Synthesis and In SituBehavior of 1,4- and 2,5-(13C) Isotopomers of p-Phenylenediamine in Reconstructed Human Epidermis Using High Resolution Magic Angle Spinning NMR

p-Phenylenediamine (PPD) has been classified as a strong skin allergen, but when it comes to toxicological concerns, benzoquinone diamine (BQDI), the primary oxidation derivative of PPD, is frequently considered and was shown to covalently bind nucleophilic residues on model peptides. However, tests in solution are far from providing a reliable model, as the cutaneous metabolism of PPD is not covered. We now report the synthesis of two 13C substituted isotopomers of PPD, 1,4-(13C)p-phenylenediamine 1and 2,5-(13C)p-phenylenediamine 2, and the investigation of their reactivity in reconstructed human epidermis (RHE) using the high resolution magic angle spinning (HRMAS) NMR technique. RHE samples were first treated with 1or 2and incubated for 1 to 48 h. Compared to the control, spectra clearly showed only the signals of 1or 2gradually decreasing with time to disappear after 48 h of incubation. However, the culture media of RHE incubated with 1for 1 and 24 h, respectively, showed the presence of both monoacetylated- and diacetylated-PPD as major products. Therefore, the acetylation reaction catalyzed by N-acetyltransferase (NAT) enzymes appeared to be the main process taking place in RHE. With the aim of increasing the reactivity by oxidation, 1and 2were treated with 0.5 equiv of H2O2prior to their application to RHE and incubated for different times. Under these conditions, new peaks having close chemical shifts to those of PPD-cysteine adducts previously observed in solution were detected. Under such oxidative conditions, we were thus able to detect and quantify cysteine adducts in RHE (maximum of 0.2 nmol/mg of RHE at 8 h of incubation) while no reaction with other nucleophilic amino acid residues could be observed.