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.

Emerging metrology for high-throughput nanomaterial genotoxicology

“…All these assays have been successfully applied in genetic toxicology ( in vitro and in vivo ), population biomonitoring, eco-toxicology, DNA damage and repair research and/or in cancer risk assessment for potential chemical and biological genotoxins. One of the most recent additions to the current suite of genotoxicity assays is the ToxTracker reporter assay (43). This assay provides a distinct, alternative measurement of genetic damage by identifying cell signalling pathways that are activated following chemical exposure.…”

A novel toxicogenomics-based approach to categorize (non-)genotoxic carcinogens

“…Furthermore, another group of chemicals, comprising DEM, MEN, but also the non-genotoxic carcinogens LAC and SAR induced a similar gene expression response. These four chemicals have in common that they all induce oxidative stress (Aragon et al 2006;Beyersmann and Hartwig 2008;Hendriks et al 2012;Yamauchi et al 2011). In both cell lines, we detected three out of these four substances as chemicals with a common gene expression pattern: LAC, SAR, and DEM were recognized in PMH, whereas DEM, MEN, and SAR were detected in mESC.…”

Webinar | Getting the best data from your cells: From tissue culture to final analysis

“…Whole genome transcription data of mES cells after exposure to various genotoxic and non-genotoxicc arcinogens clearly demonstrate thed ifferential responsiveness of the selected biomarker genes( Figure 1B) ( Hendriks et al, 2012, Schaap et al, 2014. Bscl2 and Rtkn are preferentially induced by DNA damaging agents, while Srxn1 and Blvrb are induced upon oxidative stress.…”