Salen Kuresepi

Bertrand Vileno

Philippe Turek

Jean-Pierre Lepoittevin

Elena Giménez-Arnau

Potential of EPR spin-trapping to investigate in situ free radicals generation from skin allergens in reconstructed human epidermis: cumene hydroperoxide as proof of concept

Keywords: skin sensitizers, radicals, EPR spectroscopy, spin-trapping, reconstructed human epidermis

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Introduction

Allergic contact dermatitis (ACD) is the clinically relevant outcome of skin sensitization, one of the most important occupational and environmental health issues. It is a delayed-type hypersensitivity reaction, and the most frequent expression of immunotoxicity in humans [START_REF] Pesonen | Patch test results of the European baseline series among patients with occupational contact dermatitis across Europe - analyses of the European Surveillance System on Contact Allergy network, 2002-2010[END_REF]. Prevalence is rising worldwide, and exposure to allergens and the risk of skin sensitization have become an essential regulatory issue within industry. Thus it is nowadays crucial to predict the sensitization potential of chemicals before their marketing authorization to perform reliable risk assessment. In this context, more upstream investigations are required aiming at understanding molecular mechanisms involved in allergic-inflammatory reactions induced by sensitizers to cover all categories of molecules for risk assessment.

Chemical skin allergens (haptens) are low molecular weight compounds unable to stimulate by themselves an adaptive immune response after penetration into the epidermis. Immunogenicity is attained by their reaction with skin proteins, forming stable antigenic conjugates recognized and processed for presentation to the immune system [START_REF] Lepoittevin | Molecular aspects in allergic and irritant contact dermatitis[END_REF]. The usual mechanism for the hapten-protein interaction is the formation of covalent bonds via two electrons mechanisms. However, some allergens do not fit this model. In this case, there is a real belief that radical-mediated mechanisms are involved in the antigen formation [START_REF] Karlberg | Allergic contact dermatitisformation, structural requirements, and reactivity of skin sensitizers[END_REF].

Many natural terpenes widely used as fragrances in consumer goods are excellent targets for autoxidation and form allylic hydroperoxides (ROOHs) on air exposure. It has been shown that these ROOHs are frequent causes of ACD, form specific antigens and act as skin sensitizers [START_REF] Bråred Christensson | Hydroperoxides form specific antigens in contact allergy[END_REF]. We believe that sensitizing ROOHs form antigens via radical mechanisms starting with the cleavage of the O-O bond of weak dissociation energy (175 kJ mol -1 ). Such cleavage is an easy process allowing unstable alkoxyl (RO • )/peroxyl (ROO • ) radicals to efficiently convert to longer half-life carbon centered radicals (R • ) via intramolecular cyclization, fragmentation and/or hydrogen abstraction.

Our studies are focused on the identification of radical species derived from sensitizing ROOHs and on the study of their involvement in ACD for risk assessment purposes. We reported previously free radical generation from a range of ROOHs studied by EPR spin-trapping, specifically employed for the characterization of transient radicals in chemical and biological systems. Basically, the formed radical (either too short-lived or of too low concentration to be directly detected) reacts with a diamagnetic reagent (spin-trap) to form a more persistent radical (spin-adduct), whose EPR signature might be function on the trapped transient reactive species. The use of spin-traps 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) allowed the identification of different oxygen and carbon centered free radicals derived from ROOHs [START_REF] Giménez-Arnau | Identification of radical species derived from allergenic 15-hydroperoxyabietic acid and insights into the behaviour of cyclic tertiary allylic hydroperoxides in Fe(II)/Fe(III) systems[END_REF][START_REF] Kao | Synthesis of allylic hydroperoxides and EPR spin-trapping studies on the formation of radicals in iron systems as potential initiators of the sensitizing pathway[END_REF][START_REF] Johansson | Carbon-and oxygen-centered radicals are equally important haptens of allylic hydroperoxides in allergic contact dermatitis[END_REF]. Such studies revealed that several reactive radical intermediates could issue from the ROOHs depending on their chemical structure, suggesting that different immunogenic protein chemical modifications could lead to sensitization. However, such studies were carried out in solution (aqueous buffer or semiorganic), far from mimicking real life scenario.

The aim here was to transfer the EPR spin-trapping methodology developed in solution to the potential investigation of free radicals formation issued from ROOHs in a reconstructed human epidermis (RHE) 3D model, thus much closer to what may happen in vivo. RHE consist of normal multi-layered keratinocyte cultures, being the major cell type in the epidermis and playing a key role in skin inflammatory reactions. EPR has been used for dermatological purposes and skin research mainly for the detection of free radicals in UV-irradiated skin, melanoma investigation and in vivo EPR imaging directly in skin of human volunteers [START_REF] He | In vivo EPR imaging of the distribution and metabolism of nitroxide radicals in human skin[END_REF][START_REF] Jurkiewicz | EPR detection of free radicals in UV-irradiated skin: mouse versus human[END_REF][START_REF] Plonka | Electron paramagnetic resonance as a unique tool for skin and hair research[END_REF], but never in the skin allergy field. In this report we describe the technology transfer by using cumene hydroperoxide (CumOOH) as proof of concept and model for ROOHs. CumOOH is a common reference compound in EPR investigations [START_REF] Davies | Studies on the photolytic breakdown of hydroperoxides and peroxidized fatty acids by using electron spin resonance spectroscopy. Spin trapping of alkoxyl and peroxyl radicals in organic solvents[END_REF][START_REF] Ueda | ESR spin trapping studies on the reactions of hydroperoxides with Cu (II) complex[END_REF][START_REF] Taffe | Generation of free radicals from organic hydroperoxide tumor promoters in isolated mouse keratinocytes[END_REF], and it has also been reported as a significant sensitizer in guinea-pigs, with positive specific responses reported in allergy cross-reactivity studies with allergenic cyclohexene hydroperoxides [START_REF] Bråred Christensson | Hydroperoxides form specific antigens in contact allergy[END_REF].

Materials and Methods

Studies in Solution

Chemicals and Reagents

CumOOH, PBN (98%) and DMPO were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France) and used as received. DEPMPO was synthesized as reported in the literature [START_REF] Barbati | Minisci F. Ed. Free Radicals in Biology and Environment[END_REF]. HEPES (≥ 99.5 %), ferrous sulfate heptahydrate FeSO 4 .7H 2 O and acetonitrile CH 3 CN (99.8%) were acquired from Sigma-Aldrich (Saint-Quentin Fallavier, France). Aqueous solutions were prepared with deionized water.

EPR Experiments

EPR spectra were recorded on an EPR X-band spectrometer (EMXplus, Bruker Biospin GmbH, Germany), equipped with a high sensitivity resonator (HSW, Bruker Biospin GmbH, Germany). The g calibration was performed using Bruker standard (strong pitch) with known isotropic g factor of 2.0028.

The principal experimental parameters values were: microwave power 4.5 mW, modulation amplitude 0.5 to 1 G, for a resulting sweep time of ca. 120 s for a single scan. Up to 2 scans were accumulated to improve the signal-to-noise ratio. Spectra were recorded at room temperature (295K±1K). As fast as possible after reagents mixing, the resulting sample was introduced into a glass capillary (Hirschmann, 25 µL), sealed at both ends and rapidly transferred into the EPR cavity for measurement.

General Procedure in Solution

HEPES buffer solution (10 mM, pH 6.8) was prepared by dissolving 1.19 g HEPES in 400 mL deionized water, with additional 4 g NaCl and 0.1 g KCl. To attain pH 6.8 NaOH pellets were added. If the pH went too high, it was lowered back by carefully adding HCl until pH remained stable to 6.8.

Finally, deionized water was added for a final volume of 500 mL.

Stock solutions were prepared for CumOOH (5.4 mM, HEPES/CH 3 CN 9/1), the spin-traps (100 mM in HEPES) and FeSO 4 .7H 2 O (10 mM in deionized water). 12.5 µL of spin-trap solution were mixed with FeSO 4 .7H 2 O (i. e. 0.5 µL for a final concentration in the reaction mixture of 0.1 mM), 5 µL CumOOH solution added and final volume completed to 50 µL. This way, final concentrations in the reaction mixture were 25 mM spin-trap and 0.54 mM CumOOH. The reaction mixture was subjected to stirring, further introduced into the EPR quartz capillary tube, sealed on both ends, and EPR spectra registered.

Reconstructed Human Epidermis Studies

Chemicals and Reagents

Episkin TM (Lyon, France) is an in vitro reconstructed human epidermis (RHE) from normal human keratinocytes cultured for 13 days on a collagen matrix at the air-liquid interface. It is a 3D epidermis model histologically similar to human epidermis. The small 0.38 cm 2 format was chosen for the studies. Immediately after arrival in the laboratory, the 3D reconstructed epidermis were removed from the agarose-nutrient solution in the shipping multiwell plate under a sterile airflow. Then, they were immediately placed in a plate in which each well was previously filled with 2 mL Episkin TM maintenance or growth medium at room temperature. It was necessary to act quickly as the tissue cultures dry out rapidly when not in contact with medium. Samples were placed in the incubator at 37 °C, 5% CO 2 and saturated humidity, at least 24 h before incubation. Episkin TM (Lyon, France) furnished the assay medium used for incubations.

