show moreChemical exposure of cells may damage biomolecules, cellular structures and organelles thereby jeopardising cellular homeostasis. A multitude of defence mechanisms have evolved that can recognise specific types of damaged molecules and will initiate distinct cellular programs aiming to remove the damage inflicted and prevent cellular havoc. As a consequence, quantitative assessment of the activity of the cellular stress responses may serve as a sensitive reporter for the induction of specific types of damage. We have previously developed the ToxTracker assay, a mammalian stem cell-based genotoxicity assay employing two GFP reporters specific for DNA damage and oxidative stress, respectively. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signalling pathways. This panel of six GFP reporters is able to discriminate between different primary reactivity of chemicals being their ability to react with DNA and block DNA replication, induce oxidative stress, activate the unfolded protein response or cause a general P53-dependent cellular stress response. Extensive validation using the ECVAM-suggested compound library and a large panel of reference chemicals shows that the ToxTracker assay has an outstanding sensitivity and specificity. In addition, we developed Toxplot, a dedicated software tool for automated data analysis and graphical representation of the test results. Rapid and reliable identification by the ToxTracker assay of specific biological reactivity can significantly improve in vitro human hazard assessment of chemicals.Show less

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The extended ToxTracker assay discriminates between induction of DNA damage, oxidative stress and protein misfolding