The use of MI across European countries is extensive. In view of the ongoing epidemic of MI contact allergy, an evaluation of the safety of MI in paints is needed.

Limonene is one of the most commonly used fragrance compounds in western countries today. When exposed to air, it autoxidises, forming hydroperoxides that are strong contact allergens. To cause allergic contact dermatitis (ACD), the hydroperoxides are considered to bind covalently to proteins in the skin via a radical pathway. Consequently, the nature and reactions of the radicals formed from the hydroperoxides are important. We have examined the radical formation from, and sensitizing potential of, three allylic hydroperoxides. Two of these are found in the oxidation mixture of limonene, while the third is a synthetic structural analogue. The identity of the radicals formed from these hydroperoxides has been studied in radical trapping experiments. Chemical trapping experiments were performed using 5,10,15,20-tetraphenyl-21 H,23 H-porphine iron(III) chloride [Fe(III)TPPCl 3] as an initiator and 1,1,3,3-tetramethylisoindolin-2-yloxyl as a radical trapper. Electron paramagnetic resonance experiments using photolysis for initiation were performed with and without 5-diethoxy-phosphoryl-5-methyl-1-pyrroline N-oxide. Our results demonstrate the ability of the studied hydroperoxides to form peroxyl, allyloxyl, and oxiranylcarbinyl radicals. These radicals can potentially react with proteins to form immunogenic hapten-protein complexes relevant for ACD. The sensitizing potency of the hydroperoxides was studied in the murine local lymph node assay. All three hydroperoxides were found to be potent sensitizers with some variations, which can be related to the identity and quantity of the radicals formed. The results indicate that both carbon- and oxygen-centered radicals are important intermediates in the formation of hapten-protein complexes and that the sensitizing potency of the hydroperoxides is related to their structures.

Many terpenes used as fragrance compounds autoxidize when exposed to air, forming allylic hydroperoxides that have the potential to be skin contact allergens. To trigger the immunotoxicity process that characterizes contact allergy, these hydroperoxides are supposed to bind covalently to proteins in the skin via radical pathways. We investigated the formation of reactive radical intermediates from 7-hydroperoxy-3,7-dimethylocta-1,5-dien-3-ol and 2-hydroperoxylimonene, responsible for the sensitizing potential acquired by autoxidized linalool and limonene. Both compounds were synthesized through new short and reproducible synthetic pathways. The hydroperoxide decomposition catalyzed by Fe(II)/Fe(III) redox systems, playing a key role in degradating peroxides in vivo, was examined by spin-trapping-EPR spectroscopy. Alkoxyl and carbon-centered free radicals derived from the hydroperoxides were successfully trapped by the spin-trap 5,5-dimethyl-1-pyrroline N-oxide, whereas peroxyl radicals were characterized by spin-trapping studies with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. Using liquid chromatography combined with mass spectrometry, we demonstrated the formation of adducts, via radical mechanisms induced by Fe(II)/Fe(III), between the hydroperoxides and N-acetylhistidine methyl ester, a model amino acid that is prone to radical reactions. Free radicals derived from these hydroperoxides can thus induce amino acid chemical modifications via radical mechanisms. The study of these mechanisms will help to understand the sensitizing potential of hydroperoxides.

Our findings suggest that mixtures of allergens increase the primary response that potentiates the generation of memory T cells in response to the specific allergen. Thus, allergen mixtures enhance both induction and elicitation of contact allergy.

In addition to pure synthetic fragrance materials several natural extracts are still in use in the perfume industry. Among them oak moss absolute, prepared from the lichen Evernia prunastri (L.) Arch., is considered a major contact sensitizer and is therefore included in the fragrance mix used for diagnosing perfume allergy. The process of preparing oak moss absolute has changed during recent years and, even though several potential sensitizers have been identified from former benzene extracts, its present constituents and their allergenic status are not clear. In the study reported here, we applied a method developed for the identification of contact allergens present in natural complex mixtures to oak moss absolute. The method is based on the combination of bioassay-guided chemical fractionation, gas chromatography-mass spectrometry analysis and structure-activity relationship studies. Our first results showed that atranol and chloroatranol, formed by transesterification and decarboxylation of the lichen depsides, atranorin and chloroatranorin, during the preparation of oak moss absolute, are strong elicitants in most patients sensitized to oak moss. Methyl-beta-orcinol carboxylate, a depside degradation product and the most important monoaryl derivative of oak moss from an olfactory standpoint, was also found to elicit a reaction in most patients.

No abstract

MI and other isothiazolinones are widely used in paints available in Europe. Their use does not seem to be decreasing.

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.