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Updated: Mar 28, 2026
Assays for the Degradation of Misfolded Proteins in Cells
Published on: August 28, 2016
The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding
Giel Hendriks1, Remco S Derr2, Branislav Misovic2
1Department of Human Genetics, Leiden University Medical Center, 2300RC Leiden, The Netherlands g.hendriks@lumc.nl.
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.
Insights
The expanded ToxTracker assay uses six reporter cell lines to detect chemical damage, improving cellular stress response assessment. This enhanced genotoxicity test offers sensitive and specific results for chemical hazard identification.
Area of Science:
- Cellular Biology
- Toxicology
- Biochemistry
Background:
- Chemical exposure can disrupt cellular homeostasis by damaging biomolecules and organelles.
- Cellular defense mechanisms have evolved to detect and repair molecular damage, preventing cellular dysfunction.
- Quantifying cellular stress responses provides a sensitive method for identifying specific chemical-induced damage.
Purpose of the Study:
- To expand the ToxTracker assay with additional reporter cell lines for broader chemical reactivity monitoring.
- To develop a comprehensive panel of reporters for diverse cellular stress pathways.
- To enhance the in vitro human hazard assessment of chemicals through improved reactivity identification.
Main Methods:
- Development of four new reporter cell lines to complement the existing ToxTracker assay.
- Utilizing a panel of six green fluorescent protein reporters to monitor DNA damage, oxidative stress, unfolded protein response, and P53-dependent stress.
- Validation using the European Centre for the Validation of Alternative Methods (ECVAM) compound library and reference chemicals.
- Development of Toxplot software for automated data analysis and visualization.
Main Results:
- The expanded ToxTracker assay effectively discriminates between various chemical reactivities, including DNA reactivity, DNA replication blockage, oxidative stress induction, unfolded protein response activation, and P53-dependent stress.
- Extensive validation demonstrated outstanding sensitivity and specificity of the assay.
- The Toxplot software facilitates rapid and reliable data interpretation.
Conclusions:
- The enhanced ToxTracker assay provides a sensitive and specific tool for identifying distinct chemical-induced cellular stress responses.
- This assay significantly improves the in vitro human hazard assessment of chemicals by enabling rapid and reliable identification of specific biological reactivity.
- The integrated approach of reporter cell lines and dedicated software enhances the efficiency and accuracy of toxicological evaluations.
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