Cinnamyl alcohol oxidizes rapidly upon air exposure

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1 Contact Dermatitis riginal Article CD Contact Dermatitis Cinnamyl alcohol oxidizes rapidly upon air exposure Ida B. Niklasson 1, Tamara Delaine 1, M. Nurul Islam 1, Roger Karlsson 1, Kristina Luthman and Ann-Therese Karlberg 1 1 Department of Chemistry and Molecular Biology, Dermatochemistry and Skin Allergy, University of Gothenburg, SE Gothenburg, Sweden and Department of Chemistry and Molecular Biology, Medicinal Chemistry, University of Gothenburg, SE Gothenburg, Sweden doi:1.1111/cod.19 Summary Background. Cinnamyl alcohol and cinnamal are frequent fragrance contact allergens. Both are included in the European baseline fragrance mix I, which is used for screening of contact allergy in dermatitis patients. bjectives. The aim of this study was to investigate the autoxidation of cinnamyl alcohol and to identify the oxidation products formed on air exposure. We also wanted to evaluate the effect of autoxidation on the sensitization potency of cinnamyl alcohol. Methods. Samples of commercially available cinnamyl alcohol with and without purification were exposed to air, and the autoxidation was followed by chemical analysis. The analysis was performed with mass spectrometry (LC/MS/MS). Sensitization potencies of compounds were determined with the murine local lymph node assay (LLNA) in mice. Results. Chemical analysis showed that the concentration of cinnamyl alcohol in the air-exposed samples decreased rapidly over time, and that autoxidation products were formed. Cinnamal, epoxy cinnamyl alcohol and cinnamic acid were identified as oxidation products. According to our study, cinnamal and epoxy cinnamyl alcohol were the first autoxidation products formed. The epoxy cinnamyl alcohol was shown to be the oxidation product with the highest sensitization potency. The analysis of our samples of commercially available cinnamyl alcohol showed that there was already a content of 1.5% cinnamal at the start of the autoxidation experiments. Conclusion. Cinnamyl alcohol readily autoxidizes upon air exposure, and forms strong sensitizers as determined by the LLNA. Cinnamal was formed in the largest amounts, showing that cinnamal is not only formed via bioactivation, as has previously been shown. A highly sensitizing epoxide was also identified and quantified in the oxidation mixture. Key words: autoxidation; bioactivation; cinnamal; cinnamyl alcohol; concomitant reactions; epoxides; fragrance; local lymph node assay (LLNA); mass spectrometry; prehapten; prohapten. Cinnamyl alcohol (cinnamic alcohol; CAS ) (Fig. 1) is found naturally in the leaves and the inner bark of several trees of the genus Cinnamomum. It is Correspondence: Ann-Therese Karlberg, Department of Chemistry and Molecular Biology, Dermatochemistry and Skin Allergy, University of Gothenburg, SE Gothenburg, Sweden. Tel: karlberg@chem.gu.se Conflicts of interest: The authors have declared no conflicts. Accepted for publication 15 September 1 also present in balms such as styrax and Myroxylon pereirae resin (balsam of Peru). Cinnamyl alcohol has the odour of hyacinth, and is frequently used as a fragrance ingredient in shampoos, soaps, fine fragrances, and other toiletries. The worldwide annual amounts of cinnamal (cinnamaldehyde; CAS ) (Fig. 1) and cinnamyl alcohol used industrially have been estimated to be 159 and 7 metric tons, respectively (1). Fragrances are common causes of contact allergy, owing to widespread use and frequent exposure (, 3). Cinnamyl alcohol and cinnamal are frequent contact allergens, causing Contact Dermatitis, 68,

2 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNETAL. H Cinnamic acid air exposure H Epoxy cinnamyl alcohol Epoxy cinnamal H Cinnamyl alcohol air exposure Cinnamal Epoxy cinnamyl alcohol air exposure H Epoxy cinnamal Fig.. Chemical structures of synthesized compounds. Materials and Methods Fig. 1. Plausible autoxidation products after air exposure of cinnamyl alcohol. Red structures denote strong sensitizers according to the local lymph node assay. allergic reactions in a substantial number of individuals sensitized from contact with cosmetics. They are therefore two of eight constituents of the fragrance mix I used in the baseline series for screening of contact allergy in dermatitis patients. Moreover, they