EPR Experiments

EPR spectra were recorded on an EPR X-band spectrometer (ESP300E, Bruker Biospin GmbH, Germany), equipped with a standard TE102 rectangular resonator (Bruker Biospin GmbH, Germany).

The g calibration was performed using Bruker standard (strong pitch) with known isotropic g factor of 2.0028. The principal experimental parameters values were: microwave power of 5-10 mW, modulation amplitude 1 to 2 G, and resulting sweep time of ca. 164 s to 328 s for a single scan. Up to 4 scans were accumulated to improve the signal-to-noise ratio. Spectra were recorded at room temperature (295K±1K) as soon as possible after incubating the reagents. RHE were placed in an EPR tissue cell equipped with a silica window (Willmad, #ER162TC-Q) and resulting EPR spectra recorded in situ right afterwards.

General Procedure in RHE

Incubation in the Assay Medium: CumOOH (200 µL of 5.4 mM HEPES/CH 3 CN 9/1 stock solution)

and DEPMPO (100 µL of 500 mM stock solution in HEPES) were introduced in a mixture of 300 µL assay medium and 1400 µL of HEPES buffer. The RHE model was then placed in a well containing this mixture. After 2 h incubation, RHE was placed in the EPR tissue cell and EPR spectra recorded.

Incubation by Topical Application to the RHE: RHE were topically treated with CumOOH in acetone

(1 mM, 20 µL) and post incubated (37 °C, 5% CO 2 ) different time periods (1 min to 2 h). After the incubation time, the RHE were placed in the EPR tissue cell and DEPMPO (50 mM in HEPES buffer, 20 µL) was applied to the epidermis taking care to ensure that the solution was only applied to it. EPR spectra were then recorded.

EPR Simulations

All experimental EPR spectra were analyzed by means of computer simulation using labmade scripts based on Easyspin toolbox (http://www.easyspin.org) under Matlab (Mathworks) environment [START_REF] Stoll | EasySpin, a comprehensive software package for spectral simulation and analysis in EPR[END_REF].

Results

Studies in Solution

Studies in solution were carried out to set up the optimal experimental conditions providing a good signal-to-noise ratio while keeping low CumOOH concentrations, i.e. suitable with physiological conditions. α-Phenyl-N-tert-butylnitrone (PBN), DMPO and DEPMPO were used as complementary spin-traps to obtain a comprehensive picture of all radicals being formed. Spin-adducts are formed by addition of transient short-lived radicals to the α-C of the nitronyl group of the spin-traps. PBN and DMPO are commonly used in biological media, but their low resistance to bio reductions of their spinadducts limit potential in vivo applications. DEPMPO produces spin-adducts with longer lifetime, particularly for the superoxide and alkylperoxyl radicals adducts [START_REF] Clement | Use of a phosphorylated spin trap to discriminate between the hydroxyl radical and other oxidising species[END_REF][START_REF] Liu | Evaluation of DEPMPO as a spin trapping agent in biological systems[END_REF].

Radical initiation was triggered by Fe(II) induced Fenton reaction. Spin-traps, in 10 mM buffer, were employed in an excess concentration (25 mM) with respect to CumOOH (0.54 mM). The spintrap solution was mixed with aqueous ferrous sulfate (0-1.7 mM), CumOOH added and the mixture subjected to conventional field-swept continuous wave-EPR spectroscopy (cw-EPR) in a glass capillary after different reaction times (1 min to 2 h). Initial tests were carried out in phosphate buffer (pH 7.4) but we observed that Fe(II) concentration was a limiting factor in the generation of radical species as Fe(II)-catalyzed CumOOH oxidation was inhibited at low Fe(II) concentrations [START_REF] Pazos | Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study[END_REF] (see Supplementary Material for experimental data). The use of HEPES buffer of better stability at physiological pH resolved this problem: (i) similar EPR spectra were observed in HEPES solutions and (ii) Fe(II) initiation was efficient instantly at catalytic concentrations (0.1 mM).

Relevant 14 N, 1 H, 31 P hyperfine coupling constants (hfccs) of detected spin-adducts are listed in Table 1. Scheme 1 suggests mechanisms explaining 1-8 formation. (Table 1 and Scheme1 around here) Experiments with PBN and DMPO provided an initial basis of radicals trapped while DEPMPO gave the most complete information. Very briefly, we assigned adduct 1 to a PBN-carbon centered radical adduct, probably the • CH 3 generated by β-scission of CumO • radicals [START_REF] Pazos | Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study[END_REF]. Adduct 2 with hfccs also characteristic of trapped carbon radicals was observed too but at higher Fe(II) concentrations (≥ 0.5 mM) (see Supplementary Material). In the literature, 2 has been reported only in presence of amino acids or ethanol in the reaction mixtures, which was not the case here [START_REF] Pazos | Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study[END_REF][START_REF] Davies | Protein hydroperoxides can give raise to reactive free radicals[END_REF][START_REF] Augusto | Spin-trapping of free radicals formed during microsomal metabolism of ethylhydrazine and acetylhydrazine[END_REF]. A hypothetical explanation could be trapping of oxaspiro radicals in equilibrium with CumO • , which are suspected intermediates in known O-neophyl rearrangement processes [START_REF] Salomone | Phenyl bridging in ring-substituted cumyloxyl radicals. A product and time-resolved kinetic study[END_REF][START_REF] Dilabio | Isomerization of triphenylmethoxyl: the Wieland free-radical rearrangement revisited a century later[END_REF]. DMPO-OCH 3 spin-adduct 3 was detected at catalytic Fe(II) levels (0.1 mM), and • CH 3 /HO • radicals also formed and trapped (4, 5) at higher Fe(II) concentrations [START_REF] Guo | Separation and identification of DMPO adducts of oxygencentered radicals formed from organic hydroperoxides by HPLC-ESR, ESI-MS and MS/MS[END_REF] (see Supplementary Material).

DEPMPO was undoubtedly more valuable when compared to PBN or DMPO as a broad view of all sort of radicals deriving from CumOOH were observed all at once with catalytic Fe(II) concentration as low as 0.1 mM (Fig. 1b). Higher Fe(II) concentrations resulted simply in ratio changes of radicals formed (Fig. 1a). We attributed the EPR spectrum shown in Fig. 1b to a mixture of spinadducts formed by trapping hydroxyl HO • (6, Fig. 1d), carbon centered (7a/7b, Fig. 1e/1f), CH 3 O • and/or possible peroxyl CumOO • radicals (8, Fig. 1g) [START_REF] Karoui | DEPMPO: an efficient tool for the coupled ESR-spin trapping of alkylperoxyl radicals in water[END_REF]. (Figure 1 around here) In the experimental spectra it is shown two low-field/high-field overlapping EPR lines that correspond to carbon centered radicals. These could be two stereoisomers (7a/7b) since it is known that DEPMPO can produce stereospecific adducts [START_REF] Clément | Deuterated analogues of the free radical trap DEPMPO: synthesis and EPR studies[END_REF]. The same should be observed theoretically for 8-like adducts, having stereoisomers when trapping CH 3 O • and/or CumOO • radicals [START_REF] Dikalov | Characterization of the high resolution ESR spectra of the methoxyl radical adducts of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO)[END_REF]. However, in our case, to differentiate between these two radicals was hardly possible as hfccs could correspond to both upon literature depending on the experimental conditions [START_REF] Karoui | DEPMPO: an efficient tool for the coupled ESR-spin trapping of alkylperoxyl radicals in water[END_REF].

Reconstructed Human Epidermis Studies

DEPMPO was chosen for the development of the EPR spin-trapping methodology with RHE based upon (i) the results of the studies in solution where an extensive view of all sort of radicals deriving from CumOOH were observed all at once with catalytic Fe(II) concentration and (ii) the high persistency of its superoxide and alkylperoxyl radicals spin-adducts when compared to DMPO [START_REF] Frejaville | 5-(Diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide: a new efficient phosphorylated nitrone for the in vitro spin trapping of oxygen-centered radicals[END_REF]. Also, DEPMPO allows a clear distinction between oxygen and carbon centered radicals [START_REF] Stolze | Spin trapping of lipid radicals with DEPMPO-derived spin traps: detection of superoxide, alkyl and alkoxyl radicals in aqueous and lipid phase[END_REF].

Episkin TM RHE (Lyon, France) (0.38 cm 2 ) were stored at arrival in 2 mL provided maintenance medium (at 37°C, in 5% CO 2 saturated humidity atmosphere). After 24 h, the maintenance medium was replaced by 2 mL of assay medium.

