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

Abstract

A major research topic consists of revealing the contribution of radical-mediated reactions in dermatological diseases related to xenobiotic-induced stress to succeed risk-assessment procedures protecting producers and consumers. Allergic contact dermatitis is the clinically relevant consequence of skin sensitization, one of the most critical occupational and environmental health issues related to xenobiotics exposure.The first key event identified for the skin sensitization process to a chemical is its aptitude to react with epidermal proteins and form antigenic structures that will further trigger the immune response. Many chemical sensitizers are suspected to react through mechanisms involving radical intermediates. This review focuses on the recent progress we have accomplished over the last few years studying radical intermediates derived from skin-sensitizing chemicals by electron paramagnetic resonance in combination with the spin-trapping technique. Our work is carried out "from the molecule", performing studies in solution, "to the tissue", by the development of a methodology on a reconstructed human epidermis model, very close in terms of histology and metabolic/enzymatic activity to real human epidermis, that can be used as suitable biological tissue model. The benefits are to test chemicals under conditions close to human use and real-life sensitization exposures and benefit from the threedimensional (3D) microenvironment.

Autoxidized citronellol: Free radicals as potential sparkles to ignite the fragrance induced skin sensitizing pathway

“…2. Simulation and deconvolution afforded spectra of spin adducts corresponding to the trapping of hydroxyl (DEPMPO-OH spin adduct), C-centered (DEPMPO-R1 and DEPMPO-R2 spin adducts) and potentially peroxyl/alkoxyl radicals (DEPMPO-OOR/OR spin adducts), with distinctive hyperfine coupling constants (hfccs) reported in Table 1 and in agreement with previous investigations (Dikalov et al, 2003;Frejaville et al, 1995;Karoui et al, 2011;Kuresepi et al, 2020;Vileno et al, 2022). A very similar profile to that of Citr-7-OOH was observed with the mixture Citr-6-OOH/Citr-7-OOH, yet with distinct spin adduct proportions (Supplementary Material S.3).…”

“…In this case, spin adducts are formed by addition of transient radicals to the a position of the nitronyl group of DEPMPO (Supplementary Material S.1). In the case of ACD, an EPR-ST method was established "from the molecule to the tissue" using EpiSkin TM RHE as skin model (Kuresepi et al, 2020(Kuresepi et al, , 2018Lichter et al, 2021;Sahli et al, 2019a;Vileno et al, 2022). Prior to the RHE investigation, a preliminary study was carried out in solution aiming at determining the nature of the radicals formed in a minimal system when compared to the skin environment.…”

“…We have demonstrated by electron paramagnetic resonance (EPR) combined to the spin trapping technique (ST) that easy cleavage of the O-O bond (dissociation energy ca. 175 kJ.mol -1 ) of the hydroperoxides topically applied to a 3D model of reconstructed human epidermis (RHE) affords oxygen (O)-centered radicals (i.e., alkoxyl, peroxyl), further undergoing radical rearrangements to form more stable carbon (C)-centered radicals in the epidermis (Kuresepi et al, 2020;Lichter et al, 2021; For a review see Vileno et al, 2022). Evaluation of the reactivity in solution of these radicals toward amino acids prone to radical reactions showed that this intrinsic chemical reactivity could be potentially the needed trigger for the formation of the immunogenic antigen through covalent linking involving radical intermediates.…”

Antioxidant Activity and Skin Sensitization of Eugenol and Isoeugenol: Two Sides of the Same Coin?

“…EPR-ST investigations in solution point toward EPR fingerprints as testimony of radical intermediates issued from PhO • , which are very similar for Eug, IsoEug, and guaiacol (Figure ; See Supporting Information S1 for control experiments). Two types of spin adducts were identified on the basis of the analysis and deconvolution of the hyperfine coupling constants ( hfccs ), compared with existing databases and previous ST studies carried out in the laboratory (see Figure for example on Eug). , Taking Eug as an example, up to four spin adducts were identified. On the one hand, the hfccs of two spin adducts were typical of two C-radicals (Figure b; a H = 20.7 G, a N = 14.7 G, a P = 47.3 G; a H = 21.2 G, a N = 14.2 G, a P = 46.2 G) that are consistent with electronic delocalization of PhO • along the carbon skeleton of the molecules.…”

“…Following the studies in solution, similar investigations were carried out in RHE to highlight the formation of reactive radical intermediates induced by skin exposure to Eug and IsoEug. The general methodology had been developed elsewhere . Briefly, RHE was placed in 1.5 mL of HEPES, pretreated topically with DEPMPO in dimethyl sulfoxide/HEPES 1:1 (400 mM, 30 μL, dose/surface area 20 μmol.cm –2 ), and incubated for 15 min (37 °C, 5% CO 2 ) to allow a homogeneous diffusion of the spin trap within the skin model.…”