are among the 6 fragrance substances for which, according to EU legislation, information should be provided to consumers about their presence in cosmetic products, in order to reduce the risk of sensitization and eczema in the population. For products that are intended to stay on the skin for a prolonged time and not to be rinsed off, the limit for labelling is set at.1% (e.g. lotions and deodorants). For those compounds that are rinsed off directly after application, the limit is.1% (e.g. shampoos) (4). Some fragrances are not electrophilic and proteinreactive themselves; they need to be activated first, either via autoxidation (prehapten) or bioactivation (prohapten) (5). The toxicological and dermatological properties of cinnamyl alcohol have been extensively reviewed (6). As cinnamyl alcohol lacks structural alerts for protein reactivity, it has been shown to act as a prohapten by forming the hapten cinnamal via metabolic oxidation in the skin (7, 8). Cinnamyl alcohol is frequently used as the model prohapten in mechanistic studies on skin metabolism (9, 1). The aim of the present study was to investigate the stability of cinnamyl alcohol upon air exposure. In the case of autoxidation, we wanted to investigate whether specific oxidation products with known (cinnamal) or suspected (epoxy cinnamyl alcohol and epoxy cinnamal) sensitizing effects were formed in the oxidation mixture, and if so, to determine their sensitization potential and their impact on the sensitization potential of the autoxidation mixture. Chemicals Cinnamyl alcohol and cinnamal (Fig. 1) were purchased from Aldrich Chemicals (Stockholm, Sweden). Acetone was purchased from Merck (Darmstadt, Germany) and olive oil from Apoteket AB (Gothenburg, Sweden). Unless otherwise indicated, reagents were obtained from commercial suppliers and used without further purification. Thin-layer chromatography was performed with unmodified silica gel-coated ( μm) aluminium plates. Column chromatography was performed with Merck silica gel 6 (3 4 mesh ASTM). The purity of both synthesized and purchased test compounds was >98% according to gas chromatography/mass spectrometry (GC/MS) before testing of sensitization potential. Synthesis Epoxy cinnamyl alcohol. The synthesis was performed as previously described (11), producing the compound with a yield of 85% (Fig. ). The product was purified with column chromatography on silica gel (% ethyl acetate in hexanes). 1 H nuclear magnetic resonance (NMR) δ 1.9 (1H, dd, J = 5.3, 7.5 Hz, H), 3. (1H, ddd, J =.,,3, 4.1 Hz, H), 3.8 (1H, ddd, J = 4.1, 7.5, 11.8 Hz, H1a), 3.93 (1H, d, J =. Hz, H3), 4.5 (1H, ddd, J =.3, 5.3, 11.8 Hz, H1b), (5H, m, H5a, 5b, 6a, 6b, 7). 13 CNMRδ 55.5 (C3), 61.3 (C), 6.5 (C1), 15.6 (C5a, 5b), 18.4 (C7) 18.6 (C6a, 6b), (C4). Electron ionization mass spectrometry (EI-MS) (7 ev), m/z (%) 13 (7) (M + ), 119 (8), 17 (96), 91(1), 79 (49), 71 (5), 57 (69). Epoxy cinnamal. The synthesis was performed as previously described (1) with cinnamal as starting material, producing the compound with a yield of 6% (Fig. ). The product was purified with column chromatography on silica gel (17% ethyl acetate in pentane). 1 HNMRδ 3.44 (1H, dd, J = 1.8, 6. Hz, H) 4.16 (1H, d, J = 1.8Hz, H3), (5H, m, H5a, 5b, 6a, 6b, 7), 9. (1H, d, J = 6.Hz,H1). 13 CNMRδ 56.8 (C3), 63. (C), Contact Dermatitis, 68,

3 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNET AL. (C5a, 5b), 18.9 (C6a, 6b), 19.3 (C7), (C4), 197. (C1). EI-MS (7 ev), m/z (%) 147 (36) (M + ), 131 (6), 119 (35), 15 (1), 91(1), 77(14), 65 (14), 51 (1). Air exposure procedure Autoxidation I. Before the start of the experiment, cinnamyl alcohol was purified with column chromatography. No impurities were detected according to chemical analysis with GC/MS performed after the purification. Cinnamyl alcohol (5 g) dissolved in ethanol (1 ml) was airexposed in an Erlenmeyer flask, covered with aluminium foil to prevent contamination. It was gently stirred for 1 hr four times a day, as previously described, and illuminated with a daylight lamp (Philips Master TL-D 9 De Lux, 18 W/95) for 1 hr a day to minimize the difference in light caused by annual variations (13). ver a period of 56 weeks, 5 samples were taken with only a few days between sampling occasions from the beginning, and with several weeks between them at the end of the time period. The samples were flushed