Preliminary trials were conducted by incubating CumOOH (0.54 mM) and DEPMPO (25 mM in HEPES) in the assay medium. The aim was to avoid the epidermal barrier function and check at a first sight using absorption from the bottom of RHE if CumOOH-mediated radicals could be formed within the epidermis. Even though Episkin TM RHE barrier function has been described to be less developed than that of normal skin, regarding its different lipids composition and organization, it may not be neglected [START_REF] Ponec | Characterization of reconstructed skin models[END_REF]. For control purposes, a sample from assay medium was regularly taken (20 µL) during incubation (37°C, 5% CO 2 , 1 min to 2 h), inserted into a capillary, sealed and measured by EPR at room temperature. Whatever the incubation time, no EPR signal for spin-adducts was observed in the assay medium (Fig. 2a). (Figure 2 around here) After 2 h incubation in the assay medium, the RHE was placed in a commercial EPR tissue cell (Willmad) equipped with a silica window and EPR spectra recorded. Although being noisy, a signal was then already observed, increasing with time (Fig. 2c-2e).

Major peaks could be assigned to a carbon centered radical DEPMPO adduct (spin-adduct 7-like) based on the following set of hfccs a N =15.4 G, a H =22.6 G and a P =48.2 G (Fig. 2f; Table 2) and comparison with our previous data in solution (Fig. 1b). It was not possible to distinguish between isomers 7a/7b due to the noise level. Control experiments without CumOOH were performed, i.e. after only DEPMPO incubation in the RHE and showed no EPR signal, corroborating that most probably spinadduct 7 came from trapping a carbon centered radical issued from the hydroperoxide (Fig. 2b). As shown in Fig. 2f, hydroxyl HO • was also trapped in the RHE at a lower ratio. Worthy of note, after penetrating the RHE, CumOOH radical initiation should have been induced by RHE itself as no Fe(II) was included.

Episkin TM RHE is a very good replicate of human epidermis architecture, and is currently used for irritation and penetration tests of cosmetic and chemical compounds as replacement of in vivo testing.

In recent OECD guidelines on in vitro skin irritation assessment by using such RHE system it is specified that test chemicals must be applied topically to the RHE model [START_REF]vitro skin irritation: RHE test method[END_REF]. Also, the stratum corneum of the RHE used, and its lipid composition, must simply resist to the penetration of SDS or Triton X-100 cytotoxic benchmark chemicals. It is thus supposed that allergens can penetrate the epidermis. In consequence, with the acquired knowledge that CumOOH radicals can be formed within RHE as described above, and in order to get closer to real life sensitization, further experiments were based on a topical application procedure. With this purpose, RHE were treated with CumOOH in acetone (1 mM, 20 µL) on the top and incubated for different time periods (37°C, 5% CO 2 , 1 min to 2 h). RHE negative controls were treated only with acetone (20 µL). After post incubation, RHE were placed in the EPR tissue cell, DEPMPO was then added whilst ensuring that the solution was applied to the epidermis top (50 mM in HEPES) and EPR spectra recorded immediately afterwards. After 5 min of incubation, the fingerprint of a carbon centered radical DEPMPO adduct was detected as previously observed during incubation protocol (spin-adduct 7-like), with hfccs a N =15.4 G, a H =22.7 G, a P =48.1 G, and yet with a better signal-to-noise ratio (Fig. 3b; Table 2). Negative controls (incubation of the sole acetone, followed by DEPMPO addition) gave no signal, indicating that the carbon radical arose from CumOOH and not by induction of radical formation on a RHE biomolecule (Fig. 3a). Best computer simulation obtained from Fig. 3b is shown in Fig. 3c where spin-adduct 7-like seems to be predominant.

Higher incubation times resulted in a weaker signal-to-noise ratio pointing to carbon radicals formed in the epidermis shortly after hydroperoxide topical application. (Figure 3 and Table 2 around here)

Discussion

CumOOH is used in the chemical industry in the manufacturing of organic peroxides, the production of phenol and acetone, and as initiator and catalyst in polymers production and fiber-reinforced plastics. It is also a strong oxidizing agent, which makes it an important intermediate in the manufacture of epoxy and polyester resin coatings. Thus, dermal exposure to CumOOH during manufacturing processes has been for many years of concern, and its toxicity and ability to promote skin tumor and induce epidermal hyperplasia have been described. As free radicals are considered key factors contributing to skin tumor promotion by organic peroxides, it is not surprising that CumOOH became an EPR reference compound regarding oxidative stress investigations. EPR spin-trapping has been used to study the production of free radicals in isolated murine keratinocytes and in murine skin treated with CumOOH and other organic peroxides [START_REF] Timmins | Free radical formation in murine skin treated with tumour promoting organic peroxides[END_REF][START_REF] Timmins | Free radical formation in isolated murine keratinocytes treated with organic peroxides and its modulation by antioxidants[END_REF]. Furthermore, the ability to trigger free radicals has also been suggested to be critical for the carcinogenic properties. By studying lipid extracts of mouse skin exposed to CumOOH, it was proved the formation of in vivo lipid-derived free radicals triggered by the hydroperoxide and causing severe oxidative stress in murine skin [START_REF] Shvedova | Antioxidant balance and free radical generation in vitamin E-deficient mice after dermal exposure to cumene hydroperoxide[END_REF][START_REF] Shvedova | Pro/antioxidant status in murine skin following topical exposure to cumene hydroperoxide throughout the ontogeny of skin cancer[END_REF]. CumOOH has also been described to cause allergic and irritant dermatitis, being reported as a significant sensitizer in guineapigs [START_REF] Bråred Christensson | Hydroperoxides form specific antigens in contact allergy[END_REF].

To be a sensitizer, ROOHs need to covalently bind to skin proteins to form the antigenic entity that will trigger the immunotoxic process, and this via radical processes. These start with the cleavage of the O-O bond, allowing unstable RO • /ROO • radicals formed to rearrange to longer half-life reactive carbon centered radicals R • . It has been argued that the hydroxyl radical HO • might act as a hapten, thus affording an unspecific allergic response. However, no such unspecific responses were observed when performing cross-reactivity studies with even structurally closed related ROOHs in guinea-pigs and mice, and in clinical studies [START_REF] Bråred-Christensson | Hydroperoxides form specific allergens in contact allergy[END_REF][START_REF] Bråred-Christensson | Limonene hydroperoxide analogues differ in allergenic activity[END_REF][START_REF] Deza | Contact sensitization to limonene and linalool hydroperoxides in Spain: a GEIDAC prospective study[END_REF]. Most probably, specific carbon radicals R • that depend on the chemical structure of the ROOH must be at the origin of the antigen formation. This said, the relative contribution in skin sensitization processes of R • radicals and, in parallel, of also produced radical oxygen species (ROS) needs still to be elucidated. In the work we report herein, the aim was to develop an EPR spin-trapping methodology using CumOOH as reference hydroperoxide to elucidate if radicals can be formed in 3D RHE human epidermis model directly issued from the peroxide (RO • /ROO • /R • /ROS). The objective was to clarify which radical intermediates could be involved in the antigen formation in a system closer to what may happen in in vivo sensitization.

Studies in solution were initially carried out to establish the best experimental conditions transferable to the RHE matrix (i.e. concentrations, solvent, spin-trap). Radical initiation was performed via a Fe(II) Fenton reaction and DEPMPO was taken as the optimum spin-trap when compared to DMPO or PBN. Carbon centered, alkoxyl, and HO • radicals were identified. Interestingly, the developed EPR spin-trapping methodology in the Episkin TM RHE model, either by incubating in the assay medium or by topical application, indicated that carbon radicals (adducts 7-like) are mainly formed, with slight HO • radical (adduct 6), suggesting again a key role of carbon radicals in the antigen formation process. Control experiments without CumOOH always produced no EPR signal, suggesting that DEPMPO trapped radicals most probably originate from the hydroperoxide rather than through biomolecules present in RHE. The assignment of DEPMPO-OH 6 could leave open interpretation regarding the origin of this adduct. Aside from CumOOH, it could arise from superoxide radical adduct, the superoxide production being induced in skin cells by CumOOH. Additional tests to decipher the HO • radical origin could be preincubation of RHE with polyethylene glycol-conjugated superoxide dismutase, a powerful free radical scavenger that acts by reducing superoxide anions. This was out of the scope of the work presented here, but will need to be addressed in a near future. It is also important to stress that in both, solution and RHE investigations, control experiments without CumOOH did not exhibit any detectable signal from Fe(II) autoxidation in HEPES or in the assay medium, perhaps because of the catalytic concentrations of Fe(II) applied.

No Fe(II) was used for radical initiation when using RHE, to mimic real life skin allergy situation.

