3Rs INFO HUB

AOP

After understanding the processes that happen in our body during skin sensitization, the targeting of alternative test methods becomes clearer.
Scheme of Adverse Outcome Pathway - Skin Sensitization

Nrf2/ARE-Pathway

It has been shown that skin sensitizers activate the ARE-pathway (=antioxidant response element). An activation of the pathway protects a cell from oxidative-induced stress. How does this work?

First of all – oxidative stress can have many causes for example UV-light which can itself cause the build-up of reactive oxygen species (ROS). To avoid the interaction of reactive molecules with cell proteins or DNA our body produces more metabolizing enzymes to eliminate the substance.

As we already know, skin sensitizers can activate the pathway too. The induction of enzymes is what we can finally measure in our test method via Luciferase Genes.

Small electrophilic substances can bind to Keap1 which is the sensor protein of the pathway. Now Nrf2 can dissociate from Keap1 which it was bound to. Nrf2 is a transcription factor that causes the transcription of ARE-dependent genes which are
coding detoxifying enzymes.

Luciferase

Luciferases are enzymes that catalyze a reaction which causes light. Bioanalytically they are used as so called “Reporter Genes”. We are able to insert the luciferase-gene behind a certain promotor region on our DNA. If the gene is induced – for example because of a skin sensitizer – we can measure the amount of expressed genes by measuring the power of the light.

Mutual Acceptance of data

In 1981 the OECD established the principle of Mutual Acceptance of Data, which states that…

“the test data generated in any member country in accordance with OECD Test Guidelines and Principles of Good Laboratory Practice (GLP) shall be accepted in other member countries for assessment purposes and other uses relating to the protection of human health and the environment”.

This principle has various advantages like saving huge amounts of money and less animal-testing.

Sources

OECD:
Guidance document on Good In Vitro Method Practice (GIVIMP)

This is a lexicon, written by the OECD to explain important terms such as “mutual acceptance of data” (MAD), which is an important principle to reduce animal testing worldwide.
Link to OECD website

Keratinosens/ ARE-dependent pathway:
Link to pdf from Eurofins.

Eurofins provides different analytic testing services for clients all around the globe. In this abstract they informed about the KeratinoSens as an alternative test method and gave the reader some extra information about the Nrf2-ARE-dependent pathway for better understanding.
The role of transcription factor NrF2 in skin cells metabolism

Luciferase genes
The wikipedia article explains why Luciferase genes can be used as a biochemical marker in analytics and helps understanding the evaluation of KeratinoSens.