with argon to evaporate the ethanol and to keep an inert atmosphere in the vials. The samples were stored at 7 C prior to analysis by liquid chromatography coupled to high resolution mass spectrometry (LC/MS/MS) to determine the decreasing concentration of cinnamyl alcohol and the formation of oxidation products over time. Autoxidation II. Cinnamyl alcohol was purified not only with column chromatography but also with preparative high-performance liquid chromatography (HPLC) before the study of autoxidation was started. A round-bottomed flask containing cinnamyl alcohol (1. g) dissolved in ethanol ( μl), covered with aluminium foil to prevent contamination, was placed on the bench in the middle of a laboratory; that is, no stirring and no daylight lamp were used. ver a period of weeks, 1 samples were withdrawn from the flask, flushed with argon to evaporate the ethanol, and directly analysed with LC/MS/MS to determine the decreasing concentration of cinnamyl alcohol and the formation of oxidation products with time. Autoxidation III. Cinnamyl alcohol was not purified before the study of autoxidation, but was used at commercially available purity (98%). Analyses on two samples exposed to different conditions were performed. A round-bottomed flask containing cinnamyl alcohol (1. g) dissolved in ethanol ( μl) was (i) stirred for 1 h four times a day, as previously described, or (ii) covered with aluminium foil to prevent contamination, and placed on the bench in the middle of a laboratory; that is, no stirring and no daylight lamp were used. ver a period of weeks, 1 samples were withdrawn from each of the two flasks, and flushed with argon to evaporate the ethanol. The samples were stored at 7 C prior to analysis with LC/MS/MS to determine the decreasing concentration of cinnamyl alcohol and the formation of oxidation products over time. Instrumentation and mode of analysis 1 Hand 13 C NMR spectroscopy. These were performed on a Jeol Eclipse 4 spectrometer at 4 and 1 MHz, respectively, with CDCl 3 solutions (residual CHCl 3 δ 7.6 and CDCl 3 δ 77. as internal standards). Gas chromatography/mass spectrometry. EI-MS analysis (7 ev) was performed on a Hewlett-Packard 5973 mass spectrometer connected to a gas chromatograph (Hewlett-Packard 689) equipped with a cool on-column capillary inlet and an HP-5MSi fused silica capillary column (3 m.5 mm,.5 μm; Agilent Technologies, Palo Alto, CA, USA). Helium was used as carrier gas, and the flow rate was 1. ml/min. The temperature started at 35 C for 1 min, was increased by 1 C/min, and ended at 5 C for 5 min. For mass spectral analysis, the mass spectrometer was used in the scan mode, detecting ions with m/z values ranging from 5 to 15. Preparative HPLC. This was performed with a Gilson pump model 35, a Gilson ultraviolet/visible detector model 119, and a Zorbax semi-preparative column (5 mm 9.4 mm, 5-μm particles; Agilent Technologies); the flow rate was 6. ml/min, and the compounds were monitored at 3 nm. Aliquots of 1 μl were injected onto the column and eluted with acetonitrile (35%) in Milli-Q water. The LC/MS/MS system. The system used for analysis (1 series; Agilent Technologies, Waldbronn, Germany) consisted of a high-pressure pump, an autosampler, and a mass spectrometer (Triple Quad LC-MS 641; Agilent Technologies, Palo Alto, CA, USA) equipped with an electrospray interface and a triple-quadrupole mass analyser. The electrospray triple-quadrupole mass spectrometer was operated in the positive ionization mode. The positive ions of the analytes were detected by following transitions in multiple reaction monitoring (MRM) mode. Mode of analysis for autoxidation I and II with LC/MS/MS. The separation was performed on a phenyl column (4.6 5 mm, 5 μm, FRTIS Phenyl; Fortis Technologies Ltd, Neston, UK). A guard column (FRTIS Phenyl, mm, 5 μm) was used to reduce column degradation. The mobile phases consisted of Milli-Q Contact Dermatitis, 68,

