Antioxidant Response Element Reporter Cell Line (AREc32)
The antioxidant response element (ARE) is a transcriptional regulatory element involved in the activation of genes coding for a number of antioxidant proteins and phase I & II detoxifying enzymes. These enzymes work in concert to protect tissues from oxidative insults and are collectively referred to as the "Phase 2 protein battery" (see table).
Protective Phase 2 protein “battery” under ARE promoter control |
Phase I enzymes - Inducible Heme oxygenase I
- aldehyde dehydrogenase
- dihydrodiol dehydrogenase
- leukotriene B4 dehydrogenase
Phase II enzymes - Glutathione S-transferase
- UDP glucuronyl transferase
- NADH quinone oxidoreductase 1
- Epoxide hydrolase
- γ-glutamylcysteine ligase
glutathione reductase
Others / stress proteins - Ferritin
- Glutathione S-conjugate efflux pumps
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Oxidative chemicals such as phenolic antioxidants and electrophilic compounds induce gene transcription via the transcription factor Nrf2 (NF-E2-related factor 2) which is the key central protein that binds to the ARE to activate gene transcription across the battery. Cytoprotective agents can also directly induce Nrf2 and act to upregulate Phase2 induction to protect against oxidative stress and maintain cellular homeostasis.
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Chemicals or drugs that induce the ARE may therefore be oxidative or cytoprotective.
1. Oxidative stress Assay: Oxidative stress is linked to a number of toxicities (see below), and the activation of ARE provides a screen to identify potentially toxic or carcinogenic compounds that induce oxidative stress.
- hepatotoxicity
- cardiotoxicity
- skin sensitization
2. Cytoprotective Assay: The ARE cell based assay can also be configured to identify drugs or chemicals that induce ARE and hence have potential as cytoprotective and chemoprotective agents.
The AREc32 Cell Line
Features
AREc32 is a cell-based assay reporting on the induction of the ARE on a stable MCF7 cell line background. These cells are transfected with copies of the rat GST ARE linked to a luciferase gene, such that the induction of the ARE results in luciferase activity.
Validation of AREc32
Validation has demonstrated an inducible level of up to 70-fold higher than basal luciferase activity following treatment with ter-butylhydroquinone (tBHQ), a phenolic antioxidant and known inducer of Phase 2 drug metabolising enzymes. In addition, a recent publication, using this cell line, by Natsch & Emter in the Toxicological Sciences journal has also validated the response of the cell line to a variety of oxidative stressors, and demonstrated the value of the AREc32 assay as an in vitro screen for skin sensitizers compared to the LLNA.
Natsch A and Emter R Skin sensitizers induce antioxidant response element dependent genes: Application to the in vitro testing of the sensitization potential of chemicals. Published on-line October 11th, 2007 Toxicological Sciences. Access the paper here.
Benefits
Allows screening of compounds for activation of protective phase 2 gene battery
Can be used in two modes
potential for use in automated medium throughput screening
Rapid and cost effective
Good signal to noise (70-fold increase in luciferase activitty)
Applications
Toxicity screen for oxidative stress inducers
Screen for chemopreventative agents
Screen for cytoprotective agents
Nutraceutical industry
Cosmetics industry
Pharmaceutical industry
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Toxicity Reporter cell lines
In vitro models for the rapid assessment of mechanisms of toxicity. Gene promoters associated with specific toxic insults have been linked to biomarker reporter systems. These models provide a rapid & cost effective means to assess modes of toxicology during e.g. drug discovery.
