MutaTracker
MutaTracker is a unique mammalian in vitro assay that predicts mutagenicity and genotoxicity of compounds and chemicals with exceptional accuracy and sensitivity. It seamlessly combines ToxTracker, a stem cell-based reporter assay that discriminates between direct and secondary genotoxicity mechanisms such as aneugenicity and oxidative stress, with single-molecule mutation sequencing (SMM-seq), which detects gene mutations at ultra-low frequencies. Together, the two methods offer unmatched insights into mutagenic properties, covering all genotoxicity endpoints in one assay, delivering fast results. MutaTracker provides a single workflow that captures genotoxicity at single nucleotide resolution, thanks to our partnership with:
Key Features
- Unmatched accuracy in genotoxicity prediction
- Mutations detected: as low as 1 in 107 bases
- Genotoxic and mutagenic mode-of-action (MoA)
- All genotoxicity endpoints captured in one assay
MutaTracker is an all-in-one mammalian cell-based genotoxicity and mutagenicity assay and combines ToxTracker with SMM-seq. ToxTracker is used to detect the genotoxic potential of substances and provides insight into the mode of action. SMM-seq is used to determine the mutagenic potential of substances.
ToxTracker
ToxTracker combines six fluorescent reporter genes that are specifically activated by different cellular signaling responses associated with genotoxicity and carcinogenicity. It includes markers not only for DNA damage but also for non-genotoxic MoA, including oxidative stress, protein misfolding, and general cellular stress. Activation of the different GFP-reporters is determined by flow cytometry.
SMM-seq
SMM-seq is an error-corrected next-generation sequencing (ecNGS) technique that uses rolling circle amplification (RCA): hairpin-like adapters are ligated to each end of a double-stranded DNA fragment forming a circle, multiple independent copies of each strand are generated, a true mutation appears on every copy made from the parental strands, while others are marked as artifacts with the highest confidence.
MutaTracker protocol
First, a dose range finding is performed to determine the relevant concentrations for ToxTracker and SMM-seq. Next, the 6 reporter cell lines are exposed to 5 concentrations for 24h in the absence or presence of rat S9 liver extract. GFP reporter induction is assessed by flow cytometry. The differential activation of the reporter genes indicates the genotoxic MoA. For SMM-seq, ToxTracker cells are exposed to 3 concentrations of the test substance for 4h. Cells are harvested for DNA isolation after 5 days. DNA is fragmented, ligated with a custom adaptor, amplified by RCA, indexed, and sequenced. The consensus sequence, mutation frequency, and mutation spectrum are then determined for mutagenic MoA.
Assay readouts with known substances and representative data
Mutagenic substances (clastogen, aneugen, and oxidizing agent) are tested at different concentrations. Differential activations of the six ToxTracker reporters predict their genotoxic MoAs and the mutational spectra from SMM-seq provide their mutagenic signatures.
Project report
The project report contains the protocol, technical details, cytotoxicity testing/dose range finding results, biomarker expression with test compounds, morphological analysis with test compounds, Lowest Observed Adverse Effect Level (LOAEL) concentrations of the compounds, and mutation spectra. We are happy to share a sample report with you. Please click here to request a sample report.
Dissemination meeting
Upon sharing the draft report, we will invite you for a dissemination meeting where the study director will go over the conclusions in the report. We will make sure you understand the results and provide our expertise on the results and potential next steps.
Flyers
What endpoints are reported?
MutaTracker combines the six ToxTracker endpoints for genotoxicity with the mutagenicity endpoints from SMM-seq. ToxTracker consists of two biomarkers (Bscl2/Rtkn) for direct genotoxicity and four biomarkers (Srxn1/Blvrb/Btg2/Ddit3) for non-genotoxic effects that can indirectly cause DNA damage or can cause misleading positive results in the standard battery of in vitro genotoxicity assay. SMM-seq reports the mutation frequency and the single base mutation spectrum to determine the mutagenicity of compounds.
How do you classify compounds as genotoxic/mutagenic?