One can hypothesize that reaction of organic peroxides in the skin in the presence of one-electron donor agents (i.e. amino and thiol groups present in amino acids, metal complexes, enzymes) may start the electron transfer processes and further skin proteins haptenation [START_REF] Moriarty-Craige | Extracellular thiols and thiol/disulphide redox in metabolism[END_REF][START_REF] Fujimoto | Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation controls[END_REF][START_REF] Grossi | The mechanism of oxidation of aniline by peroxy-compounds: can EPR spectroscopy help to discriminate between one-electron transfer and a nucleophilic substitution process?[END_REF]. Also, the potential presence in the skin of Fe(III) haem-containing enzymes might be responsible for the peroxide O-O bond cleavage and start radical processes. Reactions of model Fe(III) porphyrin complexes with oxidants, such as peroxyacids and hydroperoxides, have been extensively studied as models. The cleavage has been confirmed but a clear mechanistic consensus has not evolved on the homolytic or heterolytic nature of the O-O bond cleavage by these species, both kinds of mechanisms being possible [START_REF] Traylor | Reactions of hydroperoxides with iron (III) porphyrins: heterolytic cleavage followed by hydroperoxide oxidation[END_REF][START_REF] Traylor | High-yield epoxidations with hydrogen peroxide and tert-butyl hydroperoxide catalyzed by iron(III) porphyrins: heterolytic cleavage of hydroperoxides[END_REF][START_REF] Almarsson | A Homolytic mechanism of O-O bond scission prevails in the reactions of alkyl hydroperoxides with an octacationic tetraphenylporphinato-iron(III) complex in aqueous solution[END_REF][START_REF] Labeque | Homolytic and heterolytic scission of organic hydroperoxides by (meso-tetraphenylporphinato)iron(III) and its relation to olefin epoxidation[END_REF].

Conclusion

To our knowledge, EPR spectroscopy had never been used before for the study of radicals derived from skin allergenic substances in RHE. In this study, EPR spin-trapping allowed us for the first time to monitor the formation of carbon radicals issued from CumOOH in a 3D epidermis model. This is an important achievement opening new perspectives and mechanistic insights for the molecular understanding of sensitization processes to precursors of radical intermediates (i.e. allylic hydroperoxides derived from autoxidation of natural terpenes, aromatic phenols, aromatic amines).

Since the 1980s, RHE models have been developed successfully as alternative methods to animal experiments to assess skin penetration of chemicals, and for predicting epidermal responses to irritants and sensitizers. The development and use of EPR methodologies to follow radical production and stress induction of allergens in RHE models described in this work could be further used as an alternative method and an ex vivo indication of induced free radical processes occurring in skin and would be valuable afterwards to study other dermato-oxidation events. These novel studies on the reactivity understanding through radical mechanisms, and the influence on the epidermis cell environment, should help for the development of specific alternative methods for skin sensitization assessment adapted to sensitizers reacting through radical mechanisms.

Figure 3 LegendsScheme 1 .

 31 Figure 3

Figure 1 .

 1 Figure 1. EPR spectra obtained with CumOOH (0.54 mM) and DEPMPO (25 mM) dissolved in

Figure 2 .

 2 Figure 2. Preliminary trials incubating CumOOH (0.54 mM) and DEPMPO (25 mM in HEPES) in the

Figure 3 .

 3 Figure 3. EPR spectra obtained in RHE topical application experiments by using CumOOH (1 mM in

Table 1

 1

Table 1 .

 1 Coupling constants of spin-adducts identified in solution studies

	Spin-trap		Coupling constants		Assignment
		aN (G)	aH (G)	aP(G)	g
	PBN	15.3	3.5		2.0060	1 PBN-carbon R [18]*
	PBN	16.6	3.6		2.0059	2 PBN-carbon R [18,19,20]*
	DMPO	14.6	10.8 (1.4)		2.0055	3 DMPO-OCH3 [23]*
	DMPO	16.4	23.7		2.0053	4 DMPO-carbon R [23]*
	DMPO	15.3	15.0		2.0057	5 DMPO-OH [23]*
	DEPMPO	14.1	13.4	47.5	2.0059	6 DEPMPO-OH [24]
	DEPMPO	15.3	22.6	47.9	2.0057	7a DEPMPO-carbon R [24]
	DEPMPO	15.3	19.9	45.8	2.0059	7b DEPMPO-carbon R [24]
	DEPMPO	13.1	9.4	48.0	2.0059	8 DEPMPO-OCH3 and/or
						DEPMPO-OOCum [24]
	* See Supplementary Material for experimental data.

Acknowledgements

This research was supported by the International Center for Frontier Research in Chemistry (icFRC, Strasbourg, France, grant number JLE-FRC-0001). The authors thank the French Ministry of Research, the University of Strasbourg, the CNRS and the REseau NAtional de Rpe interDisciplinaire (RENARD, Fédération IR-RPE CNRS #3443).


Matthieu Lang

Elena Giménez-Arnau

Jean-Pierre Lepoittevin

Is it possible to assess the allergenicity of mixtures based on in chemico methods? Preliminary results on common fragrance aldehydes

Keywords: Fragrance aldehydes, mixtures, allergenicity, hapten-peptide reactivity

Most perfume compositions and cosmetics contain mixtures of fragrance ingredients, synthetic or extracted from natural sources. Many of these are known to be weak or moderate allergens when tested individually. However, fragrances are one of the most common causes of allergic contact dermatitis to consumer products, ranking second after nickel. As consumers are exposed to mixtures of ingredients when using products that contain perfume, it was suggested that mixtures could enhance induction and elicitation of skin allergy to fragrances. Prediction of skin sensitization to chemicals is crucial for the cosmetics industry. Nowadays, methods to assess skin sensitization hazard replacing the use of animals have been developed and validated for the testing of pure substances. However, there is practically no experience with testing of mixtures. Here we evaluated in a very preliminary approach, and from a chemical reactivity perspective, if the validated DPRA, hapten-peptide reactivity based assay, could be used at a first sight to evaluate the chemical reactivity of mixtures. Two simple binary combinations of fragrance aldehydes were studied, hydroxycitronellal-citral and citral-cinnamaldehyde. Their reactivity towards DPRA peptides was compared with that of the single constituents. In general, the chemical described as the most potent sensitizer was driving the reactivity in the mixtures. Therefore, it could be hypothesised that the DPRA would estimate the mixture chemical reactivity similar to that of the stronger sensitizer component. This will be reinforced by conditions described for the DPRA where the tested chemical or mixture of chemicals will be added in large excess to the peptides.

Introduction

Approximately 3000 fragrance ingredients, of synthetic or natural origin, are currently available to the perfumer for compounding a fragrance formula. [1] Thus, a single perfume or fragrance composition may consist of 10-300 different ingredients. Many of these are known to be weak or for some of them moderate skin sensitizers when tested individually. [START_REF] Rastogi | Fragrances: beneficial and adverse effects[END_REF][START_REF] Johansen | Contact Dermatitis[END_REF] However, in most dermatological centres fragrances rank second after nickel as the most common source of contact allergy. The main tools for diagnosing allergic contact dermatitis (ACD) to fragrances are Fragrance Mix I (amyl cinnamal, cinnamal, cinnamyl alcohol, eugenol, isoeugenol, geraniol, hydroxycitronellal, oak moss) and Fragrance Mix II (hydroxyisohexyl-3-cyclohexene carboxaldehyde, citral, farnesol, coumarin, citronellol, α-hexyl cinnamal), both included in the European base-line patch test series. [START_REF] Larsen | [END_REF]5] A response rate of 6-11% to Fragrance Mix I, and of 2.1-4.6% to Fragrance Mix II has been reported in dermatological patients suffering from ACD. [6,7] As most perfumes contain mixtures of fragrance ingredients, and many of them have a skinsensitizing capacity, consumers are almost certainly exposed to mixtures of allergens, also called cocktails, when they use products that contain perfume. Very often, at least one of the 26 individual fragrance ingredients identified as sensitizers that need to be labelled on the packaging of cosmetic products according to EU regulations (Annex 3 Cosmetics Directive 76/768/EEC) is present in consumer products. It might be thus possible that the existence of several fragrance compounds together in a consumer product could lead to increased sensitization potency. Recently, we investigated if there is any influence when mixing fragrance allergens on the sensitization process to the individual compounds. [8] Mice were sensitized with three different concentrations of three fragrance compounds, hydroxyisohexyl 3-cyclohexene carboxaldehyde, isoeugenol and cinnamaldehyde, alone or as a mixture. The sensitization and elicitation responses were measured by ear thickness plus infiltration of B and T cells, and T cell proliferation in the draining lymph nodes. Results showed a dose-dependent sensitization response for each compound and an increased response when they were mixed. A stronger challenge response to cinnamaldehyde was observed in mice sensitized with the mixture than in mice sensitized with cinnamaldehyde alone. We conducted similar studies with atranol and chloroatranol, main allergens of oak moss absolute, showing that the mixture in the natural extract was more potent at both sensitization and challenge than atranol and chloroatranol alone. [9] These findings suggested that allergen mixtures could enhance both induction and elicitation of ACD.

Assessment of the skin sensitizing potential of chemicals is crucial for the cosmetics industry. Following the adoption of REACH, [START_REF] Anon | Regulation (EC) No 1907/2006 of the European Parliament and of the Council of the 18 December 2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), establishing a European Chemicals Agency[END_REF] but even more with the 7th amendment to the EU Cosmetics Directive banning from the European market cosmetic products containing ingredients tested on animals, [START_REF] Anon | Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003 amending Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products -7[END_REF] a great deal of progress has been made in the development of alternative methods to animal tests to assess skin sensitization to chemicals. [START_REF] Adler | [END_REF] However, a major drawback of these methods is their inability to assess the sensitizing potential of mixtures or cocktails of chemicals.

