LuSens Assay

The LuSens assay supports the discrimination between sensitizing and non-sensitizing chemicals. It is based on the same concepts as the OECD adopted KeratinoSens™.

The LuSens assay 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 LuSens assay is performed using genetically modified keratinocytes derived from HaCaT cells. It employs the reporter gene for luciferase under the control of an antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the up-regulation of the luciferase activity after 48 h incubation with test substance. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway by ‘electrophilic stress' caused by cystein reactive substances.