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Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress
Sander C van der Linden1, Anne R M von Bergh2, Barbara M A van Vught-Lussenburg1
1BioDetection Systems BV, Amsterdam, The Netherlands.
Abstract:
The lack of toxicological information on many of the compounds that humans use or are exposed to, intentionally or unintentionally, poses a big problem in risk assessment. To fill this data gap, more emphasis is given to fast in vitro screening tools that can add toxicologically relevant information regarding the mode(s) of action via which compounds can elicit adverse effects, including genotoxic effects. By use of bioassays that can monitor the activation of specific cellular signalling pathways, many compounds can be screened in a high-throughput manner. We have developed two new specific reporter-gene assays that can monitor the effects of compounds on two pathways of interest: the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress. To exclude non-specific effects by compounds influencing the luciferase reporter-gene expression non-specifically, a third assay was developed to monitor changes in luciferase expression by compounds in general (Cytotox CALUX). To facilitate interpretation of the data and to avoid artefacts, all three reporter-gene assays used simple and defined reporter genes and a similar cellular basis, the human U2OS cell line. The three cell lines were validated with a range of reference compounds including genotoxic and non-genotoxic agents. The sensitivity (95%) and specificity (85%) of the p53 CALUX was high, showing that the assay is able to identify various types of genotoxic compound, while avoiding the detection of false positives. The Nrf2 CALUX showed specific responses to oxidants only, enabling the identification of compounds that elicit part of their genotoxicity via oxidative stress. All reporter-gene assays can be used in a high-throughput screening format and can be supplemented with other U2OS-based reporter-gene assays that can profile nuclear receptor activity, and several other signalling pathways.
Insights
New reporter-gene assays offer fast in vitro screening for toxicological effects. The p53 CALUX assay effectively identifies genotoxic compounds, while the Nrf2 CALUX assay detects oxidative stress, improving chemical risk assessment.
Area of Science:
- Toxicology and Environmental Health
- Molecular Biology and Genetics
Background:
- Significant data gaps exist regarding the toxicological profiles of many chemicals, hindering accurate risk assessment.
- In vitro screening tools are crucial for rapidly generating toxicologically relevant data on compound modes of action, including genotoxicity.
- High-throughput screening using bioassays that monitor cellular signaling pathways is essential for addressing these data gaps.
Purpose of the Study:
- To develop novel, specific reporter-gene assays for high-throughput screening of chemical toxicity.
- To establish assays for monitoring the p53 pathway (p53 CALUX) for genotoxicity and the Nrf2 pathway (Nrf2 CALUX) for oxidative stress.
- To create a general cytotoxicity assay (Cytotox CALUX) to exclude non-specific effects and facilitate data interpretation.
Main Methods:
- Development of three distinct reporter-gene assays (p53 CALUX, Nrf2 CALUX, Cytotox CALUX) utilizing the human U2OS cell line.
- Validation of the assays using a diverse set of reference compounds, including known genotoxic and non-genotoxic agents.
- Employing simple, defined reporter genes and a consistent cellular basis across all assays to ensure reliable data.
Main Results:
- The p53 CALUX assay demonstrated high sensitivity (95%) and specificity (85%) in identifying genotoxic compounds while minimizing false positives.
- The Nrf2 CALUX assay specifically responded to oxidants, enabling the detection of compounds inducing toxicity via oxidative stress pathways.
- All developed assays proved suitable for high-throughput screening and can be integrated with other U2OS-based assays for broader pathway profiling.
Conclusions:
- The developed reporter-gene assays provide a robust and efficient method for assessing chemical genotoxicity and oxidative stress.
- These assays significantly contribute to filling toxicological data gaps, aiding in chemical risk assessment.
- The platform allows for high-throughput screening and can be expanded to profile additional cellular signaling pathways.
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