A key step in the sensitization process to any compound is the formation of a covalent adduct by its reaction with skin proteins, generally through a nucleophile-electrophile mechanism, forming this way an immunogenic complex. [START_REF] Lepoittevin | Contact Dermatitis[END_REF] The formation of the hapten-protein complex, able to be fully recognized by naïve T cells, is one of the mandatory conditions for skin allergy to occur. In chemico alternative tests, such as the Direct Peptide Reactivity Assay (DPRA), are based on this capacity of chemical sensitizers to react with amino acids. To develop the DPRA, Gerberick et al. evaluated 82 chemicals, including allergens of different potencies and non-allergenic chemicals, for their ability to react with glutathione and two synthetic peptides having cysteine (Cys) or lysine (Lys) as the reactive amino acid (N-Ac-Arg-Phe-Ala-Ala-X-Ala-Ala; Pep-Cys if X=Cys; Pep-Lys if X=Lys), and measured peptide depletion using UV detection. [START_REF] Gerberick | [END_REF] Peptide reactivity data were compared with existing in vivo local lymph node assay (LLNA) data using recursive partitioning methodology. In general, moderate, strong and extreme sensitizers showed moderate to high peptide reactivity, while weak and non-sensitizers showed minimal to low reactivity. A classification tree model was then built based on a Pep-Cys and Pep-Lys depletion pattern. Classifying minimal reactivity as non-sensitizers and low, moderate and high reactivity as sensitizers, it was determined that the model based on cysteine and lysine gave a prediction accuracy of 89%. [15] The DPRA was first endorsed by the European centre for validation of alternative methods (ECVAM) [START_REF] Anon | EURL-ECVAM recommendation on the peptide reactivity assay (DPRA) for skin sensitization testing[END_REF] and was recently adopted as an OECD-guideline for the testing of chemicals under section 4-health effects. [START_REF] Anon | Test No. 442C: In Chemico Skin Sensitisation Direct peptide Reactivity Assay (DPRA). OECD guidelines for the testing of chemicals[END_REF] As most toxicological methods, the DPRA was developed for the evaluation of the skin sensitizing potential of chemicals when taken individually. For many years we conducted studies in order to validate the peptide reactivity assay concept, and to complement depletion data observed for those chemicals. [START_REF] Merckel | [END_REF][19][20] However, the behaviour of the DPRA to evaluate the reactivity of chemical mixtures is not known. In the work detailed herein we studied the reactivity of binary mixtures of fragrance sensitizers towards DPRA peptides, and we compared this reactivity with that of the constituents alone. The aim was to perform mechanistic based studies in order to evaluate, in a very first approach, if the reactivity towards DPRA peptides of allergens in a mixture was modified compared to the one of the single compounds, and if this was significant enough to help for the prediction of skin sensitization. Compounds tested were fragrance aldehydes hydroxycitronellal 1, citral 2 and cinnamaldehyde 3 (Figure 1).

Materials and Methods

Chemistry

Hydroxycitronellal [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-substituted at position 1, 1-( 13 C)-1, and citral [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-substituted at positions 1, 1-( 13 C)-2, and 3, 3-( 13 C)-2, were synthesized using a procedure we reported previously. [START_REF] Merckel | [END_REF] Synthesis of cinnamaldehyde [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-substituted at positions 1, 1-( 13 C)-3, and 3, 3-( 13 C)-3 is reported herein. 1-( 13 C)Acetonitrile, 1-( 13 C)benzaldehyde and deuterated solvents were purchased from Euriso-Top (Saint Aubin, France). All other chemicals were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Chemicals and solvents were used as delivered. Air-or moisture-sensitive reactions were conducted in flame-dried glassware under an atmosphere of dry argon. Reactions were followed by thin layer chromatography on 0.25 mm silica gel plates (60F 254 ; Merck, Darmstadt, Germany). After migration, the plates were inspected under UV light (254 nm) or sprayed with a solution containing phosphomolybdic acid (5 g), cerium (IV) sulfate (2 g) and sulphuric acid (12 mL) in water (188 mL), followed by heating. Column chromatography purifications were performed on silica gel 60 (Merck, Geduran ® , 40-63 µm). 1 H and [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR spectra were recorded on a Bruker Avance 300 spectrometer at 300 MHz and 75 MHz, respectively. The chemical shifts (δ) are reported in ppm and are indirectly referenced to TMS via the solvent signal (CDCl 3 : δ 1 H = 7.26, δ 13 C = 77.16). In the 1 H NMR spectra multiplicities are denoted as d (doublet) and m (multiplet). The different types of carbon in the structures were identified by the DEPT-135 technique.

1-( 13 C)Cinnamaldehyde, 1-( 13 C)-3. To a solution of freshly distilled diisopropylamine (1.06 mL, 7.52 mmol, 4 equiv) in dry tetrahydrofuran (6 mL) was slowly added a solution of n-butyllithium (1.64 M in hexane, 4.59 mL, 7.52 mmol, 4 equiv) at -20°C. The reaction medium was stirred for 30 min at -20°C, then cooled down to -70°C. A solution of 1-( 13 C)acetonitrile (197 µL, 3.76 mmol, 2 equiv) in dry tetrahydrofuran (3 mL) was then added. After 30 min, the reaction medium was allowed to warm up to 0°C, and a solution of diethylchlorophosphate (575 µL, 3.76 mmol, 2 equiv) in dry tetrahydrofuran (3 mL) was added slowly. After 1 h of stirring at 0°C, a solution of freshly distilled benzaldehyde (192 µL, 1.88 mmol) in dry tetrahydrofuran (3 mL) was added. The mixture was kept under stirring at room temperature for an additional 3 h and then hydrolysed with water (30 mL). The aqueous layer was extracted with diethyl ether (3 × 30 mL) and the combined organic layers washed with brine (50 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The crude oil was directly used without purification for the next step.

To a solution of the previously obtained crude oil in dry dichloromethane (20 mL), was slowly added a solution of diisobutylaluminium hydride (1 M in hexane, 7.52 mL, 7.52 mmol, 4 equiv) at -78°C drop by drop. After 15 min stirring at -78°C, the reaction medium was allowed to warm up to room temperature and then stirred for an additional 2 h. The reaction mixture was hydrolysed with tartrate buffer (45 mL) and kept under stirring for 12 h. The aqueous layer was extracted with dichloromethane (4 × 20 mL) and combined organic layers washed with brine (60 mL), dried over MgSO4, filtered, and concentrated under reduced pressure. The crude oil was purified by distillation under reduced pressure (90°C at 4 mbar) to give 1-( C)benzaldehyde (98%, 4.57 mmol), 50 mL dry dichloromethane, 18.3 mL diisobutylaluminium hydride (1 M in hexane, 18.4 mmol, 4 equiv) and 112 mL tartrate buffer. The crude oil was purified by distillation under reduced pressure (90°C at 4 mbar) to give 3-( 13 C)-3 (265 mg, 1.99 mmol, 44% yield) as a yellow oil (E/Z > 98%). 1 H NMR (300 MHz, CDCl3): δ 6.69 (dd, 1H, 3 JHH=15.9 Hz, 3 JHH=7.7 Hz, -CHCHO), 7.42-7.45 (m, 3H, 2 × m-CH(Ar), p-CH(Ar)), 7.55-7.60 (m, 2H, 2 × o-CH(Ar)), 7.60 (dd, 1H, 1 JHC=152.2 Hz, 3 JHH=16.3 Hz, -13 CHCHCHO), 9.72 (dd, 1H, 3 JHH=7.7 Hz, 3 JHC=0.9 Hz, -CHO). [START_REF] Lepoittevin | Contact Dermatitis[END_REF]

Peptides for Reactivity Studies

Synthetic peptides Pep-Cys and Pep-Lys were furnished by Genepep SA (Prades le Lez, France) and purity (> 95%) was certified by HPLC.

Reaction of Aldehydes with Pep-Cys

For the reaction of each single aldehyde with Pep-Cys, the chemical (6 µmol) was dissolved in a mixture of deuterated acetonitrile (200 µL) and phosphate buffer pH 7.4 (400 µL). Then Pep-Cys ( 6µmol, 1 equiv) was added to the medium and the whole mixture introduced into a NMR tube to follow the reaction by [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR. For the reaction of binary mixtures, the two chemicals (6 µmol each) were dissolved in a mixture of deuterated acetonitrile (200 µL) and phosphate buffer pH 7.4 (400 µL). Pep-Cys (6 µmol, 1 equiv) was added to the medium and the whole mixture introduced into a NMR tube to follow the reaction by [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR. Kinetic studies were carried out with the same experimental conditions adding tert-butanol (10 µL) as internal standard for the calculation of the relative intensities of 13 C NMR peaks.

