Antioxidant Response Element Reporter Cell Line (ARE32)

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

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.

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 ARE 32 Cell Line

Features

ARE32 is 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 ARE32

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 ARE32 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
• efficacy target to identify compunds with chemoprotective properties
• toxicity reporter to screen out compounds that induce oxidative stress
• 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