Hapten-protein binding: from theory to practical application in the in vitro prediction of skin sensitization.

In view of the forthcoming European Union ban on in vivo testing of cosmetic and toiletry ingredients, following the publication of the 7th amendment to the Cosmetics Directive, the search for practical, alternative, non-animal approaches is gathering pace. For the end-point of skin sensitization, the ultimate goal, i.e. the development and validation of alternative in vitro/in silico assays by 2013, may be achieved through a better understanding of the skin sensitization process on the cellular and molecular levels. One of the key molecular events in skin sensitization is protein haptenation, i.e. the chemical modification of self-skin protein(s) thus forming macromolecular immunogens. This concept is widely accepted and in theory can be used to explain the sensitizing capacity of many known skin sensitizers. Thus, the principle of protein or peptide haptenation could be used in in vitro assays to predict the sensitization potential of a new chemical entity. In this review, we consider some of the theoretical aspects of protein haptenation, how mechanisms of protein haptenation can be investigated experimentally and how we can use such knowledge in the development of novel, alternative approaches for predicting skin sensitization potential in the future.

Effect of glutathione on the covalent binding of the 13C-labeled skin sensitizer 5-chloro-2-methylisothiazol-3-one to human serum albumin: identification of adducts by nuclear magnetic resonance, matrix-assisted laser desorption/ionization mass spectrometry, and nanoelectrospray tandem mass spectrometry.

The covalent binding of 4-[(13)C]- and 5-[(13)C]-5-chloro-2-methylisothiazol-3-one (MCI) toward human serum albumin (HSA) was followed by (13)C and (1)H[(13)C] NMR spectroscopy. MCI was found to react with histidine through an addition-elimination at position 5, leading to stable substitution adducts, and with lysine to form open adducts of the thioamide or amide type. No other modification could be detected on either cysteine or tyrosine. In the presence of glutathione (GSH), we observed an increased covalent binding to lysine residues. This could be explained by the rapid reaction of GSH with MCI to form a chlorothioacyl intermediate very reactive toward primary amino groups of lysine residues. To further confirm these observations and map covalent binding sites, HSA samples modified by MCI with or without GSH were analyzed by matrix-assisted laser desorption/ionization mass spectrometry of tryptic digests and electrospray tandem mass spectrometry of modified peptides purified by reverse phase HPLC. About 80% of the HSA sequence was mapped, and several modified peptides were identified. When HSA was incubated with MCI without GSH, three peptides modified at histidine residues were characterized while when HSA was incubated in the presence of GSH, five peptides modified at histidine and lysine residues were identified. These experiments confirmed that modifications on lysine residues were of the amide and thioamide types. Observed modifications were in accordance with mass increases corresponding to structures identified by NMR, and an extra adduct corresponding to a double modification of His 338 was observed. Comparison of HSA-MCI and HSA-MCI-GSH samples confirmed that the presence of GSH increased the modification of lysine residues.