Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept

Abstract

The first step in the development of skin sensitisation to a chemical, and in the elicitation of further allergic contact dermatitis (ACD), is the binding of the allergen to skin proteins after penetrating into the epidermis. The so-formed antigenic adduct is then recognised by the immune system as foreign to the body. Sensitising organic hydroperoxides derived from autoxidation of natural terpenes are believed to form antigens through radical-mediated mechanisms, although this has not yet been established. So far, in vitro investigations on reactive radical intermediates derived from these skin sensitisers have been conducted in solution, yet with experimental conditions being far away from real-life sensitisation. Herein, we report for the first time, the potential use of EPR spin-trapping to study the in situ generation of free radicals derived from cumene hydroperoxide CumOOH in a 3D reconstructed human epidermis (RHE) model, thus much closer to what may happen in vivo. Among the undesirable effects associated with dermal exposure to CumOOH, it is described to cause allergic and irritant dermatitis, being reported as a significant sensitiser. We considered exploiting the usage of spin-trap DEPMPO as an extensive view of all sort of radicals derived from CumOOH were observed all at once in solution. We showed that in the Episkin^TM RHE model, both by incubating in the assay medium and by topical application, carbon radicals are mainly formed by redox reactions suggesting the key role of CumOOH-derived carbon radicals in the antigen formation process.

Keywords:

• Skin sensitisers
• radicals
• EPR spectroscopy
• spin-trapping
• reconstructed human epidermis

Acknowledgements

The authors thank the French Ministry of Research, the University of Strasbourg, the CNRS and the REseau NAtional de Rpe interDisciplinaire (RENARD, Fédération IR-RPE CNRS #3443).

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Additional information

Funding

This research was supported by the International Center for Frontier Research in Chemistry (icFRC, Strasbourg, France, grant number JLE-FRC-0001).