Reaction of Aldehydes with Pep-Lys

For the reaction of each single aldehyde with Pep-Lys, the chemical (6 µmol) was dissolved in a mixture of deuterated acetonitrile (200 µL) and ammonium acetate buffer pH 10.2 (400 µL). Then Pep-Lys (6 µmol, 1 equiv) was added to the medium and the whole mixture introduced into a NMR tube to follow the reaction by [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR. For the reaction of binary mixtures, the two chemicals (6 µmol each) were dissolved in a mixture of deuterated acetonitrile (200 µL) and ammonium acetate buffer pH 10.2 (400 µL). Pep-Lys (6 µmol, 1 equiv) was added to the medium and the whole mixture introduced into a NMR tube to follow the reaction by [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR. Kinetic studies were carried out with the same experimental conditions adding tert-butanol (10 µL) as internal standard for the calculation of the relative intensities of 13 C NMR peaks.

NMR Experiments and Structure Assignment

The reactions were followed by mono-dimensional [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR on a Bruker Avance 300 spectrometer at 75 MHz. Chemical shifts (δ) are reported in ppm in comparison to TMS, using the residual signal of acetonitrile ( 1 H, δ = 21.94 ppm; [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C, δ = 118.26 ppm) as internal standard. [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C peak integrations were calculated using the signal of tert-butanol (δ = 29.23 ppm) or deuterated tert-butanol (δ = 68.46 ppm) as internal standard with NMRTEC/NMR notebook software (version 2.0, NMRtec ® , Illkirch-Graffenstaden, France). The structure of the products formed during the reactions was assigned by heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments, and by combining all information from the different [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-substituted positions. Bi-dimensional 1 H- [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C heteronuclear experiments were carried out on a Bruker Avance 400 spectrometer ( 1 H, 400 MHz; [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C, 100 MHz). Chemical shifts (δ) were compared with those calculated using ACD/CNMR and ACD/HNMR Predictor software (version 6.0, ACD/Labs, Toronto, Canada).

Results and Discussion

Fragrance aldehydes chosen for the study were hydroxycitronellal 1, citral 2 and cinnamaldehyde 3 (Figure 1). Hydroxycitronellal and cinnamaldehyde are constituents of Fragrance Mix I, and citral is included in Fragrance Mix II. The three of them belong to the 26 fragrance ingredients list (Annex 3 Cosmetics Directive 76/768/EEC) identifying sensitizers that need to be labelled on the packaging of cosmetic products. Hydroxycitronellal and citral are classified as weak sensitizers according to the LLNA, [21,[START_REF] Basketter | Contact Dermatitis[END_REF] but are of important clinical relevance due to the high exposure of consumers. Cinnamaldehyde is classified as a moderate sensitizer up to the LLNA, [21,[START_REF] Basketter | Contact Dermatitis[END_REF] but for long time it was considered as a top-ranking fragrance allergen, [START_REF] Enders | [END_REF]24] though a decline in reactions has been seen from the beginning of the 2000s. [25] We used two binary mixtures of these aldehydes. A mixture hydroxycitronellal-citral was chosen to represent two weak sensitizers together, and a mixture citralcinnamaldehyde to exemplify a pair weak-moderate sensitizers, according to LLNA data.

The use of [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-substituted molecules in association with NMR techniques is a powerful tool we have developed for the investigation of hapten-protein interactions. [26][27][28] Best results are obtained using direct substitution at the reactive sites of the molecule. Thus, hydroxycitronellal was prepared [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C substituted at the reactive electrophilic aldehyde chemical function (1-( 13 C)-1). Citral and cinnamaldehyde were prepared [START_REF] Lepoittevin | Contact Dermatitis[END_REF]

Binary Mixture Hydroxycitronellal-Citral

The behaviour towards Pep-Cys and Pep-Lys was studied using two binary mixtures, 1-( 13 C)-1/1-( 13 C)-2 and 1-( 13 C)-1/3-( 13 C)-2. Reactions were followed by mono-dimensional [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR, and the chemical structure of the products formed was elucidated by using a combination of bi-dimensional 1 H and [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C data obtained from all [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C substituted positions.

Reaction of hydroxycitronellal and citral towards Pep-Cys

We first studied the reactivity of hydroxycitronellal and citral separately with Pep-Cys in order to determine structures of potential adducts formed and their NMR δ characteristics. The adducts characterized were in accordance with those we had observed in previous studies. [START_REF] Merckel | [END_REF][START_REF] Mutschler | Study of the chemical reactivity of sensitizing molecules towards peptides as part of the development of alternative methods for the detection of allergens[END_REF] We used these data to elucidate the structure of adducts formed when hydroxycitronellal and citral were in an equimolar mixture. already at the first day of the reaction. The spectra were strictly the same one-month later, showing no more evolution of the reaction. This was also the reactivity observed for citral when treated alone with Pep-Cys. Contrariwise, hydroxycitronellal was found reactive when treated separately with Pep-Cys affording classical adducts derived from addition of the thiol group of Cys on the carbonyl chemical function.

Reaction of hydroxycitronellal and citral towards Pep-Lys

According to the DPRA protocol, the pH of the Pep-Lys reaction was set at 10.2 (ammonium acetate buffer solution) in order to increase the reactivity of the amino group of the lateral chain of Lys (pKa 10.43 ± 0.1). Results were strictly the same with both mixtures 1-( 13 C)-1/1-( 13 C)-2 and 1-( 13 C)-1/3-( 13 C)-2. Adducts characterized are shown in Figure 3. Their characteristic δ were compared with those of adducts formed by reaction of each constituent alone with Pep-Lys. Hydroxycitronellal and citral formed the same adducts separately with Pep-Lys or when mixed. At the first day of the reaction, the hydrated form 4 of hydroxycitronellal was formed together with adduct 6, resulting from a nucleophilic attack of the amino group of Lys on the carbonyl function, followed by displacement of a water molecule to form imine 7. Also, through a similar mechanism, citral reacted with the amino group of Lys to form a Schiff base, together with addition of a water molecule on position 3, affording this way adducts 8-10. After 8 days of reaction, citral was completely consumed in the mixture.

Kinetic competition results

The percentage over time of hydroxycitronellal and citral in the binary mixtures in the presence of the peptides is shown in Figure 4. In the reaction with Pep-Cys hydroxycitronellal was found essentially non-reacting compared to citral that was more reactive. The mixture behaved basically as if citral was the only reagent present. In other words, hydroxycitronellal reacted with Pep-Cys individually but seemed to be non reactive in presence of citral. Looking at the loss of citral in the mixture over time, only around 20% was consumed after 25 days (Figure 4). If reactivity was directly related to adduct formation with Pep-Cys, theoretically 80% of Pep-Cys would still be available for reaction with hydroxycitronellal. However it has been reported that several fragrance aldehydes included citral are able to oxidise Pep-Cys and other test peptides containing Lys and Cys residues by some unknown mechanisms. [START_REF] Natsch | [END_REF] Ideally, a quantitative analysis of unreacted Pep-Cys should indicate if the peptide was still present in the mixture in the parent form and thus available for reaction or if it did oxidize. Reactivity with Pep-Lys showed a complete disappearance of citral after 8 days of reaction, the reaction being faster than with Pep-Cys, and a low consumption of hydroxycitronellal. Citral reacted preferentially with Pep-Lys, with only 50% still present in the mixture after 24 h of reaction.

Discussion of the results

Hydroxycitronellal and citral are both categorized as weak sensitizers in the LLNA with average EC3 (%) values of 20 and 13, respectively. Recently, based on human data, hydroxycitronellal has indeed been classified as an infrequent cause of contact allergy in relation to level of exposure. However, citral has been classified as a common cause of contact allergy but with a substantial degree of exposure necessary. [31] Based on clinical data citral seems therefore to have a higher relative skin sensitizing potency. This is also supported by DPRA results, hydroxycitronellal being classified as a low reactivity chemical with Pep-Cys and Pep-Lys depletions of 17.5% and 6.5% respectively, whereas citral was classified as a moderate reactivity chemical with depletion values of 85.7% and 16.9%, respectively. Consistent with these data, in our reactivity studies the mixture behaved basically as if citral, which could be considered as a stronger sensitizer than hydroxycitronellal, was the unique reagent present, and thus the main sensitizer in the mixture. It could then be concluded that the reactivity of the mixture is similar to that of the stronger sensitizer constituent that is also the most reactive. Thus it could be expected that the DPRA would predict this mixture to have a moderate chemical reactivity. For a sensitizer to be classified as having a moderate chemical reactivity in the DPRA it needs to deplete Pep-Cys > 22.6% and Pep-Lys ≤ 42.5%, being depletion directly correlated with reactivity (Figure 5). However, in the binary mixture citral had a higher reactivity with Pep-Lys than with Pep-Cys (Figure 4) but experimental conditions were not the same as DPRA. In our approach, equimolar mixtures were used for the studies in order to assess the possible competition between the two allergens towards the peptides, whereas in the DPRA there is a peptide to chemical ratio of 1:10 for Pep-Cys and 1:50 for Pep-Lys. Also, even if adducts were formed, reactivity of the aldehydes could result in other processes too, such as degradation of the molecule itself or oxidation of the peptide catalysed by the test compound as mentioned earlier. [START_REF] Natsch | [END_REF] The DPRA would most probably consider the mixture as having a weak sensitizing activity.

