High-throughput screening for mitochondrial toxicity.
Mitochondrial toxicity is known as a common cellular mechanism behind many adverse drug reactions, which can lead to drug attrition and post-market drug withdrawals. However, mitochondrial toxicity assessment in vivo is complex and resource-intensive, making early in vitro screening essential for efficient chemical or compound development.
Widely used methods such as Seahorse XF, MitoTracker, and MitoSOX provide valuable mitochondrial readouts, but their suitability may be limited by throughput and/or workflow complexity. Additionally, current in vitro assays are often overly sensitive to general cellular stress, which can result in misleading positive results and the unnecessary loss of promising candidates.
MitoToxScreen addresses these limitations with a scalable, high-throughput assay designed for early-stage mitochondrial toxicity screening. By integrating a unique reporter assay with the established glucose/galactose metabolic switch approach, it enables more specific identification of true mitochondrial dysfunction while reducing false-positive findings.
BENEFITS
- High sensitivity and specificity
- Screen up to 40 test articles per week
- Get your results in 10 working days
- Low amount of test article required
- Technology How does it work?
- Readouts A unique combination
- Validation What's the accuracy?
- Applications How are others using MitoToxScreen?
- FAQ Learn more
- Resources
Predicting mitochondrial toxicity with high accuracy
MitoToxScreen integrates two complementary approaches: a fluorescent reporter gene (CHOP‑GFP) that is activated as part of the endoplasmic reticulum (ER) stress response, and the glucose/galactose (Glu/Gal) metabolic switch assay. Combining these readouts within a single platform minimizes misleading positives while maintaining high sensitivity for detecting mitochondrial toxicity.
The Glu/Gal readout is a sensitive indicator of altered cellular dependence on mitochondrial energy production. However, it is not fully specific for direct mitochondrial toxicity. Increased cytotoxicity under galactose conditions can also result from general cytotoxicity, impaired glycolysis, disrupted nutrient uptake, or redox imbalance. In such cases, the Glu/Gal shift alone does not reliably indicate mitochondrial dysfunction.
The CHOP‑GFP reporter adds mechanistic depth by capturing activation of the PERK/ATF4/CHOP pathway, which is well‑documented to respond to direct mitochondrial toxicants. Therefore, concurrent activation of CHOP and a Glu/Gal shift provides stronger evidence that the observed cellular stress reflects genuine mitochondrial toxicity, whereas a Glu/Gal shift in isolation should be interpreted with caution.
Steps in MitoToxScreen
Key assay features
- 384-well plate format
- Live-cell high-content imaging
- Combination of 2 readouts:
- CHOP-GFP reporter activation, reflecting induction of endoplasmic reticulum (ER) stress
- Glu/Gal shift, derived from propidium iodide (PI) staining to assess cell survival under glucose and galactose culture conditions
- Differentiates direct mitochondrial dysfunction from general cellular stress and cytotoxicity
Do you want to get more detailed information?
CHOP-GFP Induction
The CHOP-GFP reporter is used to monitor induction of endoplasmic reticulum stress by quantifying nuclear GFP fluorescence intensity.
The mitochondria and the endoplasmic reticulum work closely together to maintain normal cell function. When mitochondria are disrupted, they can send signals that activate part of the endoplasmic reticulum stress response system, including CHOP activation [1,2]. This connection helps explain why mitochondrial damage can trigger broader cellular stress pathways that are relevant for toxicity assessment.
Differential Cytotoxicity under Glucose versus Galactose Conditions
Replacing glucose with galactose in the culture medium increases cellular dependence on oxidative phosphorylation instead of glycolysis as main source of energy. As a result, mitochondrial toxicants are expected to cause a stronger reduction in cell survival under galactose conditions than under glucose conditions.
In our validation set, combining CHOP-GFP activation with the Glu/Gal metabolic switch achieved 93% accuracy for mitochondrial toxicity detection.
References:
- Vlasveld, M. et al. (2024). https://doi.org/10.1111/liv.15822
- Carta, G. et al. (2023). https://doi.org/10.1007/s10565-023-09816-7
93% accuracy in correctly predicting mitochondrial toxicity of 42 reference compounds
MitoToxScreen was validated using a reference panel of 21 mitochondrial and 21 non-mitochondrial toxicants.
Compounds were characterized based on the combined interpretation of CHOP-GFP induction and Glu/Gal metabolic shift. The assay correctly identified 18 of 21 mitochondrial toxicants and all 21 non-mitochondrial toxicants, resulting in:
- 93% overall accuracy
- 86% sensitivity
- 100% specificity
The MitoToxScreen Advantage: Minimize misleading positive results
With MitoToxScreen, misleading positive results due to cytotoxicity are minimized by combining evidence of both cellular stress pathway activation and mitochondrial functional impairment. This combination provides a more selective assessment of mitochondrial toxicity.
As an example, cadmium chloride and brefeldin A are frequently reported as positive in mitochondrial toxicity assays, likely due to their ability to trigger oxidative or general cellular stress responses. In MitoToxScreen, both compounds are identified as non-mitochondrial toxicants.
Left panel: concentration-response curve CHOP-GFP induction (blue line with shaded variability) with reporter-specific threshold (horizontal red line). Significant CHOP-GFP induction is defined as a concentration-dependent increase exceeding the GFP threshold. If it crosses the threshold, we calculate benchmark concentration lower confidence limit (BMCL) and upper confidence limit (BMCU) values. Cell survival under glucose conditions (black) is also shown to assess whether changes in GFP signal coincided with cytotoxicity.
