TP MAX name

ToxProfiler™ is a unique human in vitro assay that combines seven stable fluorescent reporter genes and can provide an extensive toxicological profile of novel drugs and chemicals. ToxProfiler MAX (Mitotox Assessment Xtension) is an extended version of the ToxProfiler assay that combines the differential activation of the seven major stress pathway responses of ToxProfiler with mitochondrial toxicity assessment. This assay is a unique and powerful means to discriminate between compounds that directly affect mitochondrial function from those that induce other types of cellular stress and toxicity. The information generated is particularly useful in Weight of Evidence (WoE), read-across, and Adverse-Outcome Pathway (AOP) approaches.

A ToxProfiler MAX assay under a microscope

Key Features

    • Discriminates between direct and indirect mitochondrial toxicants
    • Highly sensitive and specific in detecting mitochondrial toxicity
    • Rapid and cost effective
    • Low amount of compound required

ToxProfiler MAX (Mitochondrial toxicity Assessment Xtension) is an extended version of the ToxProfiler assay, which in addition to oxidative stress, ER stress, cell cycle stress, ion stress, protein stress, autophagy, and inflammation also includes assessment of mitochondrial toxicity. This unique combination identifies the toxicological Mode-of-Action (MoA) and mitochondrial toxicity of novel drugs and chemicals in a single assay. The ToxProfiler MAX is able to discriminate between compounds that directly affect mitochondrial function from those that induce other types of cellular stress (e.g. oxidative stress, autophagy, or ER stress).

A panel of 7 genetically engineered HepG2 cell lines in the ToxProfiler assay with high content images at unstressed and chemically stressed states. Next to these cell lines, there is also a depiction of chemically unstressed and stressed cells in glu/gal medium.

Highlights of ToxProfiler MAX

  • Human fluorescent biomarkers are expressed at physiological levels, reflecting endogenous cellular stress responses.
  • Image-based high throughput screening platform with a single cell resolution (high content imaging).
  • Quantifying stress response signaling and cytotoxicity with point of departure (POD) determination.
  • Quantitatively measure mitochondrial toxicity with high sensitivity and specificity.

Assessment of mitochondrial toxicity in ToxProfiler MAX

ToxProfiler MAX uses the glucose/galactose medium switch principle to assess the mitochondrial toxicity of drugs and chemicals. By replacing the glucose medium with a galactose variant, cells switch their metabolism from glycolysis to oxidative phosphorylation, relying solely on mitochondria for ATP/energy.

Two graphs per ETC complex inhibitors (fenpyroximate, mepronil, and antimycin A). The graphs on top show GFP expression per ToxProfiler MAX reporter and the graphs below show fraction of PI positive cells in increasing concentrations of glucose and galactose medium.ToxProfiler MAX correctly identified mitochondrial toxicity of known ETC complex inhibitors (fenpyroximate, mepronil, and antimycin A). These chemicals induced a higher level of cytotoxicity in the galactose medium when compared to the glucose medium and activated the ToxProfiler reporter for ER stress (CHOP).

Typical applications of the ToxProfiler MAX

  • Early-phase in vitro safety screening: ToxProfiler MAX is a rapid and reliable assay and will provide a detailed and quantitative toxicological profile, which can be used to make informed decisions for the compound selection and development process.
  • Read-across approaches: ToxProfiler MAX provides mechanistic insight into the toxic properties of chemicals that are applied to clustering structures with similar reactivity.
  • Weight-of-evidence approach: ToxProfiler MAX can be applied in screening as a mechanistic follow-up of the regulatory in vitro tests to provide insight into the MoA of compounds.

Meet the study director for ToxProfiler MAX

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 ToxProfiler MAX in understanding where and how ToxProfiler MAX 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?

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Will you also take me through the data after a project in a TC or meeting?

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Dr. Bas ter Braak
Dr. Bas ter Braak
Study director for ToxProfiler

Bas ter Braak obtained his PhD at the Leiden Academic Centre for Drug Research (LACDR).  He co-developed the HepG2 BAC-GFP reporter platform that is central to ToxProfiler. Bas joined Toxys in 2020 as a senior scientist to run ToxProfiler and further expand the assay with his knowledge in compound-induced stress signalling processes in differentiated reporter cell lineages.

Can you help with a study design with different types of solvents?

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Click here to send your question!

The ToxProfiler protocol consists of three steps. First, a broad concentration range-finding is performed to select the compound concentrations that will be applied in the reporter assay. Next, the ToxProfiler cells are exposed to 7 selected concentrations of the test compound. GFP reporter activity is measured by live-cell confocal imaging to determine stress pathway activity. Finally, in the data acquisition and analysis step, image segmentation to identify sub-cellular localization and quantification of the GFP reporter activations is done. Concentration-response graphs are created, the point of departure (PoD) is calculated, and hierarchical clustering provides a total overview of the generated data.

For the mitochondrial toxicity assessment, the genetically unmodified cell line is exposed to 7 selected concentrations of the test compound in either high glucose or galactose medium (Glu/Gal assay). By replacing the glucose medium with a galactose variant, cells switch their metabolism from glycolysis to oxidative phosphorylation, relying solely on mitochondria for ATP/energy. Next, the PoD for cytotoxicity of compounds in both galactose and glucose medium is calculated. If the determined PoD in the galactose medium is 2 times lower than that of the high glucose medium, the compound is considered as a mitochondrial toxicant.

