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.

Keywords: DNA damage response; genotoxicity; mechanisms of toxicity; oxidative stress; reporter cell lines.

FIG. 1.

Biomarkers for cancer-associated biological damages. A, Overview of the three major types of biological damage and the respective cellular consequences that have been associated with increased carcinogenicity hazard. B, Heatmap indicating the preferential induction of six selected biomarker genes at 8 h after exposure to various genotoxic and nongenotoxic carcinogens as determined by whole-genome transcription profiling of mouse embryonic stem cells.

FIG. 2.

Selective activation of the ToxTracker reporter cell lines. A, green fluorescent protein (GFP) reporter cells were exposed to increasing concentrations of the DNA damaging agents cisplatin and etoposide, the alkylating agent methyl methanesulfonate (MMS), the oxidative stress-inducing agent diethyl maleate (DEM), and the unfolded protein response-activating compounds tunicamycin and nitrophenol. GFP induction levels in intact cells were determined by flow cytometry at 24 h after initiation of the exposure. B, Cell survival was determined by flow cytometry after 24-h exposure as the relative decrease in cell concentration compared with untreated controls. C, Reporter cell lines were exposed to 10 μM cisplatin in the presence of an increasing concentration of the NF-κB inhibitor BMS-345541.

FIG. 3.

Validation of the differential activation of the ToxTracker reporter cell lines. A, Induction levels of the different GFP reporters were calculated for all compounds at an equitoxic concentration that induced 50% cytotoxicity. GFP levels were determined by linear regression of the GFP induction data points of the two doses encompassing 50% cytotoxicity. B, Induction of the GFP reporter upon exposure to a selection of ECVAM-recommended carcinogens that are established positives in an in vitro genotoxicity assay. Cisplatin (red), diethyl maleate (blue), and tunicamycin (green) are considered as positive controls for induction of DNA damage, oxidative stress and activation of the unfolded protein response and color-coded in all heatmaps and graphs. C, Induction of ToxTracker reporters following exposure to a selection of ECVAM-recommended noncarcinogens that are established negatives in an in vitro genotoxicity assay. D, Activation of ToxTracker reporters after exposure to ECVAM-recommended compounds that are noncarcinogens or nongenotoxic carcinogens but that occasionally scored positive in a conventional in vitro/in vivo genotoxicity test.

FIG. 4.

Extended validation of the GFP reporter cell lines. A, ToxTracker reporter cell lines were exposed to a collection of reference nongenotoxic carcinogens. GFP induction levels were calculated for a compound concentration that induce 50% cytotoxicity. Cisplatin, DEM, and tunicamycin were used as positive controls for the induction of DNA damage, cellular oxidative stress, and protein damage, respectively. B, Induction of the GFP reporters following exposure to a selection of established genotoxic and nongenotoxic chemicals.

FIG. 5.

Specificity of the GFP reporters is largely unaffected by cytotoxicity. A, GFP reporter induction levels were calculated at compound concentrations that induce 10%, 25%, 50%, or 75% cytotoxicity using the panel of ECVAM-selected genotoxic carcinogens. Clustering of compounds was based on the similarity in reporter activation at 50% cytotoxicity. B, Kinetics of GFP induction was determined by flow cytometric analysis of intact cells following 4-, 8-, 12-, 16-, and 24-h exposure to the DNA damaging agents cisplatin and doxorubicin, the microtubule disrupting agent taxol and the control compounds DEM and tunicamycin for induction of oxidative stress and protein unfolding, respectively.

FIG. 6.

Differential responses of the BSCL2 and RTKN-GFP reporters discriminate between clastogenic and aneugenic compounds. A, ToxTracker reporter cell lines were exposed to a selection of 11 established microtubule disrupting agents. GFP induction levels were determined after 24-h exposure by flow cytometry. B, Kinetics of BSCL2-GFP and RTKN-GFP following exposure to the DNA damaging compounds cisplatin, etoposide, mitomycin C, and doxorubicin or the mitotic spindle poisons colcemid, nocodazole, vinorelbine and taxol. GFP induction was determined after 4-, 8-, 12-, 16-, and 24-h exposure. C, Exposure times that resulted in a 1.5-fold increase in GFP signal for the BSCL2-GFP and RTKN-GFP reporters after exposure to the tested clastogenic and aneugenic compounds were calculated by linear regression of the GFP induction data points of the two time points encompassing 1.5-fold induction.