4 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNETAL. water with.1% formic acid (solvent A) and acetonitrile with.1% formic acid (solvent B). For separation, a starting concentration of 3% solvent B was used for 5 min, followed by a linear gradient up to 48% solvent B for 4 min. To re-equilibrate the column between the runs, 15 min of isocratic elution (3% solvent B) was used. The flow rate was.4 ml/min and the injection volume was 5 μl. Mode of analysis for autoxidation III with LC/MS/MS. The separation was performed on a phenyl column (.1 15 mm, 3 μm, FRTIS Phenyl; Fortis Technologies Ltd). The mobile phases consisted of Milli-Q water with.1% formic acid (solvent A) and acetonitrile with.1% formic acid (solvent B). For separation, a starting concentration of 3% solvent B was used for 1 min, followed by a linear gradient up to 5% solvent B for 5 min, and the gradient programme then continued to 75% solvent B for 1 min. To re-equilibrate the column between the runs, 1 min of isocratic elution (3% solvent B) was used. The flow rate was.3 ml/min and the injection volume was 5 μl. A representative LC/MS/MS chromatogram and MRM transitions are shown in Fig. 3. Quantification of cinnamyl alcohol and its oxidation products with LC/MS/MS The degradation of cinnamyl alcohol and the formation of oxidation products were quantified with LC/MS/MS. The sample concentrations were 5 μm (autoxidation I and II) and 6.5 μm (autoxidation III) in acetonitrile for analysis of the depletion of cinnamyl alcohol, and μm (autoxidation I and II) and 13 μm (autoxidation III) in acetonitrile for analysis of the formation of the Fig. 3. LC/MS/MS chromatogram showing all six time segments from a sample of autoxidation III of cinnamyl alcohol (t = 8 days). Multiple reaction monitoring showing 4-hydroxybenzoic acid at.3 min, epoxy cinnamyl alcohol at 4.1 min, cinnamyl alcohol at 5.6 min, cinnamic acid at 5.9 min, and cinnamal at 6.9 min. According to analysis with standard solutions, epoxy cinnamal would have been seen in time segment 6 if present. MSD, Mass Selective Detector. 13 Contact Dermatitis, 68,

5 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNET AL. oxidation products of specific interest. The quantification was performed with external standard curves from pure reference compounds with 4-hydroxybenzoic acid ( μm) as an internal standard to determine the relative response factors. Reference compounds were purchased or synthesized, and their chromatographic and spectral properties were compared with those in the autoxidation mixture. The purchased compounds were purified using HPLC (cinnamyl alcohol) and column chromatography (cinnamal and cinnamic acid). Epoxy cinnamyl alcohol and epoxy cinnamal were synthesized. Sensitization experiments in mice Experimental animals. Female CBA/Ca mice, 8 or 9 weeks of age, were purchased from B&K Sollentuna. The mice were housed in HEPA-filtered air flow cages, and kept on standard laboratory diet and water ad libitum. The local ethics committee in Gothenburg approved the study. Sensitization potential of oxidation products in mice. The murine local lymph node assay (LLNA) (14) was used to assess the sensitization potential. Mice in six groups of 3 animals each were treated by topical application on the dorsum of both ears with the test compound (5 μl) dissolved in acetone/olive oil (4:1 vol/vol) or with the vehicle control. All solutions were freshly prepared for every application. Each compound was tested at five different concentrations. Treatments were performed daily for three consecutive days (, 1, and ). Shamtreated control mice received vehicle alone. n day 5, all mice were injected intravenously via the tail vein with [ 3 H]methylthymidine (. Ci/mmol; Perkin-Elmer Biosciences, Waltham, MA, USA) ( μci) in phosphatebuffered saline (PBS) containing 137 mm NaCl,.7 mm KCl, and 1 mm phosphate buffer (ph 7.4) (5 μl). After 5 hr, the mice were killed, the draining lymph nodes were excised and pooled for each group, and single-cell suspensions of lymph node cells in PBS were prepared with cell strainers (Falcon; BD Labware, Franklin Lakes, NJ, USA; pore size, 7 μm). Cell suspensions were washed twice with PBS, precipitated with trichloroacetic acid (TCA) (5%), and left in the refrigerator overnight. The samples were then centrifuged, resuspended in TCA (5%) (1 ml), and transferred to scintillation cocktail (1 ml) (EcoLume INC Radiochemicals, Solon, H, USA). The [ 3 H]methylthymidine incorporation into DNA was measured by β-scintillation counting on a Beckman LS 6TA instrument. Results are expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI) (15), that is, test group/control group ratio. Test materials that, at one or more concentrations, caused an SI of >3 were considered to be positive in the LLNA. EC3 