Skin sensitizers differentially regulate signaling pathways in MUTZ-3 cells in relation to their individual potency.

BACKGROUND: Due to the recent European legislations posing a ban of animal tests for safety assessment within the cosmetic industry, development of in vitro alternatives for assessment of skin sensitization is highly prioritized. To date, proposed in vitro assays are mainly based on single biomarkers, which so far have not been able to classify and stratify chemicals into subgroups, related to risk or potency. METHODS: Recently, we presented the Genomic Allergen Rapid Detection (GARD) assay for assessment of chemical sensitizers. In this paper, we show how the genome wide readout of GARD can be expanded and used to identify differentially regulated pathways relating to individual chemical sensitizers. In this study, we investigated the mechanisms of action of a range of skin sensitizers through pathway identification, pathway classification and transcription factor analysis and related this to the reactive mechanisms and potency of the sensitizing agents. RESULTS: By transcriptional profiling of chemically stimulated MUTZ-3 cells, 33 canonical pathways intimately involved in sensitization to chemical substances were identified. The results showed that metabolic processes, cell cycling and oxidative stress responses are the key events activated during skin sensitization, and that these functions are engaged differently depending on the reactivity mechanisms of the sensitizing agent. Furthermore, the results indicate that the chemical reactivity groups seem to gradually engage more pathways and more molecules in each pathway with increasing sensitizing potency of the chemical used for stimulation. Also, a switch in gene regulation from up to down regulation, with increasing potency, was seen both in genes involved in metabolic functions and cell cycling. These observed pathway patterns were clearly reflected in the regulatory elements identified to drive these processes, where 33 regulatory elements have been proposed for further analysis. CONCLUSIONS: This study demonstrates that functional analysis of biomarkers identified from our genomics study of human MUTZ-3 cells can be used to assess sensitizing potency of chemicals in vitro, by the identification of key cellular events, such as metabolic and cell cycling pathways.

Investigation of the sunscreen octocrylene's interaction with amino acid analogs in the presence of UV radiation.

Octocrylene is an organic UV filter, commonly used in sunscreens and cosmetics, which can give rise to both contact and photocontact allergy. Our aim was to investigate octocrylene's interaction with amino acid analogs in the presence of UV radiation to better understand the reason for octocrylene's photoallergenic capacity. The amino acid analogs were photolysed in presence and absence of octocrylene for 1 h in cyclohexane. The rate of degradation was considerably slower for all amino acid analogs when octocrylene was present in the mixture. Benzylamine, the lysine analog, did react with octocrylene during the photolysis and the corresponding amide was formed in an acylation reaction. By varying the benzylamine concentration and keeping the octocrylene concentration fixed the reaction rate was shown to be independent of the amine concentration. The same type of acylation reaction took place when octocrylene alone was photolysed in ethanol in which the ethyl ester was formed from octocrylene and ethanol. Our results suggest that octocrylene's ability to cause photocontact allergy could be due to its photoinduced reactivity toward primary amines and alcohols.

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,ß-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.

Clinical and experimental studies of octocrylene's allergenic potency.

BACKGROUND: Reports of positive patch test and photopatch test reactions to the chemical ultraviolet filter octocrylene have increased during the last decade. Little is known about the reason for octocrylene's allergenic activity. OBJECTIVES: To present and discuss the results of patch tests and photopatch tests with octocrylene, and to investigate the possible cause of its allergenic properties. METHODS: Results of patch tests and photopatch tests with octocrylene in patients with adverse skin reactions to sunscreen products and/or ketoprofen were collected. The allergenic potency of octocrylene was investigated in the murine local lymph node assay (LLNA). Chemical reactivity assays were used to mimic octocrylene's interaction with biomolecules. RESULTS: We report 23 cases of positive test reactions to octocrylene (5 patch test and 18 photopatch). Notably, many of these patients also had positive photopatch test reactions to ketoprofen and benzophenone-3. Octocrylene was shown to be a moderate sensitizer in the LLNA, and it reacted with amines such as lysine, but not with thiols such as cysteine. CONCLUSIONS: The clinical studies show that octocrylene is both a photocontact allergen and a contact allergen. Octocrylene's ability to cause contact allergy is probably attributable to its reactivity towards lysine. To be able to understand why octocrylene causes photocontact allergy, further studies are needed.

