Limonene Hydroperoxides in Allergic Contact Dermatitis. Radical Formation, Sensitizing Capacity and Immunogenic Complex Formation

Sammanfattning: Contact allergy to fragrance compounds is an increasing problem in the western countries today. R-Limonene is one of the most common fragrance compounds; it is used in hygiene products and cosmetics as well as in industrial products such as hand cleansers and degreasers. R-Limonene is prone to autoxidation and it has been shown that 2-3% of consecutive dermatitis patients are allergic to oxidized limonene or the hydroperoxide fraction of the oxidation mixture. This thesis examines limonene hydroperoxides, what radicals they can form, their sensitizing capacities, and a possible mechanism for immunogenic complex formation. Six structurally similar hydroperoxides were studied. Two of these are naturally occurring in oxidized limonene (limonene-1-hydroperoxide and limonene-2-hydroperoxide), while the others are synthetic structural analogues used for SAR-studies. The formation of radicals was studied in radical trapping experiments using iron porphyrin as a model for enzyme-initiated radical formation. All hydroperoxides formed large amounts of radicals and the trapping experiments showed that the identity and quantity of radicals formed depend on the structure of the hydroperoxide. In combination with the sensitizing capacities, the results also indicate that the alkoxyl radicals are the most important in the immunogenic complex formation. The sensitizing capacities were studied in the local lymph node assay (LLNA) and all hydroperoxides were found to be potent sensitizers. In a modified LLNA, comprising non-pooled lymph nodes and statistical evaluation, limonene-1-hydroperoxide was significantly more sensitizing compared to two other hydroperoxides. The clinical relevancy of this result was demonstrated in a limited study where more allergic reactions to limonene-1-hydroperoxide compared to limonene-2-hydroperoxide were recorded in individuals with known contact allergy to oxidized limonene. The immunogenic complex formation of limonene-2-hydroperoxide was studied in a model using amino acids. Limonene-2-hydroperoxide forms carvone that reacts with thiyl radicals from cysteine according to the thiol-ene reaction. The identification of a carvone-cysteine adduct indicates a possible radical mechanism for the immunogenic complex formation of olefinic hydroperoxides. The combined results indicate that the immunogenic complex formation of hydroperoxides may include two phases. The formation of large amounts of radicals in the skin weakens the antioxidant defense; this facilitates the addition of a compound derived from the hydroperoxide to a protein via a radical mechanism, resulting in a specific immunogenic complex. This form of action explains why all hydroperoxides are strong sensitizers with very small differences in their sensitizing capacities. In summary, the results presented in this thesis demonstrate that the radical formation of the hydroperoxides depends on their structure and influence the sensitizing capacity of the hydroperoxide. In addition, the formation of protein radicals and addition of a compound originating from the hydroperoxide via the thiol-ene reaction is proposed as a possible mechanism of immunogenic complex formation of olefinic hydroperoxides.