values (the estimated concentration required to induce an SI of 3) were calculated by linear interpolation. The sensitization potency was classified as follows: <.1%, extreme;.1 to <1%, strong; 1 to <1%, moderate; and 1%, weak (16). Results Air exposure of cinnamyl alcohol Autoxidation I. Aliquots were withdrawn from air-exposed cinnamyl alcohol I at different time points over a period of 56 weeks, and analysed with LC/MS/MS to quantify the remaining amount of cinnamyl alcohol and to identify and quantify the autoxidation products formed. Reference compounds were purchased and purified or synthesized, and their chromatographic and spectral properties were compared with those in the autoxidation mixture. Chemical analysis of the degradation of airexposed cinnamyl alcohol showed that 33% of the cinnamyl alcohol remained after 13 weeks and only 14% remained after 56 weeks (Fig. 4a). In the oxidation mixture, we identified cinnamal, epoxy cinnamyl alcohol, and cinnamic acid (Fig. 3). After days of air exposure, the concentration of epoxy cinnamyl alcohol was 1.1%, and the concentration of cinnamal was 1.3%. After 56 weeks, the corresponding concentrations were.1% and 1.5% (Fig. 4b). Epoxy cinnamal was not detected. As the autoxidation was very fast, another, more detailed, experiment was desirable. Autoxidation II. The degradation of cinnamyl alcohol was studied, and after 14 days only 36% remained in the air-exposed sample (Fig. 5a). The quantification of the oxidation products present after weeks in the second air-exposed sample of cinnamyl alcohol is shown in Fig. 5b. After 9 days of air exposure, small amounts of epoxy cinnamyl alcohol (.11%) and cinnamal (.35%) could be detected. After 16 days, the concentration of the epoxy cinnamyl alcohol was.3% and the concentration of cinnamal was.3% (Fig. 5b). Epoxy cinnamal was not detected. Autoxidation III. The degradation of non-purified commercially available cinnamyl alcohol was performed in two different ways: (i) dissolved in ethanol with stirring and a daylight lamp; and (ii) dissolved in ethanol without stirring and a daylight lamp. Quantification showed the presence of cinnamal (1.5%), epoxy cinnamyl alcohol (.6%) and cinnamic acid (.9%) at the start of Contact Dermatitis, 68,

6 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNETAL. Concentration (%) a b Time (weeks) Fig. 4. Autoxidation I. (a) Degradation of cinnamyl alcohol during air exposure over time ( 56 weeks). (b) Formation of cinnamal ( ), epoxy cinnamyl alcohol ( ) and cinnamic acid (o) in air-exposed cinnamyl alcohol during weeks 56. Quantification was performed with LC/MS/MS. 1 a.5 b Concentration (%) Time (days) Fig. 5. Autoxidation II. (a) Degradation of cinnamyl alcohol during air exposure over time ( weeks). (b) Formation of cinnamal ( ), epoxy cinnamyl alcohol ( ) and cinnamic acid (o) in air-exposed cinnamyl alcohol during weeks. Quantification was performed with LC/MS/MS. both experiments. After 9 days of air exposure, cinnamal, epoxy cinnamyl alcohol and cinnamic acid were present in the autoxidation mixture at concentrations of.5%, 1.%, and.1%, respectively. The quantification of the oxidation products and the depletion of cinnamyl alcohol in both experiments are shown in Fig. 6A,B. Sensitization potency according to the LLNA Cinnamyl alcohol air-exposed for weeks in the autoxidation II experiment gave an EC3 value of.58 M (4.9% wt/vol), and it can be classified as a moderate sensitizer according to the classification suggested by Kimber et al. (16). Molarity was calculated on the basis of the molecular mass of cinnamyl alcohol. Cinnamal, epoxy cinnamyl alcohol and epoxy cinnamal were all strong sensitizers, with EC3 values of 57 mm (.75% wt/vol), 39 mm (.58% wt/vol), and 15 mm (.% wt/vol), respectively (Table 1; Fig. 7). Discussion The present study shows that cinnamyl alcohol autoxidizes rapidly upon air exposure, forming a highly allergenic epoxide and cinnamal. The results show that cinnamyl alcohol is not only a prohapten that is metabolically activated in the skin, but is also readily activated outside the skin by autoxidation, thus acting as a prehapten. Another fragrance compound that has been shown to act both as a prehapten and as a prohapten is geraniol (17), and such a possibility should be considered for other compounds. Air oxidation I of cinnamyl alcohol (purified with column chromatography) was performed according to previous experience from studies of other fragrance compounds within our group, for example autoxidation of geraniol (18) and linalool (19), with stirring and a daylight lamp. After 56 weeks of air exposure, only 14% of cinnamyl alcohol remained in the oxidation mixture. To allow the autoxidation process to be followed from the very beginning in more detail, cinnamyl alcohol was 134 Contact Dermatitis, 68,