Mechanistic proposal for the formation of specific immunogenic complexes via a radical pathway: a key step in allergic contact dermatitis to olefinic hydroperoxides.

The widespread use of scented products causes an increase of allergic contact dermatitis to fragrance compounds in Western countries today. Many fragrance compounds are prone to autoxidation, forming hydroperoxides as their primary oxidation products. Hydroperoxides are known to be strong allergens and to form specific immunogenic complexes. However, the mechanisms for the formation of the immunogenic complexes are largely unknown. We have investigated this mechanism for (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH) by studying the formation of adducts in the reaction between this hydroperoxide and 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) in the presence of protected cysteine (NAc-Cys-OMe) or glutathione (GSH). Isolated adducts originate from the addition of the thiol group of NAc-Cys-OMe over the carbon-carbon double bonds of carvone. Furthermore, adducts between NAc-Cys-OMe and carveol as well as between GSH and carvone have been identified. The formation of these adducts most likely proceeds via the radical thiol-ene mechanism. The addition of a terpene moiety to cysteine offers an explanation of the specificity of the immune response to structurally different hydroperoxides. These results also explain the lack of cross-reactivity between carvone and Lim-2-OOH. In conclusion, we propose that immunogenic complexes of olefinic hydroperoxides can be formed via the radical thiol-ene mechanism. These complexes will be specific for the individual olefinic hydroperoxides due to the inclusion of a terpene moiety derived from the hydroperoxide.

Lavender oil lacks natural protection against autoxidation, forming strong contact allergens on air exposure.

BACKGROUND: Lavender oil is an essential oil frequently used as a fragrance ingredient and in traditional herbal medicine. We have previously studied the effect of air oxidation on the skin sensitizing potency of the monoterpenes linalyl acetate, linalool and beta-caryophyllene, the main constituents of lavender oil. OBJECTIVE: The aim of this study was to investigate if the autoxidation observed for the single synthetic terpenes, resulting in strong contact allergens, will take place also in lavender oil. METHODS: Lavender oil was exposed to air and the autoxidation was followed by chemical analysis. The sensitizing potency before and after air exposure was investigated in mice using the local lymph node assay. Patients with patch test reactions to oxidized linalool were tested to investigate if air-exposed lavender oil could elicit dermatitis in these individuals. RESULTS: The terpenes oxidized in air-exposed lavender oil at the same rates as the pure compounds exposed to air, and the same oxidation products were identified. The sensitizing potency of lavender oil increased accordingly on air exposure. Patch testing showed positive reactions to air-exposed lavender oil and also to oxidized linalyl acetate in patients with contact allergy to oxidized linalool. CONCLUSION: This study shows that lavender oil lacks natural protection against autoxidation, and that air-exposed lavender oil can be an important source of exposure to allergenic hydroperoxides.

Strain-induced substitutional lability in a Ru(II) complex of a hypodentate polypyridine ligand.

The ruthenium(II) complex of heptadentate N,N,N',N'-tetrakis(2-pyridylmethyl)-2,6-bis(aminomethyl)pyridine (tpap) was isolated as the hexafluorophosphate complex Ru(tpap)(PF6)2. The crystal structure has been determined for the triflate salt Ru(tpap)(CF3SO3)2.2H2O, which crystallizes in the monoclinic space group P2(1)/n. The structure was refined to a final R value of 0.0549 for 5894 observed reflections. The heptadentate ligand coordinates with six nitrogens, i.e. with two tertiary nitrogens and four pyridine nitrogens, one of the pyridines remaining un-coordinated. The resulting structure is significantly distorted from octahedral geometry with an equatorial Nsp3-Ru-Npyridine angle of 120 degrees. The consequence of the above steric strain is a labilization of the system and fluxional behaviour involving exchange between equatorially coordinated and non-coordinated pyridines has been observed by 1H NMR for Ru(tpap)(PF6)2 in d6-acetone solution. The activation parameters of DeltaG(not equal to 298) = 53 kJ mol(-1), DeltaH(not equal) = 56 +/- 1 kJ mol(-1) and DeltaS(not equal) = -10 +/- 3 J mol(-1) K(-1) were determined on the basis of NMR experiments. In addition electronic structure calculations applying density functional theory (DFT) have been performed in order to identify a transition state and to estimate the activation barrier. On the basis of NMR and DFT results the mechanism of isoexchange involving a hepta-coordinated intermediate has been proposed.