Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity and activation of the innate immune system (dendritic cells) in the epidermal environment

Abstract

To improve the prediction of the possible allergenicity of chemicals in contact with the skin, investigations of upstream events are required to better understand the molecular mechanisms involved in the initiation of allergic reactions. Ascaridole, one of the compounds responsible for skin sensitization to aged tea tree oil, degrades into intermediates that evolve via different mechanisms involving radical species. We aimed at broadening the knowledge about the contribution of radical intermediates derived from ascaridole to the skin sensitization process by assessing the reactivity profile towards amino acids, identifying whether free radicals are formed in a reconstructed human epidermis (RHE) model and their biological properties to activate the immune system, namely dendritic cells in their natural context of human HaCaT keratinocytes and RHE. Electron paramagnetic resonance combined to spin-trapping in EpiSkin TM RHE confirmed the formation of C-radicals in the epidermal tissue from 10 mM ascaridole concentration, while reactivity studies toward amino acids showed electrophilic intermediates issued from radical rearrangements of ascaridole as the main reactive species. Activation of THP-1 cells, as surrogate for dendritic cells, that were cocultured with HaCaT was significantly upregulated after treatment with low micromolar concentrations based on cell surface expression of the co-stimulatory molecule CD86 and the adhesion molecule CD54. Placing THP-1 cells underneath the RHE allowed us to monitor which of the concentrations that produce radical(s) and/or protein antigens in the epidermal skin environment promote the activation of dendritic cells. We detected no significant upregulation of CD86/CD54 after topical RHE application of concentrations up to 30 mM ascaridole (t=24 h) but clear upregulation after 60 mM.

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

“…In Figure 11, experimental spin adducts and subsequent simulations/deconvolution obtained for Asc are shown for parallel studies in solution and RHE. 91 Both investigations highlight the potential involvement of mostly C-centered radicals in Asc-induced ACD.…”

“…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.…”

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

“…We recently demonstrated by electron paramagnetic resonance (EPR) spectroscopy combined with the spin trapping (ST) technique that in reconstructed human epidermis (RHE) oxygenated and carbon‐centred radicals are formed after exposure to cumene‐OOH, 12–14 ascaridole 14 and linalool‐OOHs using concentrations based on clinical patch‐test studies 13 . The formed radicals may initiate the formation of specific antigens that are taken up, processed and cause maturation of DCs.…”

Chemical Compounds Responsible for Skin Allergy to Complex Mixtures: How to Identify Them?

“…Cleavage of the O-O bond of Asc produces the loss of isopropyl radicals (iPr • ), which forms the reactive ketone. Recently we have demonstrated in our laboratory that Asc is activated in the skin to form alkoxyl radicals that further rearrange to carbon radicals and reactive electrophilic species, and that under these conditions dendritic cell activation is promoted [42]. Peaks with m/z 478 were also observed.…”