7 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNET AL. Concentration (%) A B (a) (a) (b) (b) Time (days) Fig. 6. Autoxidation III. (A) (a) Degradation of non-purified commercially available cinnamyl alcohol (as obtained, dissolved in ethanol) during air exposureovertime ( weeks). (b) Formationofcinnamal ( ), epoxycinnamylalcohol ( ) and cinnamic acid ( ) in cinnamyl alcohol during weeks. A daylight lamp and stirring were used. (B) (a) Degradation of non-purified commercially available cinnamyl alcohol (as obtained, dissolved in ethanol) during air exposure over time ( weeks). (b) Formation of cinnamal ( ), epoxy cinnamyl alcohol ( ) and cinnamic acid ( ) in cinnamyl alcohol during weeks. No daylight lamp and no stirring were used. purified with HPLC before the shorter air oxidation II was started. The air exposure was performed without stirring and direct daylight. The sample was dissolved in ethanol and left to autoxidize at room temperature for weeks. By day 9, small amounts of both epoxy cinnamyl alcohol and cinnamal could already be detected. After weeks, only 36% of non-degraded cinnamyl alcohol remained in the oxidation mixture. Interestingly, the investigated oxidation products were already found in autoxidation I and II in similar amounts after 14 days of air exposure. The oxidation rate of cinnamyl alcohol was very high as compared with the rates seen for two other fragrance alcohols, linalool and geraniol, as 95% of linalool and 98% of geraniol remained non-degraded after 3 4 weeks of air exposure, with both stirring and daylight exposure (18, 19). It should be noted that the aim of the present study was to investigate whether specific oxidation products were formed upon air exposure (Fig. 1). Thus, no attempts were made to identify other compounds of the oxidation mixture, such as dimers, although, during autoxidation I, it was obvious that polymerization took place. Furthermore, in the autoxidation studies of cinnamyl alcohol, the fragrance chemical was dissolved in ethanol, as it is a solid compound. In our previous studies, no solvent was used, as those compounds were liquids. However, even if the impact of ethanol on the present autoxidation studies has not yet been investigated, it is a most relevant solvent to use, as it is a commonly used vehicle for fragrance substances. Analysis with the developed LC/MS/MS method revealed the presence of epoxy cinnamyl alcohol, cinnamal and cinnamic acid in newly bought samples of cinnamyl alcohol (i.e. autoxidation III) with a stated purity of 98%, according to the manufacturer, thus indicating that the commercial cinnamyl alcohol had already started to autoxidize. No difference in degradation rate was seen between the two air exposure methods used: (i) stirring and a daylight lamp; and (ii) no stirring and no daylight lamp. This is to be expected, as autoxidation is caused by a radical mechanism and, once the oxidation has started, the degradation rate depends only on the inherent properties. The LLNA experiments showed the -week autoxidized sample of cinnamyl alcohol to be a moderate sensitizer, with an EC3 value of.58 M (4.9%), whereas the Contact Dermatitis, 68,

8 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNETAL. Table 1. Results from the murine local lymph node assay (LLNA) a Compound and test concentration (% wt/vol) Test concentration (M) [ 3 H]Thymidine incorporation (dpm/lymph node) SI EC3 value (% wt/vol; M) Classification Two-week air-exposed cinnamyl alcohol 4.9;.58 Moderate Control b b b b b Cinnamal.75;.57 Strong Control Epoxy cinnamyl alcohol.58;.39 Strong Control Epoxy cinnamal.;.15 Strong Control Sensitization experiments on the cinnamic compounds studied. a Groups of mice were treated with test substance at five different concentrations, on the dorsum of both ears, for three consecutive days. Sham-treated control mice received vehicle alone. n day 5, all mice were