The ToxProfiler assay logo

ToxProfilerTM is a unique New Approach Method (NAM) that provides an extensive and quantitative toxicological profile of novel pharmaceutical, (agro)chemical and cosmetic compounds. ToxProfiler is a human cell assay that contains seven fluorescent reporter genes to visualise the major cellular stress response pathways. Based on the reporter gene activation patterns, ToxProfiler provides a detailed toxicological profile for each test compound. Quantitative dose-response modelling of the toxicological responses and point-of-departure calculations allow for potency ranking of compounds, identification of their primary mode-of-action, and read-across approaches for chemicals. ToxProfiler is particularly applicable for early safety testing, adverse outcome pathway (AOP) and weight-of-evidence (WoE) methods.

A gif of HEPG2 cells with a GFP reporter being activated

Key Features

    • Unique combination of 7 human reporter cell lines covering the major toxicological stress responses
    • NAM for potency ranking, mode-of-action assessment and chemical read-across
    • Highly quantitative dose-response modelling and point-of-departure calculation
    • Toxicological profiling supports prioritisation of chemistry and support compound design

Unique biomarkers

ToxProfiler consists of 7 genetically engineered HepG2 cell lines. Each cell line contains a specific fluorescent reporter that is associated with and represents a specific cellular stress response pathway. Each reporter represents the activation of a specific cellular stress response by increased expression or change in cellular localisation. These 7 biomarkers were selected to  cover various key toxicological stress pathways. More information can be found here.

 

From biomarker to toxicological fingerprint

The ToxProfiler reporter cell lines are cultured in a 384 well format. Automated high content imaging pipelines are applied to accurately quantify induction of the various stress pathways. Quantitative dose-response modelling is applied to determine point-of-departure (PoD) with our proprietary PODplotter software. Results are visualised as an easy to read fingerprint which allows for clustering and potency ranking of compounds.

Highlights of ToxProfiler

  • Fluorescent reporters are expressed at physiological levels, reflecting the endogenous cellular stress responses
  • Image-based high throughput screening platform with a single cell resolution (high content imaging)
  • Human biomarker expression
  • Quantification of stress response signalling and cytotoxicity with point of departure (POD) determination
  • Metabolism can be included with the addition of S9 rat liver extract

Typical applications of ToxProfiler

ToxProfiler is a fast and reliable screening assay to unravel the toxicological mode-of-actions (MoA). Examples of the most commonly used applications for ToxProfiler:

  • Early-phase in vitro safety screening. ToxProfiler 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 provides mechanistic insight into the toxic properties of chemical that is applied to for clustering structures with similar reactivity.
  • Weight-of-evidence approach. ToxProfiler can be applied in screening as a mechanistic follow-up of the regulatory in vitro tests to provide insight into the MoA of compounds. The MoA information can be used in a weight-of-evidence approach during hazard assessment of novel compounds.
  • Late-phase in vitro screening. ToxProfiler can also be used to explain specific pathological outcomes from in vivo findings.

Various studies have been published on the ToxProfiler technology and you can find them here.

Meet the study director for ToxProfiler

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 in understanding where and how ToxProfiler 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!

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

ToxProfiler experimental design

Typical output generated from the ToxProfiler assay includes:

    • Broad cytotoxicity profile of each test compound
    • Assessment of the effect of the compounds on the 7 stress pathways; Oxidative stress, ER stress, Genetic stress, Autophagy, Protein stress, Ion stress and Inflammation
    • POD calculations per compound
    • Extensive toxicological profile and hierarchical clustering of compounds

Cytotoxic profiles and concentration selection

Cytotoxicity is measured with cell impermeable DNA stain Propidium Iodine (PI).  Seven concentrations of the compound, as indicated in the green area, are selected to run in the ToxProfiler assay. If no precipitation is observed, a top concentration will be selected that causes significant (>15% PI positive cell count) cell death. The red dashed line represents the threshold that we use for cytotoxicity with  15% PI positive cells. Typically, the selected concentrations include one concentration at which see significant cytotoxicity and 6 sub-cytotoxic concentrations (Cadmium Chloride and Verapamil). If no cytotoxicity is observed (Andrographolide), the seven highest concentrations are included. In some cases, the selected concentrations are limited by precipitation of the highest concentrations in the assay medium and these concentrations are also avoided for the reporter phase of the screen (Mitomycin C).

