Potential of EPR spin-trapping to investigatein situfree 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 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.

Formation of methyl radicals derived from cumene hydroperoxide in reconstructed human epidermis: an EPR spin trapping confirmation by using13C-substitution

“…Radical initiation was triggered by a catalytic amount of Computer simulation of the signal displayed a mixture of different spin-adducts formed by addition of transient short-lived radicals derived from 1 to the -carbon of DEPMPO nitronyl group. In agreement with our previous investigations on CumOOH [21], spin-adducts were identified through their hyperfine coupling constants (hfccs) [25,26] as originating from HO • (Figure 2(e)), carbon centred radicals, probably methyl (Figure 2(f)), methoxy and/or possible peroxyl radicals (Figure 2 (Table 1). As only half of methyl radicals were 13 C-substituted, it was possible to observe two spin-adducts corresponding to two types of carbon radicals trapped with similar a N , a H and a P hfccs, being either non-substituted (Figure 2(f)) or 13 C-substituted (Figure 2(g)).…”

“…In a recent work, we employed EPR-ST for the first time in the skin allergy field to study the potential of this methodology to investigate in situ the formation of free radicals from skin allergens in 3D RHE models [21]. CumOOH was used as proof of concept and we demonstrated the formation of carbon radicals in RHE.…”

“…Analyses in solution were initially carried out to evaluate radicals formed from 13 C-substitued CumOOHs in comparison with previous studies we achieved with non-substituted CumOOH [21].…”

“…DEPMPO was chosen as the spin-trap since we observed previously that it allowed a broad view of all sort of radicals deriving from CumOOH [21]. Radical initiation was triggered by a catalytic amount of Computer simulation of the signal displayed a mixture of different spin-adducts formed by addition of transient short-lived radicals derived from 1 to the -carbon of DEPMPO nitronyl group.…”

“…We have recently reported an EPR-ST methodology allowing to investigate the formation of free radicals issued from skin allergens in a reconstructed human epidermis (RHE) 3D model, thus much closer to what may happen in vivo, by using CumOOH as proof of concept [21]. RHE are multi-layered keratinocyte cultures, major cell type in the epidermis playing a key role in skin inflammatory reactions.…”