AREc32 Cell Line

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.

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.

CXR_AREc32_graphic-2

Chemicals or drugs that induce the ARE may therefore be oxidative or cytoprotective.

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

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
    • efficacy target to identify compounds 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 activity)

Applications

  • Toxicity screen for oxidative stress inducers
  • Toxicity screen for nanoparticles
  • Screen for chemopreventative agents
  • Screen for cytoprotective agents
  • Nutraceutical industry
  • Cosmetics industry
  • Pharmaceutical industry

References:

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.

The Use Of An Oxidative Stress Associated in vitro Screen For The Prediction Of Potential Nanoparticle Toxicity. The poster was presented at Nanomedicine 2013.

Implementation of a High-Throughput Screen for Identifying Small Molecules to Activate the Keap1-Nrf2-ARE Pathway. Wu, McDonald, Liu, Chaguturu, Klaassen (2012) PLOS ONE.