Assessment of the induction of the 7 stress pathways

Using live-cell confocal imaging and automated image segmentation pipelines, the GFP reporter levels in a specific subcellular localization are accurately quantified with a single cell resolution, as shown below.

 

Four example compounds that induce different cellular stress pathways. The GFP induction levels at each concentration are quantified as concentration-response plots, as seen below.

POD calculations

With our PODplotter software, the POD concentrations (the lowest concentration at which a significant reporter induction is observed) are accurately determined. The POD values for the different ToxProfiler reporters can be used to distinguish between primary and secondary toxicological responses.

Toxicological fingerprint of cellular stress responses

The GFP induction levels across all concentrations are presented in an easy-to-read heatmap with hierarchical clustering of the test compounds. This readout allows for a quick interpretation of the stress pathway inductions.

Optional: include a metabolizing system in ToxProfiler (S9 liver extract)

The assay has been validated for the inclusion of S9 rat liver extract. This can be added to your ToxProfiler project. Click here to read more.

Project report

The project report contains the protocol, technical details, dose range finding results, biomarker expression with test compounds, POD calculations and toxicological fingerprint of cellular stress responses. We are happy to share the dummy report with you. Please click here to request for a dummy report.

Toxicological profiling of cellular stress responses

The ToxProfiler assay was used for a wide selection of compounds that have been well-established to cause specific toxicological effects. The heatmap below shows an overview of the ToxProfiler stress response signalling profiles of these compounds. Hierarchical clustering with a dendrogram provides an unbiased comparison of these profiles.

The ToxProfiler profiles for 20 selected reference compounds with well-established  toxicological properties are summarized in the table below. Compounds were clustered based in their similarity to induce the various cellular stress response reporters.


Validation of the inclusion of metabolism in ToxProfiler with S9 rat liver extract

As an example, four reference compounds have been analysed in ToxProfiler in the absence and presence of S9 rat liver extract. The graphs clearly indicate that the compounds that require metabolism ( Aflatoxin B1 and Cyclophosphamide) become activated, while another compound (CDDO-me) beomes deactivated by metabolism, as is well established for these compounds.

Flyers
Publications

Wink S, Hiemstra S, Herpers B et al. High-content imaging-based BAC-GFP toxicity pathway reporters to assess chemical adversity liabilities. Arch Toxicol. 2017 Mar;91(3):1367-1383. doi: 10.1007/s00204-016-1781-0. Epub 2016 Jun 29. PMID: 27358234; PMCID: PMC5316409.

Ter Braak B, Niemeijer M, Wolters L et al. Towards an advanced testing strategy for genotoxicity using image-based 2D and 3D HepG2 DNA damage response fluorescent protein reporters, Mutagenesis, 2021 Aug 27;geab031., doi: 10.1093/mutage/geab031

Ter Braak B, Klip JE, Wink S, et al. Mapping the dynamics of Nrf2 antioxidant and NFκB inflammatory responses by soft electrophilic chemicals in human liver cells defines the transition from adaptive to adverse responses, sumitted toxicology in vitro December 2021

Wink S, Hiemstra SW, Huppelschoten S et al. Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury. Arch Toxicol. 2018 May;92(5):1797-1814. doi: 10.1007/s00204-018-2178-z. Epub 2018 Mar 3. PMID: 29502165; PMCID: PMC5962642.

Wink S, Hiemstra S, Huppelschoten S et al. Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment. Chem Res Toxicol. 2014 Mar 17;27(3):338-55. doi: 10.1021/tx4004038. Epub 2014 Feb 5. PMID: 24450961.

