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Published on: February 7, 2018
Generation of stable ARE- driven reporter system for monitoring oxidative stress
Paria Motahari1, Majid Sadeghizadeh2, Mehrdad Behmanesh1
1Faculty of Biological Sciences, Department of Molecular Genetics, School of Biological Sciences, Tarbiat Modares University, Jalal Ale Ahmad Highway, PO Box 14115-111, Tehran, Iran.
Background:
NF-E2-related factor2 (Nrf2)-antioxidant response element (ARE) signaling pathway is the major defensive mechanism against oxidative stress and is up regulated by specific antioxidants and oxidants to comprise the chemoptotective response. Detection of ARE-activating compounds helps to develop new drugs and identify/quantify the tension range of the oxidants. Important reasons promoting this work are high throughput, rapid and inexpensive experiments relative to the in vitro studies for ARE-Nrf2 pathway monitoring of chemicals and environmental samples.
Methods:
In this study hepatoma Huh7 reporter cell line was generated which contains a luciferase gene under the control of an ARE. This is the first example of ARE construct containing one copy of extended consensus response element. The cells were treated with hydroquinone (HQ) and p-benzoquinone (BQ) (oxidative stress inducers) and the antioxidant, curcumin.
Results:
The luciferase activity was induced in a concentration-dependent manner in a concentration range of 1-2 μM for BQ and HQ. Curcumin was also validated as an ARE inducer in concentration above 10 μM. In addition, this reporter cell line provides a rapid detection as early as 4 h to respond to the ARE inducers.
Conclusion:
It is a powerful tool for the sensitive and selective screening of chemicals, drugs and environmental samples for their antioxidant and oxidant activities.
Insights
A new reporter cell line rapidly detects antioxidant and oxidant activities by monitoring the NF-E2-related factor2 (Nrf2)-antioxidant response element (ARE) pathway. This tool enables sensitive screening of chemicals and environmental samples for oxidative stress responses.
Area of Science:
- Biochemistry and Molecular Biology
- Toxicology
- Drug Discovery
Background:
- The NF-E2-related factor2 (Nrf2)-antioxidant response element (ARE) pathway is crucial for cellular defense against oxidative stress.
- This pathway is activated by antioxidants and oxidants, playing a key role in chemoprotective responses.
- Monitoring ARE-Nrf2 pathway activation aids in developing new drugs and assessing oxidant levels.
Purpose of the Study:
- To develop a novel reporter cell line for high-throughput, rapid, and cost-effective monitoring of the ARE-Nrf2 pathway.
- To establish a sensitive and selective screening tool for chemicals, drugs, and environmental samples.
- To investigate the utility of this reporter system for detecting oxidative stress inducers and antioxidants.
Main Methods:
- A hepatoma Huh7 reporter cell line was engineered with a luciferase gene under the control of a single copy of an extended consensus ARE.
- The reporter cells were treated with oxidative stress inducers: hydroquinone (HQ) and p-benzoquinone (BQ).
- The antioxidant curcumin was used as a positive control for ARE induction.
Main Results:
- Luciferase activity was induced by BQ and HQ in a concentration-dependent manner within the 1-2 μM range.
- Curcumin demonstrated ARE-inducing activity at concentrations above 10 μM.
- The reporter cell line enabled rapid detection of ARE inducers as early as 4 hours post-treatment.
Conclusions:
- The developed ARE reporter cell line is a powerful tool for sensitive and selective screening.
- It facilitates the identification and quantification of antioxidant and oxidant activities in various substances.
- This system offers a significant advancement for monitoring chemical and environmental samples for their impact on the ARE-Nrf2 pathway.