Right panel: concentration-response curves for cell survival under glucose (black) versus galactose (teal) conditions. Cell survival threshold is 85%. If the curves cross this threshold, we calculate BMCL and BMCU values. If the glucose and galactose BMC ranges do not overlap, we calculate a glu/gal ratio using the BMCL. If the glu/gal ratio is more than 1.5, the outcome is positive.
The full overview of compounds and validation data is available upon request
MitoToxScreen is designed for early-stage screening of chemicals and compounds
MitoToxScreen supports toxicity screening across different stages of product development, from early toxicity screening and test article prioritization to follow-up investigation of potential mitochondrial liabilities. The assay applies to a broad range of test articles, including pharmaceuticals, (agro)chemicals, food ingredients, consumer products, and cosmetics.
Application Example: High-Throughput Mitochondrial Toxicity Screening for Agrochemical Development
A large agrochemical company uses MitoToxScreen service to support the early-stage evaluation for approximately 400 test articles per year for potential mitochondrial toxicity liabilities. Test articles are assessed in batches of 40–80 per screening cycle, enabling efficient high-throughput evaluation without delaying pipeline progression.
Following each screening cycle, we provide comprehensive reports within 2-3 weeks, depending on batch size. These reports give the client timely and reliable data to support informed go/no-go decision-making and direct resources toward the most promising candidates.
What is the link between the CHOP reporter and mitochondrial toxicity?
There are multiple reasons why ER stress is there is a strong link between ER stress and mitochondria toxicity. First, mitochondria and the ER are physically connected at mitochondria-associated membranes (MAMs). ER stores intracellular calcium, which is released to mitochondria to stimulate ATP production. Mitochondrial dysfunction impairs calcium uptake, causing calcium overload in the ER, which triggers ER stress. At last, protein folding is a process that is highly dependent on energy, the unfolded protein response is one of the first cellular stress pathways that is affected by mitochondrial dysfunction.
What does the MitoToxScreen assay detect?
The assay is designed to identify test articles that may interfere with mitochondrial function. It combines cell survival measurements under glucose and galactose cell culture conditions (the Glu/Gal metabolic switch assay) with CHOP-GFP reporter activation.
Why should I use MitoToxScreen?
This is the ideal assay if you are looking for reliable mitochondrial toxicity predictions for large libraries of chemicals or compounds in a short time. Validation data shows 86% sensitivity and 100% specificity. Results for up to 40 test substances can be delivered within 10 working days.
How does MitoToxScreen distinguish mitochondrial toxicity from general cytotoxicity?
Cytotoxicity is evaluated under both glucose and galactose conditions. If a test substance causes similar cytotoxicity in both conditions, this may indicate general toxicity rather than a mitochondria-related effect. If cytotoxicity is stronger in galactose than in glucose, this suggests that the test substance has a greater impact when cells are more dependent on mitochondrial function. Further evidence for mitochondrial toxicity is provided by the CHOP reporter gene.
How accurate is MitoToxScreen for predicting mitochondrial toxicity?
MitoToxScreen achieved strong predictive performance in our validation set of 42 compounds, with 86% sensitivity and 100% specificity for identifying mitochondrial toxicants.
How long does it take to receive a report?
For up to 40 compounds and 2 replicates, we can deliver a report within 10 working days after receiving your samples.
Which solvents are compatible with MitoToxScreen?
The appropriate solvent is discussed and agreed upon before the project starts. Typically, DMSO or H2O is used, but other solvents (e.g. PBS or EtOH) are possible.
How many concentrations are tested?
14 concentrations spaced by a 1/3 log (i.e. approximately 2.15-fold) difference
Is MitoToxScreen performed in living cells?
Yes. The assay uses live-cell confocal imaging to measure reporter activation and cytotoxicity without requiring fixation-based staining workflows.
How much test article is needed?
2 mg, assuming a molecular weight of 500 g/mol.
Can you test autofluorescent materials?
Yes, autofluorescent materials can usually be tested. In the standard protocol, the parental non-GFP HepG2 cell line is included to assess substance-related autofluorescence. Where possible, the data are corrected for autofluorescence. If autofluorescence interferes with reliable assay interpretation, the affected concentrations are excluded from the analysis.
What cell types are used in MitoToxScreen?
Parental HepG2 cells for the Glu/Gal metabolic switch and autofluorescence correction. The HepG2 CHOP-GFP reporter line for assessment of ER stress induction.
What is included in the report?
The report typically includes concentration-response curves for the CHOP-GFP and Glu/Gal readouts, benchmark concentration modeling (BMC) results, and classification calls for mitochondrial toxicity. Additional relevant information on precipitation, autofluorescence, and other observations will be included. A sample report can be shared upon request.
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Steps to get accurate mitochondrial toxicity predictions
- Project Consultation
Contact our team to discuss your project and receive a personalized quote. Our scientific experts will recommend the optimal assay setup based on your compounds and study goals.
- MitoToxScreen Execution
Once the project is confirmed, send us your test substances and we’ll start testing.
- Compound requirements are typically 2 mg, assuming a molecular weight of 500 g/mol
- Solvents compatible with the assay are DMSO and water. PBS and EtOH can be considered
- Data Review & Reporting
Two weeks after project initiation, the report is sent to you, and we schedule a scientific review meeting to walk you through the results, provide a clear interpretation, and answer any questions you may have.
Looking for a more extensive toxicological profile of your test articles?
Discover ToxProfiler Max: a unique human in vitro assay that combines seven stable fluorescent reporter genes with mitochondrial toxicity assessment.
Contact our team
Tamara Meijer, Scientific Director at Toxys
