1 Scientific RepoRts | 6:21203 | DOI: 10.1038/srep21203 www.nature.com/scientificreports peptide Reactivity of Isothiocyanates – Implications for skin Allergy Isabella Karlsson 1 , Kristin samuelsson 2 , David J. ponting 2 , Margareta törnqvist 1 , Leopold L. Ilag 1 & Ulrika Nilsson 1 Skin allergy is a chronic condition that afects about 20% of the population of the western world. This disease is caused by small reactive compounds, haptens, able to penetrate into the epidermis and modify endogenous proteins, thereby triggering an immunogenic reaction. phenyl isothiocyanate (pItC) and ethyl isothiocyanate (eItC) have been suggested to be responsible for allergic skin reactions to chloroprene rubber, the main constituent of wetsuits, orthopedic braces, and many types of sports gear. In the present work we have studied the reactivity of the isothiocyanates PITC, EITC, and tetramethylrhodamine-6-isothiocyanate (6-TRITC) toward peptides under aqueous conditions at physiological pH to gain information about the types of immunogenic complexes these compounds may form in the skin. We found that all three compounds reacted quickly with cysteine moieties. For PITC and 6-TRITC the cysteine adducts decomposed over time, while stable adducts with lysine were formed. These experimental fndings were verifed by DFT calculations. Our results may suggest that the latter are responsible for allergic reactions to isothiocyanates. The initial adduct formation with cysteine residues may still be of great importance as it prevents hydrolysis and facilitates the transport of isothiocyanates into epidermis where they can form stable immunogenic complexes with lysine- containing proteins. Skin (contact) allergy is a chronic condition that afects as many as 20% of the population in the western world. 1 Allergic contact dermatitis, which is the clinical manifestation of contact allergy, is classifed as a T-lymphocyte mediated type IV hypersensitivity reaction. Contact allergy can be divided into two phases: sensitization and elicitation. Sensitization is the induction phase, which results in an immunological memory. Te elicitation phase takes place upon renewed contact with the same compound, which results in an infammatory reaction. Contact allergy is caused by reactive chemicals (called haptens) of low molecular weight (<1000 g/mol) and appropriate lipophilicity (LogP ~ 2), which are able to penetrate into the epidermis. Te haptens themselves are too small to be recognized by the immune system; however, through their reaction with an endogenous protein they have the ability to trigger an allergic response. 2 Te biomacromolecules in our body are rich in nucleophilic residues, such as thiols (-SH) in cysteine and primary amines (-NH 2 ) in lysine. Hence, most contact sensitizers are electrophilic compounds that can modify skin proteins by forming covalent bonds with these nucleophilic moieties. Te recommended OECD method for assessing contact sensitizing potency of various compounds is the murine local lymph node assay (LLNA). However, since 2009 testing of cosmetic products and ingredients on animals is banned in the EU and since 2013 marketing of cosmetic products and ingredients that have been tested on animals is also prohibited in the EU. Tus, most companies today use a combination of in chemico, in silico, and in vitro assays. One method commonly used is the direct peptide reactivity assay (DPRA), which has been validated by the European Center for Validation of Alternative Methods (ECVAM). Te DPRA measures the reactivity of possible skin allergens toward two heptapeptides – one with a reactive lysine (Ac-RFAAKAA-COOH) and one with a reactive cysteine (Ac-RFAACAA-COOH). 3,4 Usually, a 10-fold excess of the hapten is used and the peptide depletion afer 24 h is considered to be an indication of the sensitizing capacity of the compound. 3 Isothiocyanate (-NCS) is one example of an electrophilic functional group prone to react with both amines and thiols. Phenyl isothiocyanate (PITC) and ethyl isothiocyanate (EITC) (Fig. 1) have been suggested to be 1 Department of Environmental Science and Analytical Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden. 2 Dermatochemistry and Skin Allergy, Department of chemistry and Molecular Biology, University of Gothenburg, SE-412 96 Gothenburg, Sweden. Correspondence and requests for materials should be addressed to U.n. (email: ulrika.nilsson@aces.su.se) received: 22 May 2015 Accepted: 15 January 2016 Published: 17 February 2016 OPEN