A schematic representation of the ToxProfiler assay protocol. There are three steps involved in this protocol: a broad dose range-finding, exposure of the cell lines to 7 concentrations of compounds and also performing the glu/gal medium switch, and data acquisition and analysis.

What is the added value of ToxProfiler MAX over the standard ToxProfiler assay?
The combination of the assays is able to discriminate between compounds that directly affect mitochondria function from those that induce other types of cellular stress (e.g. oxidative stress, autophagy or ER stress).
What cytotoxicity/viability measurement is used for the ToxProfiler MAX assay?
We use propidium iodide (PI) as a measurement for cytotoxicity. PI is a cell-impermeable DNA stain that will only label dead cells in which the cell membrane became permeable. In our experience, this is a very sensitive and specific measurement for late-stage apoptosis or early necrosis.
How ToxProfiler MAX identifies mitochondrial toxicants?
For the mitochondrial toxicity assessment, the ToxProfiler cells are exposed to selected concentrations of the test compound in either high glucose or galactose medium (Glu/Gal assay) and cytotoxicity is determined using the propidium iodide (PI) stain. Next, the PoD for cytotoxicity of compounds is calculated. If the determined PoD in galactose medium is 2 times lower than that of the high glucose medium, the compound is considered to be a mitochondrial toxicant.
How does the addition of the Glu/Gal assay impact some of the practical features of the ToxProfiler assay (e.g. turnaround time, throughput and compound requirement).
The turnaround time and throughput of ToxProfiler MAX is similar to that of the ToxProfiler assay. The turnaround time is about 4 weeks with the throughput of testing >20 compounds per week.
What is the advantage of ToxProfiler MAX over standard cytotoxicity or dedicated mitochondrial toxicity assays?
The integrative approach of ToxProfiler MAX provides a broad and quantitative toxicological profile of chemicals. The induction of multiple major toxicological effects, including mitochondrial damage, can be studied in a single assay.
How specific is the prediction of mitochondrial toxicity in ToxProfiler MAX?

The protocol that is applied in ToxProfiler MAX, based on the glu/gal medium switch principle, is a very specific and sensitive means to identify mitochondrial toxicants. This is in strong contrast to other dye-based mitochondrial toxicity assays (e.g. Rho123 or MitoTracker) for which the specificity is quite poor and also general cytotoxicity often leads to a false positive. The mitochondrial damage prediction in TP MAX is further increased by the unique combination of TP reporter genes.

Can you share a sample report?

Yes, please contact us for a sample report via enquiries@toxys.com

Meet the study director for ToxProfiler MAX

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 ToxProfiler MAX in understanding where and how ToxProfiler MAX 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. Bas ter Braak
Dr. Bas ter Braak
Study director for ToxProfiler

Bas ter Braak obtained his PhD at the Leiden Academic Centre for Drug Research (LACDR).  He co-developed the HepG2 BAC-GFP reporter platform that is central to ToxProfiler. Bas joined Toxys in 2020 as a senior scientist to run ToxProfiler and further expand the assay with his knowledge in compound-induced stress signalling processes in differentiated reporter cell lineages.

Can you help with a study design with different types of solvents?

Click for the answer

Click here to send your question!

Service

We perform ToxProfiler MAX as a service for our clients. You can send your compounds and receive a full report within 4 weeks. It is also possible to request a ToxProfiler MAX test via scientist.com. Are you interested in receiving a quote or do you have any questions? Please reach out!

Practical information

Compound requirement

The amount of compound that is required is between 8 – 50 mg.

Turn around time

  • About 4 weeks

Throughput

  • Testing >20 compounds per week

Type of solvents compatible with the assay

  • DMSO
  • PBS
  • Water

A standard ToxProfiler MAX test includes

  • Broad cytotoxicity profile of each test compound
  • Assessment of mitochondrial toxicity
  • Assessment of the effect of the compounds on the 7 stress pathways; Oxidative stress, ER stress, Cell cycle stress, Autophagy, Protein stress, Ion stress, and Inflammation
  • Mitochondrial toxicity assessment
  • Extensive toxicological profile and hierarchical clustering of compounds

Next steps after sharing your interest in ToxProfiler MAX

A figure showing the next steps after sharing your interest in our assay.

Meet the study director for ToxProfiler MAX

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 ToxProfiler MAX in understanding where and how ToxProfiler MAX 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. Bas ter Braak
Dr. Bas ter Braak
Study director for ToxProfiler

Bas ter Braak obtained his PhD at the Leiden Academic Centre for Drug Research (LACDR).  He co-developed the HepG2 BAC-GFP reporter platform that is central to ToxProfiler. Bas joined Toxys in 2020 as a senior scientist to run ToxProfiler and further expand the assay with his knowledge in compound-induced stress signalling processes in differentiated reporter cell lineages.

Can you help with a study design with different types of solvents?

Click for the answer

Click here to send your question!

Please contact us for more information