Moa Andresen Bergström

α-Terpinene, an antioxidant in tea tree oil, autoxidizes rapidly to skin allergens on air exposure.

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.

Cutaneous Metabolic Activation of Carvoxime, a Self-Activating, Skin-Sensitizing Prohapten

Bioactivation of low molecular weight compounds in the skin can cause contact sensitization. We have previously shown that the alpha, beta-R-unsaturated oxime R-carvoxime [1, (R)-2-methyl-5-isopropenylcyclohex-2-enone oxime] is bioactivated to two diastereomeric highly reactive and strongly sensitizing alpha, beta-epoxy oxime metabolites. To investigate if this metabolic activation is catalyzed by the major cytochrome P450 (P450) enzymes found in human skin, incubations of 1 with a skinlike P450 cocktail in the presence of glutathione were carried out. We identified three glutathione conjugates in the incubation mixture arising from two diasteomeric alpha, beta-epoxy oxime metabolites of 1, thus showing that the metabolic activation of 1 is P450-mediated. A P450 identification study using the individual P450 enzymes present in the skinlike P450 cocktail showed the involvement of P450 1A1 and 1B1 and also to some extent 2B6. P450 1B1 metabolism of 1 was found to be stereoselective as glutathione conjugates from only one of the alpha, beta-epoxyoxime metabolites were identified (metabolite 2). Additionally, 1 was found to be an inducer of P450 1B1 (but not 1A1) in human monocyte-derived dendritic cells (moDCs) and to some extent in normal human epidermal keratinocytes. A further transcriptional gene expression change observed in moDCs was a 44-fold upregulation of IL-8, a marker often used for assessment of sensitizing potential of contact allergens. The autoinduction of P450 1B1 by 1 may be a key event in the development of contact allergy to 1 and may also explain why only metabolite 2, and not 3, was found to elicit an allergic response in mice sensitized to 1. Our data show that the alpha, beta-unsaturated oxime 1 is bioactivated by human cutaneous P450, thus forming highly allergenic metabolites, and has the potential to induce its own bioactivation pathway, particularly in antigen-presenting cells.

Diphenylthiourea, a common rubber chemical, is bioactivated to potent skin sensitizers.

Diphenylthiourea (DPTU) is a known skin sensitizer commonly used as a vulcanization accelerator in the production of synthetic rubber, for example, neoprene. The versatile usage of neoprene is due to the multifaceted properties of the material; for example, it is stretchable, waterproof, and chemical- and abrasion-resistant. The wide application of neoprene has resulted in numerous case reports of dermatitis patients allergic to DPTU. The mechanism by which DPTU works as a contact allergen has not been described; thus, the aim of the present study was to investigate if DPTU is a prohapten that can be activated by skin metabolism. The metabolic activation and covalent binding of (14)C-labeled DPTU to proteins were tested using a skinlike cytochrome P450 (P450) cocktail containing the five most abundant P450s found in human skin (CYP1A1, 1B1, 2B6, 2E1, and 3A5) and human liver microsomes. The incubations were carried out in the presence or absence of the metabolite trapping agents glutathione, methoxylamine, and benzylamine. The metabolism mixtures were analyzed by LC-radiochromatography, LC-MS, and LC-MS/MS. DPTU was mainly metabolically activated to reactive sulfoxides resulting in desulfurated adducts in both enzymatic systems used. Also, phenylisothiocyanate and phenylisocyanate were found to be metabolites of DPTU. The sensitizing capacity of the substrate (DPTU) and three metabolites was tested in the murine local lymph node assay. Two out of three metabolites tested were strong skin sensitizers, whereas DPTU itself, as previously known, was negative using this mouse model. In conclusion, DPTU forms highly reactive metabolites upon bioactivation by enzymes present in the skin. These metabolites are able to induce skin sensitization and are probable causes for DPTU allergy. To increase the possibilities of diagnosing contact allergy to DPTU-containing items, we suggest that suitable metabolites of DPTU should be used for screening testing.

Oximes: Metabolic Activation and Structure?Allergenic Activity Relationships

Metabolic activation of chemicals (prohaptens) in the skin can cause allergic contact dermatitis. We have explored structure-allergenic activity relationships for seven potential oxime prohaptens using the local lymph node assay and a GSH trapping screen with liver microsomes. The general structure-allergenic activity relationships found were that an alpha,beta-unsaturation is necessary for an oxime to be a prohapten and that increased steric hindrance around this double bond leads to reduction in sensitizing capacity. We also found that sensitizing oximes can be distinguished in vitro from nonsensitizers by monitoring of mono-oxidized (+16 Da) GSH conjugates in the GSH trapping screen. However, care should be taken when interpreting data from GSH trapping screens, as nonsensitizers may also form GSH conjugates via alternative mechanisms. This investigation emphasizes the importance of considering cutaneous bioactivation in toxicity assessment of chemicals used in contact with the skin.

Synthesis of Urine Drug Metabolites: Glucuronosyl Esters of Carboxymefloquine, Indoprofen, (S)‐Naproxen, and Desmethyl (S)‐Naproxen

Abstract A general procedure for the synthesis of 1‐O‐acyl‐β‐D‐glucuronic acids using the benzyl 1-O-trichloroacetimidoyl-2,3,4-tri-O-benzyl-D-glucopyranuronate 6 as donor is exemplified by the synthesis of the urine metabolites of (S)‐naproxen, desmethyl (S)‐naproxen, indoprofen, and carboxymefloquine. The key intermediate benzyl 2,3,4‐tri‐O‐benzyl‐D‐glucopyranuronate 5 is easily accessible in four steps (29%) from the peracetylated β-D-glucuronic acid 1.