α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity

Area of Science:

• Toxicology and Ecotoxicology
• In vitro testing methods
• Dermatology

Background:

• Existing non-animal test methods like KeratinoSens™ and LuSens evaluate key event 2 (KE-2) in skin sensitization but require separate cytotoxicity tests.
• This necessitates a more integrated and efficient approach for assessing the keratinocyte response component of the Adverse Outcome Pathway (AOP).

Purpose of the Study:

• To develop a simple, accurate, non-animal test method for evaluating skin sensitization potential.
• To integrate cytotoxicity assessment with KE-2 evaluation using a dual luciferase assay.
• To introduce normalized ARE Activity (nAA) as a novel parameter for improved prediction.

Main Methods:

• Development of the α-Sens assay using a dual luciferase reporter system (Firefly and Renilla) in a single cell line.
• Establishment of a cell line containing an Antioxidant Response Element (ARE)-inducible Firefly luciferase reporter and a Thymidine kinase (TK) promoter-driven Renilla luciferase reporter.
• Testing of 28 chemicals (19 sensitizers, 9 non-sensitizers) and calculation of normalized ARE Activity (nAA).

Main Results:

• The α-Sens assay achieved high accuracy (96.4%), sensitivity (95.0%), and specificity (100%) compared to the local lymph node assay.
• The assay demonstrated 100% accuracy when compared to human data.
• α-Sens correctly identified phenyl benzoate and eugenol as sensitizers, unlike KeratinoSens™ or LuSens, by utilizing the nAA parameter.

Conclusions:

• The α-Sens assay, using normalized ARE Activity (nAA), offers superior prediction capacity for skin sensitization compared to existing KE-2 methods.
• It provides a simple, accurate, and integrated in vitro method for evaluating KE-2 in the skin sensitization process.
• α-Sens is a promising alternative for regulatory assessment of chemical skin sensitization potential.