Contact allergy is caused by a wide range of chemicals after skin contact. Its clinical manifestation, allergic contact dermatitis (ACD), is developed upon repeated contact with the allergen. This perspective focuses on two areas that have yielded new useful information during the last 20 years: (i) structure-activity relationship (SAR) studies of contact allergy based on the concept of hapten-protein binding and (ii) mechanistic investigations regarding activation of nonsensitizing compounds to contact allergens by air oxidation or skin metabolism. The second area is more thoroughly reviewed since the full picture has previously not been published. Prediction of the sensitizing capacity of a chemical is important to avoid outbreaks of ACD in the population. Much research has been devoted to the development of in vitro and in silico predictive testing methods. Today, no method exists that is sensitive enough to detect weak allergens and that is robust enough to be used for routine screening. To cause sensitization, a chemical must bind to macromolecules (proteins) in the skin. Expert systems containing information about the relationship between the chemical structure and the ability of chemicals to haptenate proteins are available. However, few designed SAR studies based on mechanistic investigations of prohaptens have been published. Many compounds are not allergenic themselves but are activated in the skin (e.g., metabolically) or before skin contact (e.g., via air oxidation) to form skin sensitizers. Thus, more basic research is needed on the chemical reactions involved in the antigen formation and the immunological mechanisms. The clinical importance of air oxidation to activate nonallergenic compounds has been demonstrated. Oxidized fragrance terpenes, in contrast to the pure terpenes, gave positive patch test reactions in consecutive dermatitis patients as frequently as the most common standard allergens. This shows the importance of using compounds to which people are exposed when screening for ACD in dermatology clinics.

Allergic contact dermatitis is a complex syndrome representing immunological responses to cutaneous exposure to protein-reactive chemicals. Although many contact sensitizers directly can elicit this disorder, others (prohaptens) require activation. Knowledge regarding the activating mechanisms remains limited, but one possibility is metabolic activation by cytochrome P450 (CYP) enzymes in the skin. We have, after quantitative reverse transcriptase-PCR studies of the CYP content in 18 human skin samples, developed an enriched skin-like recombinant human (rh) CYP cocktail using CYP1A1, 1B1, 2B6, 2E1, and 3A5. To validate the rhCYP cocktail, a prohaptenic conjugated diene ((5R)-5-isopropenyl-2-methyl-1-methylene-2-cyclohexene) was investigated using: the skin-like rhCYP cocktail, a liver-like rhCYP cocktail, single rhCYP enzymes, liver microsomes, keratinocytes, and a dendritic cell (DC) assay. The diene was activated to sensitizing epoxides in all non-cell-based incubations including the skin-like rhCYP cocktail. An exocyclic epoxide metabolite ((7R)-7-isopropenyl-4-methyl-1-oxaspiro[2.5]oct-4-ene) was found to be mainly responsible for the allergenic activity of the diene. This epoxide also induced pronounced DC activation indicated by upregulation of IL-8. The skin-like rhCYP cocktail provides a simplified alternative to using skin tissue preparations in mechanistic studies of CYP-mediated skin metabolism of prohaptens and offers the future possibility of designing in vitro predictive assays for assessment of allergenic activity of prohaptens.

A hapten causing allergic contact dermatitis binds covalently to macromolecules via nucleophilic-electrophilic reactions or radical couplings. A prohapten can be seen as a chemically inert compound without electrophilic or radical forming properties. To exert its activity, the prohapten is activated, for example, metabolically, to the hapten. We have investigated the contact allergenic properties of a diene, (5R)-5-isopropenyl-2-methyl-1-methylene-2-cyclohexene (1), as a potential prohapten, and we found it to be a sensitizer in animal studies. The activity is likely to be exerted via epoxide metabolites. Thus, two potential metabolites of the investigated diene, (4S)-1,2-epoxy-4-isopropenyl-1-methyl-6-methylene-cyclohexane (3) and (7R)-7-isopropenyl-4-methyl-1-oxa-spiro[2.5]oct-4-ene (4), were synthesized and subjected to animal tests. Both epoxides were sensitizers. They also elicited significant reactions when tested in animals induced with 1, which indicates that they are formed from the diene in the skin. Furthermore, incubation of 1 with human liver microsomes produced both epoxides. The chemical reactivity of 1, 3, and 4 was investigated in relation to a hexapeptide, H-Pro-His-Cys-Lys-Arg-Met-OH. No adducts were obtained from reactions between the peptide and 1. However, epoxide 3 bound covalently to the cysteine residue and epoxide 4 to both the cysteine and proline residues. Since it is possible to relate the sensitizing capacity of a compound to its key physicochemical properties, knowledge-based expert systems have been developed to predict the toxicity of novel compounds by comparing the structure with activity data stored in the computer database. A diene related to 1 found in the knowledge-based expert system DEREK was considered as a nonsensitizer by this system. Our study indicates that conjugated dienes can be metabolized to contact allergens in the skin. Thus, when constructing predictive test methods based on SARs, it is important to analyze not only the virtual chemical structure of a compound but also its ability to act as a prohapten.