Binary Mixture Citral-Cinnamaldehyde

The behaviour towards Pep-Cys and Pep-Lys was studied using two binary mixtures, 1-( 13 C)-2/1-( 13 C)-3 and 3-( 13 C)-2/3-( 13 C)-3. Reactions were followed by mono-dimensional [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR. A combination of bidimensional 1 H and [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C data obtained from all [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C substituted positions was used to elucidate the chemical structure of the products formed.

Reaction of citral and cinnamaldehyde towards Pep-Cys

We previously studied the reactivity of citral and cinnamaldehyde separately with Pep-Cys in order to determine structures of potential adducts formed and their NMR δ characteristics. We used these data to elucidate the structure of adducts formed when studying the citral-cinnamaldehyde mixture.

Figure 6 is representative of the chemistry observed when the mixture citral-cinnamaldehyde was reacted with the thiol chemical group from Pep-Cys. Results were identical with the two binary mixtures used. Clearly, adducts were formed by Michael addition of the thiol group of Pep-Cys on the conjugated double bond of cinnamaldehyde. No reaction with citral was observed. The signal with δ at 41.9 ppm for the C-3 position corresponded well with compound 11. This signal was in agreement with the one observed at 202.7 ppm in the reactions with [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-1 substitution and characteristic of an aldehyde chemical function (spectra not shown). Compound 11 was in equilibrium in the reaction media with the corresponding hydrated form. δ at about 44.7 ppm for C-3 and at 88.2 for C-1 were indeed distinctive of 12. Same adducts 11-12 were identified in the individual reaction of cinnamaldehyde with the peptide. Thus, at first sight, cinnamaldehyde was much more reactive than citral.

Adducts 11 and 12 appeared at the very beginning of the reaction (day 0, one hour) and disappeared completely with time. These results probably suggested that cinnamaldehyde formed, very fast, unstable adducts with Pep-Cys that could afterwards dissociate to furnish both precursors. It is known that an equilibrium can be established between α,β-unsaturated aldehydes and their Cys adducts. Previous studies showed that half-lives of the adducts and kinetics of the reverse reactions varied widely depending on the nature of the adduct and on the carbonyl structure studied. [32] The reaction of Pep-Cys with conjugated carbonyls treated here is thus most probably a chemical equilibrium between adducts and the precursors. Further investigation is needed to confirm this hypothesis. The equilibrium constants as well as the rate constants for the reverse reactions, depending on the experimental conditions used and on the chemical structure of the aldehyde, should be determined, this not being the purpose of our investigation here. At the very beginning of the reaction only citral 8 adduct (Figure 3), resulting from Schiff base formation with the amino group of Lys, was observed with δ at 160.5 ppm in the experiments with [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-1 substitution and at 152.7 ppm with [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C-3 substitution. Over time, these signals disappeared in benefit of further hydrated derivatives as the 10-like adduct at 62.5 ppm ( 13 C-1) and 50.9 ppm ( 13 C-3) and of the new observed adduct 13 with characteristic δ at 66.4 ppm ( 13 C-1) and 123.5 ppm ( 13 C-3). Figure 7 shows the mono-dimensional [START_REF] Lepoittevin | Contact Dermatitis[END_REF] C NMR spectrum of the reaction 1-( 13 C)-2/1-( 13 C)-3/Pep-Lys at day 20, where all identified adducts were formed. No more evolution was seen, with citral and cinnamaldehyde practically consumed. Besides citral adducts, a comparable reactivity was observed for cinnamaldehyde with identification of adducts 14-16 (Figure 7), previously characterized when tested individually on Pep-Lys. The Schiff base 14 with characteristic δ at 164.2 ppm ( 13 C-1) and 142.6 ppm ( 13 C-3) was formed through the reaction of the carbonyl group with the amino residue of lysine, together with intermediary and hydrolysis products 15 and 16.

Reaction of citral and cinnamaldehyde towards Pep-Lys

Kinetic competition results

The percentage over time of citral and cinnamaldehyde in the binary mixtures in the presence of the peptides is reported in Figure 8. When following the reactivity with Pep-Cys every 30 min during two days, we saw that cinnamaldehyde reacted much faster than citral during the first 24 h. But when tracing the curves of the reaction over 37 days, the concentration of cinnamaldehyde significantly increased, certainly due to the reversed equilibrium evoked in the previous section between cinnamaldehyde and its Pep-Cys adducts. No significant decrease in the concentration of citral was noticed during the first 24 h of the reaction. At first sight and comparing with cinnamaldehyde it could be argued that the presence of the methyl group in the Michael position of citral could disadvantage the addition of the thiol group of Pep-Cys on this position. However, reaction of Pep-Cys on the Michael position of citral was observed when tested alone after several days of reaction (data not shown). It is interesting to note that we observed a very slow decrease in the citral concentration in the mixture over time (till 37 days). This was in agreement with spectra in Figure 6 that showed a weakening of the characteristic signal of 3-( 13 C)-2 at 168 ppm at day 36. Even if we were unable to distinguish any new signal corresponding to a potential adduct, one could thus hypothesise that adducts formed between citral and Pep-Cys seem to be thermodynamically more stable than those formed with cinnamaldehyde.

Only around 35% cinnamaldehyde was consumed (Figure 8), and theoretically around 65% of Pep-Cys would still be available for reaction. As it was discussed for the mixture hydroxycitronellal-citral, Pep-Cys could have been chemically modified (i. e. oxidised) during the mixture experiment becoming then non reactive. [START_REF] Natsch | [END_REF] However, analysis of what became the unreacted peptide was not in the scope of the study reported herein. Citral reacted completely after 6 days in presence of Pep-Lys, and cinnamaldehyde reacted gradually during the follow-up of the reaction. But then again, at the very beginning of the reaction, reactivity of cinnamaldehyde was higher than that of citral. The practically complete consumption of both sensitizers with time did not reflect only their reactivity towards Pep-Lys. Indeed, the amount of Pep-Lys available in the mixture was not enough to completely consume citral and cinnamaldehyde (2/3/Pep-Lys in a 1:1:1 ratio). Chemicals were initially stable in the buffer. Something may thus happen in the mixture, aside from peptide reaction, that could produce secondary reactions responsible for their complete consumption. Even if we did not observe it in the NMR studies, one suggestion could be that the use of ammonium acetate buffer, in order to increase the reactivity of the amino group of the lateral chain of lysine, involved the presence in the media of ammonia that could also react with the aldehydes.

Discussion of the results

All in all, it looked like the reactivity of the mixture was driven by cinnamaldehyde during the initial hours. This was in agreement with known data for both compounds. Cinnamaldehyde has a LLNA average EC3 (%) value of 2, being categorized as a stronger sensitizer than citral (EC3 13%). [31] Also, studies based on clinical data classified cinnamaldehyde as possessing stronger intrinsic sensitizing potency. [31] The authors even expected that regular contact with moderate concentrations was likely to sensitize 1 to 10% of those so exposed. DPRA results with Pep-Cys and Pep-Lys classified cinnamaldehyde as a moderate to strong reactivity chemical with depletion values of 70.6% and 43.2% respectively, whereas citral was just a moderate reactivity chemical with depletion values of 85.7% and 16.9% (Figure 5). [START_REF] Gerberick | [END_REF] In our studies with Pep-Lys, although cinnamaldehyde showed a strong reactivity at the very beginning of the reaction, citral was more reactive at 24 h (Figure 8) and was the first compound to be completely consumed after seven days (data not shown). All adducts identified resulted from addition of Lys on the aldehyde chemical function of the compounds (Schiff base formation). Considering the reaction with Lys there was thus certainly a difference in the kinetics of Schiff base formation between both compounds when mixed. The fact that both sensitizers reacted through a similar chemical mechanism with Pep-Lys indicated probably a synergistic effect. It was thus not possible to reduce the reactivity of this mixture towards Pep-Lys to the behaviour of one single component. Moreover, as stated earlier, something else may happen in the mixture, aside from peptide reaction, that could produce other reactions explaining the complete consumption of both aldehydes in the long term, even if not observed in the NMR spectra.

Looking at the DPRA decisional tree on Figure 5, it could be expected the citral-cinnamaldehyde mixture being predicted having at least a moderate chemical reactivity. Reactivity studies with Pep-Cys showed that the mixture behaved as if cinnamaldehyde was the major reagent present, and thus the main sensitizer in the mixture. Such observations lead us to believe that in the case of testing the mixture with the DPRA, Pep-Cys depletion would be comparable to that of single cinnamaldehyde. However, Pep-Lys reactivity of both aldehydes in the mixture was expressed in an equivalent manner, although not necessarily identical. In a DPRA test, Pep-Lys depletion would result from reactivity of the two molecules in the mixture and thus the prediction would certainly be overestimated. Because of, Pep-Lys depletion of the mixture may exceed 42.5% and this would classify it as having a strong chemical reactivity. Indeed, Pep-Lys depletion values being 16.9% for citral and 43.2% for cinnamaldehyde it could be expected that Pep-Lys depletion by the mixture would be enough to classify it as having a strong reactivity. Still, for consumer protection it is probably best that the DPRA upgrades some mixtures and not the opposite.

