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2,5-[(13)C]-Dimethyl-p-benzoquinonediimine was synthesized, and its reactivity toward several nucleophilic amino acids was studied by associated (13)C and (1)H{(13)C} NMR spectroscopies, combined with HPLC in tandem with mass spectrometry. A classical electrophile-nucleophile mechanism was observed for the reaction with N-acetyl-Cys. Adducts resulted from the reaction of the amino acid thiol group with the benzoquinonediimine electrophilic positions 3 and 6 as well as with the nitrogen atom of the imino group. However, N-acetyl-Trp and N-acetyl-Lys were chemically modified in the presence of 2,5-[(13)C]-dimethyl-p-benzoquinonediimine through the involvement of oxido-reduction processes. Heteronuclear (1)H{(13)C} NMR experiments allowed the identification of known oxidation intermediates derived from N-acetyl-Trp, indicating the oxidative strength of the reaction media. An adduct resulted from the reaction between the reduced form of the benzoquinonediimine and N-acetyl-formylkynurenine, which is the most known oxidation derivative of N-acetyl-Trp. In the case of N-acetyl-Lys, 4-amino-2,5-dimethyl-[(13)C]-formanilide and its derivative with N-acetyl-Lys at position 4 were obtained. A reaction mechanism was suggested in which the epsilon-NH(2) of the amino acid reacted on the electrophilic diimine to form an enamine adduct, which could then induce an oxidative deamination of N-acetyl-Lys. Further oxido-reduction mechanisms on the N-acetyl-alpha-aminoadipate-delta-semialdehyde formed might afford N,N-acetyl-formyl glutamic semialdehyde, which was considered as the powerful reactive species toward the reduced form of 2,5-[(13)C]-dimethyl-p-benzoquinonediimine. In the presence of N-acetyl-Tyr or N-acetyl-Met, the hydrolysis of the diimine parent compound was preferred, followed by a reduction to the hydroquinone form. In this study, we have thus shown that p-benzoquinonediimines, the first oxidation derivatives of allergenic p-amino aromatic compounds, can react with nucleophilic residues on amino acids through a set of complex mechanisms and must be seriously considered as potential candidates for the formation of antigenic structures responsible for allergic contact dermatitis.

Several human skin models employing primary cells and immortalized cell lines used as monocultures or combined to produce reconstituted 3D skin constructs have been developed. Furthermore, these models have been included in European genotoxicity and sensitization/irritation assay validation projects. In order to help interpret data, Cosmetics Europe (formerly COLIPA) facilitated research projects that measured a variety of defined phase I and II enzyme activities and created a complete proteomic profile of xenobiotic metabolizing enzymes (XMEs) in native human skin and compared them with data obtained from a number of in vitro models of human skin. Here, we have summarized our findings on the current knowledge of the metabolic capacity of native human skin and in vitro models and made an overall assessment of the metabolic capacity from gene expression, proteomic expression, and substrate metabolism data. The known low expression and function of phase I enzymes in native whole skin were reflected in the in vitro models. Some XMEs in whole skin were not detected in in vitro models and vice versa, and some major hepatic XMEs such as cytochrome P450-monooxygenases were absent or measured only at very low levels in the skin. Conversely, despite varying mRNA and protein levels of phase II enzymes, functional activity of glutathione S-transferases, N-acetyltransferase 1, and UDP-glucuronosyltransferases were all readily measurable in whole skin and in vitro skin models at activity levels similar to those measured in the liver. These projects have enabled a better understanding of the contribution of XMEs to toxicity endpoints.