Common fragrance chemicals activate dendritic cells in coculture with keratinocytes.

BACKGROUND: Fragrances are important contact allergens; however, investigation of their skin sensitization potency has been challenging in new approach methods (NAMs). Many fragrance chemicals are susceptible to autoxidation or can be metabolized by enzymes constitutively expressed in skin keratinocytes. Strong sensitizers can be formed in both of these processes. Further, keratinocytes can modulate the dendritic cell (DC) activation and maturation potential, a key event in the acquisition of contact allergy. OBJECTIVES: To evaluate the 2D coculture model consisting of keratinocytes and DCs using different weak to moderate sensitizing fragrance chemicals. Further, to investigate fragrances and related oxidation products in the in vitro model and compare to in vivo data. METHODS: Chemicals were tested in the coculture activation test (COCAT), consisting of HaCaT keratinocytes and THP-1 cells. THP-1 cell surface expression of costimulatory and adhesion molecules (CD86 and CD54) collected after 24 h incubation with the chemicals was analysed using flow cytometry. RESULTS: Twenty-four molecules were tested positive, three were negative (n = 27). Four pairs were evaluated, with aldehydes showing a 6- to 13-fold stronger responses compared to their corresponding alcohols. CONCLUSIONS: Results provide insight into the activation of DC in their natural environment of keratinocytes. α,ß-Unsaturated alcohols were classified as weaker sensitizers compared to their corresponding aldehydes. In sum, testing of fragrances retrieved results in good agreement with in vivo data.

Understanding the skin sensitization capacity of ascaridole: a combined study of chemical reactivity and activation of the innate immune system (dendritic cells) in the epidermal environment.

To improve the prediction of the possible allergenicity of chemicals in contact with the skin, investigations of upstream events are required to better understand the molecular mechanisms involved in the initiation of allergic reactions. Ascaridole, one of the compounds responsible for skin sensitization to aged tea tree oil, degrades into intermediates that evolve via different mechanisms involving radical species. We aimed at broadening the knowledge about the contribution of radical intermediates derived from ascaridole to the skin sensitization process by assessing the reactivity profile towards amino acids, identifying whether free radicals are formed in a reconstructed human epidermis (RHE) model and their biological properties to activate the immune system, namely dendritic cells in their natural context of human HaCaT keratinocytes and RHE. Electron paramagnetic resonance combined to spin-trapping in EpiSkin™ RHE confirmed the formation of C-radicals in the epidermal tissue from 10 mM ascaridole concentration, while reactivity studies toward amino acids showed electrophilic intermediates issued from radical rearrangements of ascaridole as the main reactive species. Activation of THP-1 cells, as surrogate for dendritic cells, that were cocultured with HaCaT was significantly upregulated after treatment with low micromolar concentrations based on cell surface expression of the co-stimulatory molecule CD86 and the adhesion molecule CD54. Placing THP-1 cells underneath the RHE allowed us to monitor which of the concentrations that produce radical(s) and/or protein antigens in the epidermal skin environment promote the activation of dendritic cells. We detected no significant upregulation of CD86/CD54 after topical RHE application of concentrations up to 30 mM ascaridole (t = 24 h) but clear upregulation after 60 mM.

Sensitization potential and potency of terpene hydroperoxides in the cocultured activation test method.

BACKGROUND: Positive patch test reactions to mixtures of oxidized terpenes containing allergenic hydroperoxides are frequently reported. However, human sensitization data for these hydroperoxides are not available. OBJECTIVES: To analyse and evaluate the human sensitization potential and potency of hydroperoxides in vitro by using human cells. MATERIALS/METHODS: Limonene-1-hydroperoxide, limonene-2-hydroperoxide, citronellol-7-hydroperoxide, cumene hydroperoxide, 1-(1-hydroperoxy-1-methylethyl)cyclohexene and mixtures of citronellol hydroperoxides (isomers at positions 6 and 7) and linalool hydroperoxides (isomers at positions 6 and 7) were studied. All compounds were synthesized except for cumene hydroperoxide, which was commercially available. Their potential and potency to activate dendritic cells (DCs) was evaluated by measuring the upregulation of CD86 and CD54 on THP-1 cells upon exposure in the cocultured activation test (COCAT) consisting of HaCaT cells (human keratinocyte cell line) and THP-1 monocytes (as a surrogate for DCs). RESULTS: Hydroperoxides upregulated CD86 and/or CD54 on cocultured THP-1 cells in a concentration-dependent manner. The results are comparable with their sensitization potency ranking in predictive animal models. CONCLUSIONS: For the first time, the human sensitization potential and potency of several hydroperoxides were determined by the use of human cells and the COCAT method.