Schimming JP, Ter Braak B, Niemeijer M, et al. System Microscopy of Stress Response Pathways in Cholestasis Research. Methods Mol Biol. 2019;1981:187-202. doi: 10.1007/978-1-4939-9420-5_13. PMID: 31016656.

Bischoff LJM, Kuijper IA, Schimming JP et al. A systematic analysis of Nrf2 pathway activation dynamics during repeated xenobiotic exposure. Arch Toxicol. 2019 Feb;93(2):435-451. doi: 10.1007/s00204-018-2353-2. Epub 2018 Nov 20. PMID: 30456486.

Niemeijer M, Hiemstra S, Wink S et al. Systems Microscopy Approaches in Unraveling and Predicting Drug-Induced Liver Injury (DILI). 2018 In: Chen M, Will Y (eds) Drug-Induced Liver Toxicity. Springer New York, New York, NY, pp 611-625. doi:10.1007/978-1-4939-7677-5_29

Hiemstra S, Niemeijer M, Koedoot E et al. Comprehensive Landscape of Nrf2 and p53 Pathway Activation Dynamics by Oxidative Stress and DNA Damage.  Res. Toxicol.2017, 30, 4

Herpers B, Wink S, Fredriksson L et al. Activation of the Nrf2 response by intrinsic hepatotoxic drugs correlates with suppression of NF-κB activation and sensitizes toward TNFα- induced cytotoxicity. Arch Toxicol. 2016 May;90(5):1163-79. doi: 10.1007/s00204-015-1536-3. Epub 2015 May 31. PMID: 26026609; PMCID: PMC4830895.

Fredriksson L, Wink S, Herpers B et al. Drug-induced endoplasmic reticulum and oxidative stress responses independently sensitize toward TNFα-mediated hepatotoxicity. Toxicol Sci. 2014 Jul;140(1):144-59. doi: 10.1093/toxsci/kfu072. Epub 2014 Apr 20. PMID: 24752500.

Di Z, Herpers B, Fredriksson L et al. Automated analysis of NF-κB nuclear translocation kinetics in high-throughput screening. PLoS One. 2012;7(12):e52337. doi: 10.1371/journal.pone.0052337. Epub 2012 Dec 27. PMID: 23300644; PMCID: PMC3531459.

Hiemstra S, Ramaiahgari SC, Wink S et al.  High-throughput confocal imaging of differentiated 3D liver-like spheroid cellular stress response reporters for identification of drug-induced liver injury liability. Arch Toxicol. 2019 Oct;93(10):2895-2911. doi: 10.1007/s00204-019-02552-0. Epub 2019 Aug 27. PMID: 31552476.

Ramaiahgari SC, den Braver MW, Herpers B et al. A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies. Arch Toxicol. 2014 May;88(5):1083-95. doi: 10.1007/s00204-014-1215-9. Epub 2014 Mar 6. PMID: 24599296.

Fredriksson L, Herpers B, Benedetti G et al. Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis. Hepatology. 2011 Jun;53(6):2027-41. doi: 10.1002/hep.24314. PMID: 21433042.

Posters
Webinars

Meet the study director for ToxProfiler

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 in understanding where and how ToxProfiler 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!

What endpoints are reported?

The ToxProfiler assay covers 7 different endpoints: Oxidative stress, DNA damage, Endoplasmic Reticulum (ER) stress, Autophagy, Ion stress, Protein damage and Inflammation supplemented by assessment of Mitogenicity and Cytotoxicity for dose-finding.

What is a typical application of ToxProfiler?

Chemical Read-across: By clustering compounds with similar toxicological profile, structurally related chemicals can be identified. Thereby, time consuming and expensive in vivo follow-up studies can be avoided.

Compound selection and ranking: ToxProfiler ranking is based on 7 different crucial endpoints in cellular toxicity. This toxicity ranking combined with the efficacy ranking will help with prioritization in the compound selection and development process.

Problem solving: Unforeseen toxicity can be further investigated in ToxProfiler. The underlying mode-of-action can be elucidated, which can potentially rescue a chemical from dropping out of the developmental pipeline.

