title: The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding.
abstract: Chemical exposure of cells may damage biomolecules, cellular structures, and organelles thereby jeopardizing cellular homeostasis. A multitude of defense mechanisms have evolved that can recognize 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 green fluorescent protein reporters specific for DNA damage and oxidative stress. We have now expanded the ToxTracker assay with an additional four reporter cell lines to include monitoring of additional stress signaling pathways. This panel of six green fluorescent protein 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 compound library suggested by the European Centre for the Validation of Alternative Methods (ECVAM) 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.
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands g.hendriks@lumc.nl.
authors.forename: Giel
authors.initials: G
authors.lastname: Hendriks
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors.forename: Remco S
authors.initials: RS
authors.lastname: Derr
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors.forename: Branislav
authors.initials: B
authors.lastname: Misovic
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors.forename: Bruno
authors.initials: B
authors.lastname: Morolli
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors.forename: Fabienne M G R
authors.initials: FM
authors.lastname: Calléja
authors.affiliation: Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors.forename: Harry
authors.initials: H
authors.lastname: Vrieling
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004249:DNA Damage; D053595:Embryonic Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D057166:High-Throughput Screening Assays; D051379:Mice; D009152:Mutagenicity Tests / Q000379:methods*; D018384:Oxidative Stress / Q000187:drug effects* / Q000235:genetics; D017510:Protein Folding / Q000187:drug effects*; D015203:Reproducibility of Results
chemical_list: D015415:Biomarkers; D049452:Green Fluorescent Proteins
keywords: DNA damage response; genotoxicity; mechanisms of toxicity; oxidative stress; reporter cell lines