Rationale for developing the assay
Interaction of newly developed materials, chemicals and drugs with biomolecules may disrupt cellular homeostasis and can ultimately lead to severe tissue damage or induction of cancer. Inflicted cellular damage is recognised by specialised sensor proteins that trigger a complex network of cellular signalling pathways resulting in activation or inactivation of specific enzymes and altered expression of distinct gene networks. Therefore, visualization of damage-specific cellular stress response pathways that are activated upon exposure to chemicals or xenobiotics provides insight into the type and extent of cellular damage that has been induced and thus the biological (re)activity of compounds.
Identification of the genes that specifically represent certain types of damage
We have identified a panel of genes that are preferentially activated upon exposure to different classes of carcinogens following extensive whole-genome transcription profiling of mouse embryonic stem (mES) cells after exposure to over 40 different carcinogenic chemicals.
From the genes that were activated following exposure to the chemicals, we have identified the genes that specifically represent DNA damage, oxidative stress and the unfolded protein response, being the major biological damages associated with carcinogenesis. We have identified 6 genes representing these damages very specifically for which we have created green fluorescent mES reporter cell lines, which were combined to form the ToxTracker assay.
Unique biomarkers
The ToxTracker assays consist of 6 reporter cell lines which are developed for unique biomarkers that discriminate between induction of DNA damage, oxidative stress, protein damage and general cellular stress. Genotoxicity is detected by the Bscl2-GFP reporter that is activated by promutagenic DNA lesions and DNA replication stress and the Rtkn-GFP reporter that is associated with DNA double strand breaks. The Srxn1-GFP and Blvrb-GFP reporters indicate activation of the Nrf2 and Hmox1 antioxidant responses. The Ddit3-GFP reporter is directly associated with the unfolded protein response and Btg2-GFP is activated as part of a p53-mediated stress response.
Cell system
ToxTracker consists of a panel of GFP-based reporters in mouse embryonic stem (mES) cells. mES cells are genetically stable and proficient in all cellular pathways required for accurate detection of potentially carcinogenic properties of compounds, in contrast to the cancer-derived cell lines that are currently used for in vitro genotoxicity testing.
Advantage of mouse embryonic stem cells
- Untransformed, non-cancerous mammalian cell line
- Infinite lifespan
- Proficient in all major DNA damage signalling and cell cycle regulation pathways
- Dividing rapidly
Please also see FAQ for questions on the cells used.
