title: The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals.
abstract: People are exposed to an ever-increasing number of chemical compounds that are developed by industry for a wide range of applications. These compounds may harmfully react with different cellular components and activate specific defense mechanisms that provide protection against the toxic, mutagenic, and possibly oncogenic consequences of exposure. Monitoring the activation of specific cellular signaling pathways upon exposure may therefore allow reliable and mechanism-based assessment of potential (geno)toxic properties of chemicals, while providing insight into their primary mode of toxicity. By whole-genome transcription profiling of mouse embryonic stem cells, we identified genes that were transcriptionally activated upon exposure to either genotoxic compounds or pro-oxidants. For selected biomarker genes, we constructed reporters encoding C-terminal green fluorescent protein (GFP)-tagged fusion proteins. GFP reporter genes were located on bacterial artificial chromosomes, thereby enabling transcriptional regulation of the reporters by their own physiological promoter. The Bscl2-GFP reporter is selectively activated after exposure to genotoxic agents and its induction is associated with inhibition of DNA replication and activation of the ataxia telangiectasia and Rad3-related protein signaling pathway. The Srxn1-GFP reporter is preferentially induced upon oxidative stress and is part of the nuclear factor (erythroid-derived 2)-like 2-antioxidant response pathway. The novel (geno)toxicity assay (ToxTracker) that utilize the differential responsiveness of various reporter cell lines will enable prediction of the primary reactive properties of known and unknown chemicals.
authors: Department of Toxicogenetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands.
authors: Giel
authors: G
authors: Hendriks
authors: Mirna
authors: M
authors: Atallah
authors: Bruno
authors: B
authors: Morolli
authors: Fabienne
authors: F
authors: Calléja
authors: Nienke
authors: N
authors: Ras-Verloop
authors: Ilse
authors: I
authors: Huijskens
authors: Martine
authors: M
authors: Raamsman
authors: Bob
authors: B
authors: van de Water
authors: Harry
authors: H
authors: Vrieling
mesh_terms: D000818:Animals; D015415:Biomarkers; D015153:Blotting, Western; D002460:Cell Line; D004249:DNA Damage; D053595:Embryonic Stem Cells; D005434:Flow Cytometry; D044388:GTP-Binding Protein gamma Subunits; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics*; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000737:chemistry / Q000633:toxicity*; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015203:Reproducibility of Results; D014162:Transfection
chemical_list: D015415:Biomarkers; C115783:Bscl2 protein, mouse; D044388:GTP-Binding Protein gamma Subunits; D009153:Mutagens; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C529381:sulfiredoxin protein, mouse; D020962:Heterotrimeric GTP-Binding Proteins