Recent progress in N-acetyltransferase research: 7th international workshop on N-acetyltransferases (NAT): workshop report.

The 7th International Workshop on N-Acetyltransferases (NAT), held from 18 to 20 June 2016, was hosted by Brunhilde Blömeke and her team at the Trier University (Germany). The workshop addressed important aspects and latest advancements in the fields of NAT enzymes, endogenous functions of NATs, NAT gene nomenclature, genetic polymorphisms, and their associations with diseases as well as their use in diagnosis. Representatives from the leading teams performing research on NATs presented their excellent work, discussed the latest results, and created new ideas in the field of N-acetyltransferase research.

Impact of para-phenylenediamine on cyclooxygenases expression and prostaglandin formation in human immortalized keratinocytes (HaCaT).

para-Phenylenediamine, a monocyclic arylamine, is a frequently used chemical and ingredient of oxidative hair coloring products. Thus exposure occurs predominantly via skin. Cyclooxygenases, the key enzymes in prostaglandin synthesis, exhibit manifold physiological and pathophysiologial functions in skin and skin cells such as keratinocytes. We studied if para-phenylenediamine impacts on the expression of enzymes in the cyclooxygenase pathway in human immortalized keratinocytes (HaCaT) as a model for keratinocytes. We analyzed COX-1, COX-2 and cPLA(2) steady state mRNA levels for 100-400 microM PPD after 2-24 h and found clear COX-2 induction for 400 microM PPD after 24 h, while cPLA(2) and COX-1 levels were increased dose-dependently between 8 and 24 h. Increased expression was accompanied by enhanced prostaglandin E(2) and F(2alpha) formation. Specific involvement of COX enzymes was confirmed by prostaglandin analysis in the presence of exogenous arachidonic acid and inhibition experiments using COX inhibitor NS-398. In addition, para-phenylenediamine-induced prostaglandin formation was completely inhibited in cells pre-stimulated with the anti-oxidant N-acetylcysteine. N-acetylation of PPD was observed in HaCaT yielding mono-acetyl-PPD (MAPPD) and di-acetyl-PPD (DAPPD). Further investigations of MAPPD and DAPPD and the generated auto-oxidation product Bandrowski's base (BB) found that these compounds were not able to impact on COX enzyme expression and activity. In sum, these results demonstrate that para-phenylenediamine, but not its generated acetylated derivatives or BB, induces COX expression and activity in human keratinocytes likely via oxidative processes.

Expression of N-acetyltransferase in monocyte-derived dendritic cells.

Dendritic cells (DCs) are known to internalize, process, and present low-molecular-weight chemicals to T cells in the course of the sensitization and elicitation phase of allergic contact dermatitis. Thus, DCs may be involved in metabolic activation and detoxification of haptens and thereby influence the quantity of immunogens inducing sensitization. Recently, the cytochrome P-450 enzymes expressed in monocyte-derived dendritic cells (MoDCs) were characterized. In the present study, N-acetyltransferase 1 and 2 (NAT-1 and -2) mRNA expression and N-acetylation capacities of these cells were investigated. Monocytes from healthy donors were incubated with granulocyte-monocyte colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 6 d and the resulting immature MoDCs were characterized by flow cytometry. Total RNA from MoDCs was isolated, reverse transcribed, and polymerase chain reaction (PCR) for NAT-1 and NAT-2 mRNA was performed. Data showed the presence of mRNA for NAT-1 (9 of 10 donors) and NAT-2 (8 of 10 donors) in these cells. NAT-1 enzyme activities were achieved through acetylation of para-aminobenzoic acid (PABA) by MoDC cell lysates and activities varied between 23.4 and 26.6 nmol/mg/min. In addition, complete cell acetylation of para-phenylenediamine (PPD), estimated via analysis of monoacetyl-PPD (MAPPD) and diacetyl-PPD (DAPPD) in cell culture supernatants, confirmed that in vitro generated MoDCs (4 of 6 donors) express metabolic active N-acetyltransferase (NAT-1). In the case of PPD, our results emphasize that N-acetylation status may influence the amounts of immunogens available for sensitization to PPD.