Conclusion

Several in silico, in vitro, and in chemico alternative methods have been developed this last decade in order to assess skin sensitization to chemicals, following the ban on animal testing introduced by cosmetic legislation in Europe in 2013. Some of these methods, such as the in chemico DPRA, the in vitro ARE-Nrf2 luciferase assay (KeratinoSens TM ) and the human Cell Line Activation test (h-CLAT), have been recently adopted by the Organization for Economic Cooperation and Development (OECD) for testing health effects of chemicals. [START_REF] Anon | Test No. 442C: In Chemico Skin Sensitisation Direct peptide Reactivity Assay (DPRA). OECD guidelines for the testing of chemicals[END_REF][START_REF] Anon | Test No. 442D: In Vitro Skin Sensitisation, ARE-Nrf2 Luciferase Test Method, OECD guidelines for the testing of chemicals[END_REF]34] These tests were developed and validated for the testing of pure substances. However, in the cosmetic field, active ingredients are often mixtures from natural sources together with synthetic compounds, and a fragrance formula may consist of 10-300 or more different ingredients in a consumer product. Moreover, REACH regulation requires not only pure substances to be registered, but also registration of mixtures extracted from natural sources. Though, there is very little experience with testing of mixtures, and assessment of test material with more than one component has been proved to be an elusive task in existing methods. The in vivo LLNA was used nevertheless to test a number of essential oils and authors stated that in general the potency of each essential oil did not differ significantly from that of its main individual component. [START_REF] Api | [END_REF] Also, the applicability of the KeratinoSens TM assay has been evaluated recently as a proof of concept for four plant extracts used in cosmetics, spiked with different doses of citral, cinnamaldehyde and isoeugenol. [36] The extracts were negative in the test whereas they were positive when spiked with the sensitizers. In this manuscript, we evaluated in a very simple first approach, and from the chemical reactivity point of view, if the DPRA could be used at a first sight to evaluate the reactivity-allergenicity of mixtures. Two simple binary combinations of fragrance aldehydes were chosen, hydroxycitronellal-citral and citralcinnamaldehyde. Qualitative assessment of adduct formation between the aldehydes in the mixtures and Pep-Cys and Pep-Lys was carried out. Further studies on the loss of test chemicals in the mixtures with time were attempted leaving many questions open. The studies showed that aldehydes in the mixtures could react with the peptides, indicating that the DPRA could detect the presence of sensitizers in the mixtures. In general, the most reactive chemical in each mixture was described as the most important sensitizer up to LLNA and DPRA data, which is a positive point. Therefore, it could be hypothesized that the DPRA would estimate the chemical reactivity of the mixture similar to that of the stronger sensitizer component. However, several drawbacks suggested that the DPRA decisional tree could be no longer valid for the evaluation of the studied mixtures. Considering that reactivity of the chemicals towards Pep-Cys and Pep-Lys should be directly correlated to the depletions of the peptides, there were some significant differences in the behaviour of the chemicals when tested individually or in the mixtures. Citral was much more reactive in the mixture than hydroxycitronellal, but it reacted more with Pep-Lys than with Pep-Cys. The depletions of both peptides would then be overexpressed and underestimated respectively. On another hand, even if cinnamaldehyde was more reactive in the mixture than citral with Pep-Cys, both compounds reacted enough with Pep-Lys and thus we could extrapolate that reactivity of the mixture resulted from the sum of both reactivities. Pep-Lys depletion would be then overestimated. In all cases, as we were dealing with aldehydes, the Lys-containing peptide facilitated Schiff base formation resulting in greater reactivity. Second to that, attempts to calculate kinetic constants and rates by plugging the logarithm of the concentration of chemicals present in the mixtures against time resulted in bad non-exploitable outcomes. As a conclusion, from a very first sight based on a hapten-peptide reactivity approach, the assessment of the reactivity of mixtures by using the DPRA could be attempted if one component is much more reactive and thus more strongly sensitizing than the other (i. e. strong against weak sensitizers) but becomes difficult when components are of close sensitizing potential such as fragrance aldehydes (weak or moderate) having high Lys reactivity.

Our very first approach studies were based on the hypothetical assumption that loss of chemicals in the mixtures was directly related to peptide reactivity. It cannot be excluded, however, that other processes may influence the reactivity of the test chemicals and thus skew the results. Here, adduct formation and depletion of test chemicals was studied. In the DPRA the opposite is done, depletion of the peptides is analysed. In order to match results observed, further DPRA analysis of the mixtures are thus necessary, at simple routine concentrations and at the ratios used in here. Data on the behaviour of the DPRA peptides in the mixtures would indeed give added value data. Other issues that need to be further evaluated such as DPRA values for the mixtures are LLNA values, equivalents and number of ingredients, dose-response curves, time of reading, kinetic rates and peptide depletions due to redox processes. Percentage" Time(days)"

Hydroxycitronellal"

Citral"

Figure 5. DPRA decisional classification tree, and putative association with sensitization potential classes [15] Chemical

  C NMR (75 MHz, CDCl3): E isomer, δ 128.6 (d, 1 JCC=68.5 Hz, -CHCHO), 128.6 (2 × m-CH(Ar)), 129.1 (2 × o-CH(Ar)), 131.3 ( p-CH(Ar)), 134.2 (d, 1 JCC=55.8 Hz, -C(Ar)), 152.8 (-13 CHCHCHO), 193.7 (d, 2 JCC=4.7 Hz, -CHO).

  C substituted at the aldehyde chemical function position 1 (1-( 13 C)-2, 1-( 13 C)-3) and at the Michael acceptor position 3 (3-( 13 C)-2, 3-( 13 C)-3) (Figure 1). Mixtures of aldehydes were then reacted with 1 equivalent of the nucleophilic peptides Pep-Cys and Pep-Lys.

Figure 2

 2 is representative of the chemistry observed in the reaction of the mixture hydroxycitronellal-citral with the thiol chemical group from Pep-Cys. Results were analogous with both mixtures 1-( 13 C)-1/1-( 13 C)-2 and 1-( 13 C)-1/3-( 13 C)-2. When starting the reaction (day 0) the signal of the hydrated form of hydroxycitronellal 4 at 89.4 ppm was observed and only citral reacted with the thiol group of Cys on the Michael position. The δ at 46.7 ppm indicated the formation of adduct 5

  Results were similar with both mixtures 1-( 13 C)-2/1-( 13 C)-3 and 3-( 13 C)-2/3-( 13 C)-3. Adducts characterized are shown in Figure 7. Their characteristic δ were compared with those of adducts formed by reaction of each constituent alone with Pep-Lys.

Figure 1 Figure 1 .

 11 Figure 1

Figure 2 Figure 2 . 20 Figure 3 Figure 3 .

 222033 Figure 2

  Figure 4

Figure 4 .

 4 Figure 4. Evolution as a function of time of the concentration of hydroxycitronellal and citral in the mixtures in the presence of Pep-Cys and Pep-Lys

Figure 6 Figure 6 .

 66 Figure 6

Figure 7 Figure 7 .

 77 Figure 7

Figure 8 .

 8 Figure 8

13 C)-3 (177

  mg, 1.34 mmol, 71% yield) as a yellow oil. Only the E isomer was observed by NMR. 1 H NMR (300 MHz, CDCl3): δ 6.68 (ddd, 1H, 3 JHH=15.8

	Hz, 3 JHH=7.5 Hz, 2 JHC=1.2 Hz, -CH 13 CHO), 7.40 (d, 1H, 3 JHH=15.8 Hz, -CHCH 13 CHO), 7.39-7.49 (m, 3H, 2
	× m-CH(Ar), p-CH(Ar)), 7.51-7.55 (m, 2H, 2 × o-CH(Ar)), 9.67 (dd, 1H, 1 JHC=172.0 Hz, 3 JHH=7.5 Hz, -
	13 CHO). 13 C NMR (75 MHz, CDCl3): δ 128.6 (d, 1 JCC=55.4 Hz, -CH 13 CHO), 128.5 (2 × o-CH(Ar)), 129.1
	(2 × m-CH(Ar)), 131.3 (p-CH(Ar)), 134.0 (-C(Ar)), 152.8 (d, 2 JCC=4.3 Hz, -CHCH 13 CHO), 193.7 (-
	13 CHO).

3-( 13 C)Cinnamaldehyde, 3-( 13 C)-3. Same

  methodology used for the synthesis of 1-( 13 C)cinnamaldehyde but with the following reagents: 2.56 mL freshly distilled diisopropylamine (18.3 mmol, 4 equiv), 30 mL dry tetrahydrofuran, 11.40 mL n-butyllithium (1.60 M in hexane, 18.3 mmol, 4 equiv), 478 µL dry acetonitrile (9.15 mmol, 2 equiv), 1.40 mL diethylchlorophosphate (9.15 mmol, 2 equiv), 479 µL 1-( 13