Substances are classified as genotoxic in ToxTracker if one or more of the genotoxicity markers (Bscl2 and/or Rtkn) are induced 2-fold. Induction of oxidative stress (Srxn1/Blvrb), cytotoxicity/apoptosis and p53 activation (Btg2), and protein damage (Ddit3) can explain indirect genotoxicity or misleading positive results in the standard battery of in vitro genotoxicity assay. Substances are considered mutagenic using SMM-seq when the mutation frequency is significantly increased compared to the vehicle control.
Can you test N-nitrosamines in MutaTracker?
Yes, N-nitrosamines can be tested to determine mutagenicity in vitro. See the poster for an example of N-nitrosamines tested in MutaTracker.
How much compound is needed to perform the assay?
This depends on the compound and maximum concentration you wish to test. Usually, 15 mg is sufficient for the test. This can be either dry powder material or a solution.
How long does it take to receive a report?
Depending on the size of the project, it takes approximately 6-8 weeks before delivery of a final report. Please contact us in case you have specific requirements, for example for large size projects.
What type of solvent can be used?
The type of solvent shall be agreed upon before the start of the project. Typically, DMSO or water is used but other solvents are possible.
Can you test with metabolic activation?
Yes, we can add S9 liver extract to our assays for drug metabolism.
What is the top-concentration that is tested, and how is this determined?
The top concentration is determined based on the cytotoxicity of the compound. For ToxTracker, the top concentration induced at least 75% cytotoxicity in the dose-range finding. For SMM-seq, the top concentration induced approximately 50% cytotoxicity in the dose-range finding. When there is limited cytotoxicity, the maximum test concentration will be used. When solubility is limiting, the maximum soluble concentration will be used.
How many concentrations are tested?
For ToxTracker, typically 5 concentrations are tested in 2-fold dilutions. For MutaTracker, 3 concentrations are tested in 2-fold dilutions. Please contact us in case you would like to customize the protocol.
Can you test autofluorescent materials?
Autofluorescence does not interfere with the SMM-seq but can cause problems in ToxTracker. Usually, it is possible to test autofluorescent materials. In our standard protocols we run along a non-GFP cell line to check for autofluorescence. Usually, we can correct for the autofluorescence, however occasionally the autofluorescence is so strong that it interferes with the flow-cytometry measurements. If this is picked up in the dose finding study, we will revert to other technologies to measure reporter induction.
What cell type is used for the assay?
MutaTracker combines ToxTracker and SMM-seq and therefore uses the same mouse embryonic stem cells for SMM-seq that are used for our ToxTracker assay.
Can you share a sample report?
An example report is available, please contact us via the contact form or info@toxys.com for more information.
Service
We perform MutaTracker as a service for our clients. We work together on this basis with various of the top 10 pharma, chemical and cosmetics companies. We act as an extension of your R&D team by building a scientific case and thinking along with you so that you will receive a solution that goes beyond a “yes or no” answer. We advise you on what can be done to provide further evidence on the mode-of-action for your compound.
You can send your compounds and receive a full report within 8 weeks. Are you interested in receiving a quote or do you have any questions? Please reach out!
Practical information
- MutaTracker is available as a service from Toxys and Mutagentech
- The turnaround time is 6-8 weeks
- Compound requirement: 10-15 mg
- Type of solvents compatible with the assay: DMSO, PBS, Water
- Types of materials: pharmaceuticals, (agro)chemicals, cosmetics, and food ingredients
Meet the study director for MutaTracker
Our study directors are the experts in the field to whom you can ask any question about our assays. From early screening to regulatory safety assessment, our study directors are able to listen to your questions and think along with you to provide a tailored solution. Here are some typical questions they often receive on MutaTracker in understanding where and how MutaTracker can be utilized in their strategies. Please feel free to ask your questions as well.
Can I meet you to talk about my study design?
Click for the answer
Will you also take me through the data after a project in a TC or meeting?
Click for the answer
dr. Marit Hoogenboom-Geijer
Study director for MutaTracker
Marit obtained her PhD from the Erasmus Medical Center in Rotterdam where she focused on DNA damage repair and the transcription stress response. Marit joined Toxys as a scientist, using her expertise to further develop and implement novel toxicity assays and launch TubulinTracker. Marit focused on adding error-corrected next generation sequencing to our analysis pipeline to further understand the mutagenicity of genotoxic substances.
Can you help with a study design for difficult to dissolve compounds?
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Do you also have a question? Click here to send a question to Marit.
