Cytotoxicity Burst? Differentiating Specific from Nonspecific Effects in Tox21 in Vitro Reporter Gene Assays
Beate I Escher1,2, Luise Henneberger1, Maria König1
1Department of Cell Toxicology, Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.
Background:
High-throughput screening of chemicals with in vitro reporter gene assays in Tox21 has produced a large database on cytotoxicity and specific modes of action. However, the validity of some of the reported activities is questionable due to the "cytotoxicity burst," which refers to the supposition that many stress responses are activated in a nonspecific way at concentrations close to cell death.
Objectives:
We propose a pragmatic method to identify whether reporter gene activation is specific or cytotoxicity-triggered by comparing the measured effects with baseline toxicity.
Methods:
Baseline toxicity, also termed narcosis, is the minimal toxicity any chemical causes. Quantitative structure-activity relationships (QSARs) developed for baseline toxicity in mammalian reporter gene cell lines served as anchors to define the chemical-specific threshold for the cytotoxicity burst and to evaluate the degree of specificity of the reporter gene activation. Measured 10% effect concentrations were related to measured or QSAR-predicted 10% cytotoxicity concentrations yielding specificity ratios (SR). We applied this approach to our own experimental data and to chemicals that were tested in six of the high-throughput Tox21 reporter gene assays.
Results:
Confirmed baseline toxicants activated reporter gene activity around cytotoxic concentrations triggered by the cytotoxicity burst. In six Tox21 assays, 37%-87% of the active hits were presumably caused by the cytotoxicity burst () and only 2%-14% were specific with against experimental cytotoxicity but 75%-97% were specific against baseline toxicity. This difference was caused by a large fraction of chemicals showing excess cytotoxicity.
Conclusions:
The specificity analysis for measured in vitro effects identified whether a cytotoxicity burst had likely occurred. The SR-analysis not only prevented false positives, but it may also serve as measure for relative effect potency and can be used for quantitative in vitro-in vivo extrapolation and risk assessment of chemicals. https://doi.org/10.1289/EHP6664.
Insights
A new method distinguishes specific chemical effects from false positives caused by cell death in high-throughput screening. This analysis improves the reliability of in vitro toxicity testing and aids in chemical risk assessment.
Area of Science:
- Toxicology
- Computational Chemistry
- Biochemistry
Background:
- High-throughput screening (HTS) using Tox21 reporter gene assays generates extensive data on chemical cytotoxicity and specific actions.
- The "cytotoxicity burst" phenomenon, where stress responses activate non-specifically near cell death, raises concerns about the validity of some HTS results.
Purpose of the Study:
- To develop a pragmatic method for differentiating specific reporter gene activation from cytotoxicity-triggered responses.
- To establish a reliable approach for evaluating the specificity of chemical effects observed in in vitro assays.
Main Methods:
- Comparing measured chemical effects with baseline toxicity (narcosis) using quantitative structure-activity relationships (QSARs).
- Defining a chemical-specific threshold for the cytotoxicity burst using QSAR-predicted baseline toxicity.
- Calculating specificity ratios (SR) by relating 10% effect concentrations to 10% cytotoxicity concentrations.
Main Results:
- Confirmed baseline toxicants activated reporter genes near cytotoxic concentrations, indicating a cytotoxicity burst.
- In six Tox21 assays, 37%-87% of active hits were attributed to the cytotoxicity burst.
- Specificity analysis revealed 75%-97% of chemicals were specific against baseline toxicity, highlighting the impact of excess cytotoxicity.
Conclusions:
- The developed specificity analysis accurately identifies cytotoxicity bursts in measured in vitro effects.
- Specificity ratios (SR) serve as a measure of relative effect potency and can enhance quantitative in vitro-in vivo extrapolation.
- This method improves the accuracy of chemical risk assessment by preventing false positives and providing a more reliable measure of chemical potency.