What is the threshold for a positive result?

A positive result for a readout in ToxProfiler is when a Point of Departure (PoD) can be determined for the concentration-response curve. The PoD is the intersection of the fitted curve and two times the standard deviation, meaning that there must be a response higher than 2-fold the standard deviation to warrant a call.

What type of chemistry has been tested in the assay?

The ToxProfiler technology was applied in toxicology screens that were performed in industrial collaborations with the pharmaceutical, cosmetic, (agro)chemical and the food industry. In these screens, the ToxProfiler technology was used for a wide range of different chemical entities such as the following:

  • Pharmaceutical compounds
  • Cosmetics ingredients
  • Chemicals
  • Food products
  • Nanomaterials
  • Pesticides
  • Heavy metals
How is the assay validated?

The ToxProfiler reporter cell lines were extensively validated in the EuToxRisk project, for which many libraries with chemical entities were screened covering a wide chemical space. At Toxys, the ToxProfiler assay was further developed and standardized into a robust assay.

How much compound is needed to perform the assay?

We typically ask for 1-2 mg of compound.

How long does it take to receive a report?

The typical turnaround time of the ToxProfiler assay is typically 2-3 weeks. If timelines are critical, please reach out to our Sales team.

What type of solvent can be used?

The type of solvent shall be agreed upon before start of the project. Typically, DMSO, water, or PBS is used.

Can you test with metabolic activation?

ToxProfiler reporters are built in the human liver HepG2 line, which possesses only limited metabolic activity. However, we do offer an option to include S9 liver extract (rat), which contains both phase I and II metabolic enzymes.

What is the top-concentration that is tested, and how is this determined?

The top concentration that is typically applied in the assay is 1mM. In case the test substance is poorly soluble, the maximum soluble concentration is used in the assay. In the dose-finding experiment, all wells will be inspected for precipitation during compound exposure. If no precipitation is observed, a top concentration will be selected that causes significant (>15% PI positive cell count) cell death.

How many concentrations are tested?

For the concentration range finding experiments we include 14 concentrations (e.g. 1 mM – 50 nM). For the ToxProfiler reporter assay we include 7 concentrations.

Has data from ToxProfiler ever been included in regulatory dossiers?

There are cases where ToxProfiler data has been used to supplement regulatory filing.

Can you test auto fluorescent materials?

Yes. We have protocols available to test auto fluorescent materials. In case autofluorescence is detected during the concentration range-finding experiments and the image segmentation is not affected, we can subtract the auto fluorecent signal from the reporter readout and retrieve reliable results.

What cell type is used for the assay?

The ToxProfiler reporter panel is built in the human HepG2 cell line. For the concentration range-finding experiments we use the parental HepG2 line.

Can you share a sample report?

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

Meet the study director for ToxProfiler

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 in understanding where and how ToxProfiler 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 as a service for our clients. You can send your compounds and receive a full report within 2-3 weeks. It is also possible to request a ToxProfiler test via scientist.com. Are you interested in receiving a quote or do you have any questions? Please reach out!

Photos of our sales team, whom you can reach out to receive a quote or with any questions.

Practical information

Compound requirement

The amount of compound that is required for analysis in the ToxProfiler assay depends on the maximum concentration that will be applied in the project.

Turn around time

  • 2-3 weeks

Throughput

  • Testing >20 compounds per week

Type of solvents compatible with the assay

  • DMSO
  • PBS
  • Water

A standard ToxProfiler test includes

  • Broad cytotoxicity profile of each test compound
  • Assessment of the effect of the compounds on the 7 stress pathways; Oxidative stress, ER stress, Genetic stress, Autophagy, Protein stress, Ion stress and Inflammation
  • POD calculations per compound
  • Extensive toxicological profile and hierarchical clustering of compounds

Next steps after sharing your interest in ToxProfiler

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

Meet the study director for ToxProfiler

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 in understanding where and how ToxProfiler 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