We have developed several extensions to the standard ToxTracker protocol that can be applied to further assess the underlying mode-of-action of genotoxic and non-genotoxic compounds. Protocols are available for separating clastogens from aneugens and identification of kinase inhibitors. ToxTracker can be combined with conventional genotoxicity endpoints by measuring induction of gene mutations and chromosome damage in the reporter cell lines. To assess the mechanisms of oxidative stress, we can analyse the production of ROS and contribution of oxidative stress to the genotoxic and cytotoxicity profile of a compound. In addition, we offer various assays to assess the involvement of mitochondrial damage in oxidative stress.
- Unravel the mechanism of toxicity
- Pick and chose the appropriate application as follow-up from ToxTracker results
- Classify Clastogens from Aneugens
- Unravel mechanisms of oxidative stress
- Identification of kinase inhibitor
In case of a positive ToxTracker result, we can follow this up with several additional protocols to further unravel the underlying mechanism of action of genotoxic compounds. This table provides an overview of the additions we offer under ToxTrackerPlus.
Clastogens vs Aneugens
By using a kinetics protocol we are able to separate clastogens from aneugens, please read more under the tab Clastogens vs Aneugens.
We have 2 additional protocols available, one to investigate whether oxidative stress is directly caused by increase levels of reactive oxygen species (ROS) and one to investigate if mitochondria are causing the oxidative stress. Please read more under the tab oxidative stress.
Kinase inhibitors protocols
We offer a combination of 3 additional protocols that help assess whether the your compound is a kinase inhibitors which cause DNA damage (and are likely to be positive for cancer hazard) and predict whether they will give a positive micronucleus result. Please read more under the tab kinase inhibitors.
The mouse ES cells that are applied in the ToxTracker assay have been extensively used to study mutagenesis. The hprt, aprt and tk genes are typically used to study DNA damage-induced mutations. We can extend the ToxTracker assay to allow detection of promutagenic DNA lesions with the quantitative assessment of mutation induction following chemical exposure. Read more
We can use our ToxTracker cell lines for a number of standard genotoxicity assays to identify chromosomal damage. We can perform the following assays:
For assessment of a potential aneugenic mechanism of action, the kinetics of genotoxicity reporters (Bscl2-GFP and Rtkn-GFP) induction is determined. Aneugenic compounds selectively induced the Rtkn-GFP DNA strand break reporter. Furthermore, compounds with an aneugenic MOA show a significant delay in kinetics of reporter induction compared to clastogenic compounds. In the kinetic ToxTracker study we will measure reporter induction in multiple time points to confirm a non-DNA reactive, aneugenic mode-of-action. We have previously published this ability in this paper.
Validation study to separate clastogens and aneugens
In our validation study we have tested mutiple clastogens, compounds that form bulky DNA adducts or DNA crosslinkers and aneugens such as microtubule disruptors. Figure 1. demonstrates a number of the compounds we have tested and their reporter induction.
When we follow-up with kinetic studies we can differentiate between aneugens and clastogens as demonstrated in figure 2.
The assay is currently being used in a commercial setting. Please contact us with any questions on this protocol or to discuss how this assay can be applied for your compounds or could fit in your screening strategy.
We offer 2 types of studies to follow-up or further investigate oxidative stress induced by compounds.
Classifying if mode of action is cause by oxidative stress
One of the first questions one might have when there are high levels of oxidative stress and low levels of DNA damage (for example in ToxTracker) is whether oxidative stress is the primary mode of action and cause for cytotoxicity or whether there might be a dual mode of action.
To answer this questions we have created additional protocols to the standard ToxTracker. We will then introduce a ROS scavenger and measure the GFP induction for the DNA damage markers and oxidative stress markers. By introducing the ROS scavenger we expect to see a low induction of the oxidative stress markers. With this lower induction we assess the cytotoxicity and DNA damage marker induction to understand the contribution of the oxidative stress to the toxicity profile.
Case study: assesing mode of action for MMS
In this case study we started by determining the profile of MMS in the standard ToxTracker protocol (see fig 1 for results). We found that MMS gave high levels of oxidative stress, but also induced both the DNA damage markers.
Fig.1.: ToxTracker results for MMS
To follow-up whether oxidative stress was the primary mode of action causing the cytotoxicity we added the ROS scavenger NAC (write out) to the extended assay. Subsequently we measured induction of the oxidative stress reporters and DNA damage reporters (and cytotoxicity).
We found that introduction of NAC results in a significantly lower induction of oxidative stress, as expected, however we also found the same level of cytotoxicity and DNA damage. From this we concluded that the DNA damage is a secondairy mode of action next to the induction of oxidative stress. The DNA damage actually was causatative for the cytotoxicity.
Asses the role of Mitochondria in oxidative stress
We have formed a strategic partnership with Mitologics. Mitologisch has proprietary technology to assess the ROS formation by mitochondria. In this unique partnership we can assess the role of mitochondria in the ToxTracker cell lines. Click here for more information on this application.
How to access
We can offer these additional assays as fee for service. Please contact us to discuss how this can be applied for your compounds.
Pharmaceutical compounds that target a specific therapeutic kinase in the cell sometimes also affect the activity of other kinases. The Aurora kinases that play a crucial role in cell division are a known unintended target for many kinase inhibitors. Inhibition of cell cycle checkpoint kinases affect proper progression through mitoses and are thereby inducing the formation of micronuclei and polyploidy in vitro and in vivo, however are generally not carcinogenic.
These (often unanticipated) genotoxic effects are generally seen at much higher concentrations than the therapeutic dosis of the drug. To identify compounds that (unintentionally) affect cell cycle kinases we have developed additional protocols to identify kinase inhibitors.
- We start with a standard ToxTracker analysis. Kinase inhibitors are not DNA reactive and do not activate the Bscl2-GFP and Rtkn-GFP genotoxicity reporters in ToxTracker.
- We perform staining for PhosphoH3 and propidium Iodine staining to establish inhibition of phosphorylation of histone H3 by the Aurora kinases and polyploidy
- We perform a micronucleus assay in the ToxTracker cells to confirm that these compounds give rise to micronuclei
Validation of the protocols for Phospho-H3 and Propidium Iodine staining
We have applied the protocols to 2 known aurora kinase inhibitors: AMG900 and VX680. We exposed the cells to the compounds and controls such as nocodozole (a mitotic spindle poison), cisplatin and etoposide and subsequently stained with an antibody for phospo-H3 positive cells and measured antibody staining by flow-cytometry.
We found a strong decrease of phospho-H3 stained cells when they are exposed to AMG900 and VX680. Subsequently we stained for polyploidy by adding propidium iodide DNA staining and measure staining by flow cytometry.
We found strong induction of Polyploid cell when the cells were exposed to AMG900 and VX680.
Fee for service project
We can perform the assay for you at our state-of-the-art laboratory. You can then send your compounds and receive a full report, in most cases within 2 weeks. Most of our customers ask us to perform the assay as a fee-for-service project as we have all tools and equipment in house and are experienced in the data interpretation. We work together on this basis with various of the top 10 pharma, chemical and cosmetics companies.
These additional protocols are only relevant when you have accessed the ToxTracker technology for in house use. Please contact us if you would like more information on using the additional protocols for in-house use.