In Situ Metabolism of Cinnamyl Alcohol in Reconstructed Human Epidermis: New Insights into the Activation of This Fragrance Skin Sensitizer.

Chemical modification of epidermal proteins by skin sensitizers is the molecular event which initiates the induction of contact allergy. However, not all chemical skin allergens react directly as haptens with epidermal proteins but need either a chemical (prehaptens) or metabolic (prohaptens) activation step to become reactive. Cinnamyl alcohol has been considered a model prohapten, as this skin sensitizer has no intrinsic reactivity. Therefore, the prevailing theory is that cinnamyl alcohol is enzymatically oxidized into the protein-reactive cinnamaldehyde, which is the sensitizing agent. Knowing that reconstructed human epidermis (RHE) models have been demonstrated to be quite similar to the normal human epidermis in terms of metabolic enzymes, use of RHE may be useful to investigate the in situ metabolism/activation of cinnamyl alcohol, particularly when coupled with high-resolution magic angle spinning nuclear magnetic resonance. Incubation of carbon-13 substituted cinnamyl derivatives with RHE did not result in the formation of cinnamaldehyde. The metabolites formed suggest the formation of an epoxy-alcohol and an allylic sulfate as potential electrophiles. These data suggest that cinnamyl alcohol is inducing skin sensitization through a route independent of the one involving cinnamaldehyde and should therefore be considered as a skin sensitizer on its own.

In situ chemical behaviour of methylisothiazolinone (MI) and methylchloroisothiazolinone (MCI) in reconstructed human epidermis: a new approach to the cross-reactivity issue.

BACKGROUND: Methylisothiazolinone (MI) [with methylchloroisothiazolinone (MCI) in a ratio of 1:3, a well-recognized allergenic preservative] was released as an individual preservative in the 2000s for industrial products and in 2005 for cosmetics. The high level of exposure to MI since then has provoked an epidemic of contact allergy to MI, and an increase in MI/MCI allergy. There are questions concerning the MI/MCI cross-reaction pattern. OBJECTIVES: To bring a new perspective on the MI/MCI cross-reactivity issue by studying their in situ chemical behaviour in 3D reconstructed human epidermis (RHE). METHODS: MI and MCI were synthesized with (13) C substitution at positions C-4/C-5 and C-5, respectively. Their in situ chemical behaviours in an RHE model were followed by use of the high-resolution magic angle spinning nuclear magnetic resonance technique. RESULTS: MI was found to react exclusively with cysteine thiol residues, whereas MCI reacted with histidines and lysines. The reaction mechanisms were found to be different for MI and MCI, and the adducts formed had different molecular structures. CONCLUSION: In RHE, different MI/MCI reactions towards different nucleophilic amino acids were observed, making it difficult to explain cross-reactivity between MI and MCI.

Matrix Effect of Human Reconstructed Epidermis on the Chemoselectivity of a Skin Sensitizing α-Methylene-γ-Butyrolactone: Consequences for the Development of in Chemico Alternative Methods.

Adoption of new legislations and social pressure are pushing toward the development of alternative methods to the use of animals for the assessment of most toxicological end-points including skin sensitization. To that respect, much efforts have been put in the first step of the adverse outcome pathway focusing on chemical interactions taking place between sensitizing chemicals or haptens and epidermal proteins. However, these in chemico approaches have been so far only based on the use of model nucleophiles, amino acids, peptides, or proteins in water/buffer solution and focused mainly on thiol reactivity. These studies even if bringing a valuable set of information are very far from reflecting chemical interactions that may happen between a xenobiotic and nucleophiles present in a complex heterogeneous tissue such as the epidermis. Recently, we have shown that using a high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) technique it was possible to characterize chemical interactions taking place between a skin sensitizer and nucleophilic amino acids present in a 3-D reconstructed human epidermis (RHE). We have now compared the chemical reactivity and chemoselectivity of a sensitizing α-methylene-γ-butyrolactone toward human serum albumin used as a model protein and RHE. Using this technique, we showed that amino acid modifications by this hapten was different according to the model used and that in RHE histidine residues seem to have an important role in the formation of adducts. Obviously, the role of histidine in the induction of skin sensitization has been so far neglected and should probably be taken into account for the refinement of in chemico approaches for the detection and potency classification of skin sensitizers.

HR-MAS NMR spectroscopy of reconstructed human epidermis: potential for the in situ investigation of the chemical interactions between skin allergens and nucleophilic amino acids.

High-resolution magic angle spinning (HR-MAS) is a nuclear magnetic resonance (NMR) technique that enables the characterization of metabolic phenotypes/metabolite profiles of cells, tissues, and organs, under both normal and pathological conditions, without resorting to time-consuming extraction techniques. In this article, we explore a new domain of application of HR-MAS, namely, reconstructed human epidermis (RHE) and the in situ observation of chemical interactions between skin sensitizers and nucleophilic amino acids. First, the preparation, storage, and analysis of RHE were optimized, and this work demonstrated that HR-MAS NMR was well adapted for investigating RHE with spectra of good quality allowing qualitative as well as quantitative studies of metabolites. Second, in order to study the response of RHE to chemical sensitizers, the ((13)C)methyldodecanesulfonate was chosen as an NMR probe, and we compared adducts formed on human serum albumin (HSA) in solution and adducts formed in RHE. Thus, while the modification of proteins or peptides in solution takes several days to lead to a significant amount of modification, in RHE the modifications of nucleophilic amino acids were observable already at 24 h. The chemioselectivity also appeared to be different with major modifications taking place on histidine, methionine, and cysteine residues in RHE, while on HSA, significant modifications were observed on lysine residues with the formation of methylated and dimethylated amino groups. We thus demonstrated that RHE could be used to investigate in situ chemical interactions taking place between skin sensitizers and nucleophilic amino acids. This opens perspectives for the molecular understanding of the skin immune system activation by sensitizing chemicals.