The monoterpene α-terpinene is used as a fragrance compound and is present in different essential oils. It is one of the components responsible for the antioxidant activity of tea tree oil. α-Terpinene is structurally similar to other monoterpenes, e.g., limonene, known to autoxidize on air exposure and form allergenic compounds. The aim of the present study was to investigate the possible autoxidation of α-terpinene at room temperature. To investigate the sensitization potency of air-exposed α-terpinene and the oxidation products formed, the murine local lymph node assay was used. Chemical analysis showed that α-terpinene degrades rapidly, forming allylic epoxides and p-cymene as the major oxidation products and also hydrogen peroxide. Thus, the oxidation pathway differs compared to that of, e.g., limonene, which forms highly allergenic hydroperoxides as the primary oxidation products on autoxidation. The sensitization potency of α-terpinene was increased after air-exposure. The allylic epoxides and a fraction, in which only an α,β-unsaturated aldehyde could be identified, were shown to be strong sensitizers in the local lymph node assay. Thus, we consider them to be the major contributors to the increased sensitization potency of the autoxidized mixture. We also investigated the presence of α-terpinene and its oxidation products in four different tea tree oil samples of various ages. α-Terpinene and its oxidation products were identified in all of the tea tree oil samples. Thus, from a technical perspective, α-terpinene is a true antioxidant since it autoxidizes rapidly compared with many other compounds, preventing these from degradation. However, as it easily autoxidizes to form allergens, its suitability can be questioned when used in products for topical applications, e.g., in tea tree oil but also in cosmetics and skin care products.

There is a great interest in developing in vitro/in silico methods for the prediction of contact allergenic activity. However, many proposed methods do not take the activation of prohaptens to sensitizers by skin metabolism into account. As a consequence, consumer products containing potent sensitizers could be marketed. To identify prohaptens, studies regarding their structure-activity relationships and the mechanisms of their activation must be conducted. In the present investigation, we have studied the structure-activity relationships for alkene prohaptens. A series of seven alkenes (1-7), all of the same basic structure but with variation in the number and position(s) of the double bond(s), were designed and screened for sensitizing capacity using the murine local lymph node assay. Compounds 1-7 were also incubated with liver microsomes in the presence of glutathione to trap and identify reactive metabolites. The metabolic conversion of three alkenes (9-11) to epoxides (12-15) was also studied along with comparison of their sensitizing capacity. Our results show that conjugated dienes in or in conjunction with a six-membered ring are prohaptens that can be metabolically activated to epoxides and conjugated with GSH. Related alkenes containing isolated double bonds and an acyclic conjugated diene were shown to be weak or nonsensitizers. For the first time, the naturally occurring monoterpenes alpha-phellandrene, beta-phellandrene, and alpha-terpinene were demonstrated to be prohaptens able to induce contact allergy. The difference in sensitizing capacity of conjugated dienes as compared to alkenes with isolated double bonds was found to be due to the high reactivity and sensitizing capacity of the allylic epoxides metabolically formed from conjugated dienes. We recommend that these structure-activity relationship rules are incorporated into in silico predictive databases and propose that the prediction of contact allergenic activity of suspected prohaptens is based on assessment of susceptibility to metabolic activation and chemical reactivity of potential metabolites.