KeratinoSens assay for the testing of skin sensitizers

The KeratinoSens™ test method supports the discrimination between sensitizing and non-sensitizing chemicals. It addresses one of the biological mechanisms described under key event 2 (events in keratinocytes) of the skin sensitization Adverse Outcome Pathway by measuring the activation of the Keap1-Nrf2-ARE pathway.

The Keap1-Nrf2-ARE regulatory pathway is reported to be a major regulator of cyto-protective responses to electrophile and oxidative stress by controlling the expression of detoxification, antioxidant and stress response enzymes and proteins. Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g., covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nucler factor-erythroid 12-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes.

The KeratinoSens™ is performed using an immortalized adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element. The quantitative measurement by luminescence detection of the luciferase gene induction is used as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test chemicals.