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

Abstract

Dermal exposure to cumene hydroperoxide (CumOOH) during manufacturing processes is a toxicological issue for the industry. Its genotoxicity, mutagenic action, ability to promote skin tumor, capacity to induce epidermal hyperplasia and aptitude to induce allergic and irritant skin contact dermatitis are well known. These toxic effects appear to be mediated through the activation to free radical species such as hydroxyl, alkoxyl and alkyl radicals characterized basically by electron paramagnetic resonance (EPR) and spin-trapping (ST) techniques. To be a skin sensitizer CumOOH needs to covalently bind to skin proteins in the epidermis to form the antigenic entity triggering the immunotoxic reaction. Cleavage of the O-O bond allows formation of unstable CumO • /CumOO • radicals rearranging to longer half-life specific carbon-centered radicals R • proposed to be at the origin of the antigen formation. Nevertheless, it is not still clear which R • are precisely formed in the epidermis and thus involved in the sensitization process. The aim of this work was to elucidate in conditions closer to real-life sensitization which specific R • are formed in a 3D reconstructed human epidermis (RHE) model by using 13 C-substituted CumOOH at carbon positions precursors of potentially reactive radicals and EPR-ST. We demonstrated that most probably methyl radicals derived from -scission of CumO • radicals occur in RHE through a one-electron reductive pathway suggesting that these could be involved in the antigen formation inducing skin sensitization. We also describe a coupling between nitroxide radicals and  position 13 C atoms that could be of an added value to the very few examples existing for the coupling of radicals with 13 C atoms.

Limonene and linalool hydroperoxides review: Pros and cons for routine patch testing

“…To overcome this, one possibility is to carry out spin‐trapping studies with target compounds site‐specific labelled with 13 C at positions precursors of potentially reactive radicals. Recent EPR‐ST studies with cumene hydroperoxide 13 C‐substituted at methyl positions confirmed the trapping of methyl radicals released by decomposition of the hydroperoxide in the RHE model 27 …”

“…Recent EPR-ST studies with cumene hydroperoxide 13 C-substituted at methyl positions confirmed the trapping of methyl radicals released by decomposition of the hydroperoxide in the RHE model. 27 In conclusion, we can see that radicals formed from hydroperoxides are key intermediates that could explain the reactivity with skin proteins and, therefore, their sensitizing potential. Moreover, we now know that these intermediates are formed in a 3D model of the epidermis, under conditions closer to what may occur in vivo.…”

Electron paramagnetic resonance and spin trapping to detect free radicals from allergenic hydroperoxides in contact with the skin: From the molecule to the tissue

“…This needed to further optimize the concentrations of products used in order to achieve the best S/N ratio. 90 To identify radical intermediates potentially involved in skinsensitization processes, this methodology has been further successfully applied to skin sensitizers such as endoperoxide Asc, 91 and ROOHs derived from autoxidation of linalool, 85 limonene, 86 or citronellol. We quickly noticed that concentrations of spin trap and ROOH needed in the case of EPR-ST studies in RHE are, in general, at least 10-fold higher than those giving a good S/N ratio signal in solution, yet still within the typical range of clinical ACD diagnostic tests.…”

“…As an example, our EPR-ST studies in RHE with Cum-OOH with 13 C-substitution at methyl positions confirmed the presence of methyl radicals issued from decomposition of the hydroperoxide in the epidermis model (Figure 12). 90…”

Skin sensitization to fragrance hydroperoxides: interplay between dendritic cells, keratinocytes and free radicals

“…Keratinocytes could be involved by determining the effective dose and contributing maturation signals. Focusing on keratinocytes and the early events of hydroperoxide activation, we reported lately the generation of radicals by linalool hydroperoxides and cumene hydroperoxide in situ using reconstructed human epidermis (RHE) 13,40 . Here we evaluated the individual responsiveness and magnitude of DC maturation by important hydroperoxides in 2D and 3D coculture models with keratinocytes.…”