injected intravenously with phosphate-buffered saline (5 μl) containing μci of [ 3 H]methylthymidine. After 5 hr, the mice were killed, the draining lymph nodes were excised and pooled for each group, single-cell suspensions of lymph node cells were prepared, and thymidine incorporation into DNA was measured by β-scintillation counting. The increase in thymidine incorporation relative to vehicle-treated controls was derived for each experimental group, and recorded as stimulation index (SI) (15). The EC3 values (the estimated concentration required to induce an SI of 3) were calculated by using linear interpolation. The sensitizing potency was classified as follows: <.1%, extreme;.1% to <1%, strong; 1% to <1%, moderate;and 1%, weak (16). b Molarity was calculated on the basis of the molecular weight of cinnamyl alcohol. literature value of cinnamyl alcohol is 1% (i.e. a weak sensitizer) (6, ). This result indicates that autoxidation of cinnamyl alcohol increases the sensitization potency. The autoxidation products tested epoxy cinnamyl alcohol, epoxy cinnamal, and cinnamal were all sensitizers of strong potency. Cinnamic acid was not tested, for ethical reasons, as we consider that, in general, acids have a very low sensitizing capacity. The epoxy cinnamal was never detected in the autoxidation mixture. However, the detection limit for epoxy cinnamal with our method is rather high as compared with that for the other analytes, so even if it was formed in the same amounts as epoxy cinnamyl alcohol, it would be undetected. Cinnamal possesses two electrophilic sites, and could therefore react directly with proteins via two different mechanisms: via Schiff base formation with lysine residues (1), or via Michael addition with sulfhydryls (). Epoxy cinnamyl alcohol can react directly with proteins via an S N mechanism (). We consider that both epoxy cinnamyl alcohol and cinnamal contribute to the sensitization potency of air-exposed cinnamyl alcohol. Both epoxy cinnamyl alcohol and cinnamal are strong sensitizers, with EC3 values of.39 M (.58%) and.57 M (.75%), respectively. At the very beginning of the autoxidation process they are present in equal amounts, but after a longer period of time 136 Contact Dermatitis, 68,

9 AUTXIDATIN F CINNAMYL ALCHL NIKLASSNET AL. Stimulation index Concentration (M) Fig. 7. Dose response curves for epoxy cinnamal ( ), epoxy cinnamyl alcohol ( ), cinnamal ( ) and cinnamyl alcohol autoxidized for weeks ( ), tested in the murine local lymph node assay. Molarity for the oxidized cinnamyl alcohol was calculated on the basis of the molecular weight of cinnamyl alcohol. The horizontal line marks a stimulation index of 3, the cutoff limit for a compound to be considered a sensitizer, according to the method. the concentration of cinnamal is higher in the oxidation mixture, which might be a result of further oxidation of the epoxy cinnamyl alcohol. Cinnamyl alcohol and cinnamal are generally considered to be the most prominent example of a prohapten hapten pair. There are several studies in the literature that have aimed to show that cinnamal is the hapten in common for both cinnamyl alcohol and cinnamal (7, ), but only minimal metabolic transformation from cinnamyl alcohol to cinnamal has been shown (1, 8). Even so, cinnamyl alcohol has been the preferred prohapten in numerous experimental studies over the years (3 5). As bioactivation often forms epoxides, the possibility could not be excluded that epoxy cinnamyl alcohol is also formed metabolically. When cinnamyl alcohol is used in sensitization experiments, samples must be specifically analysed and purified directly before the experiment is performed, to avoid contamination by autoxidation products. As both epoxy cinnamyl alcohol and cinnamal are strong sensitizers, they may contribute to the sensitizing potency seen for commercial cinnamyl alcohol used in the in vitro studies (4, 6). Furthermore, autoxidation of cinnamal, similar to that observed for geranial, must also be considered (18, 7). Cinnamyl alcohol is almost as frequent a cause of allergic contact dermatitis as is cinnamal (8), and both are included in the fragrance mix in the baseline series for diagnosis of contact allergy. The high frequency of allergic reactions to test preparations of cinnamyl alcohol might be explained by a higher degree of exposure to cinnamyl alcohol than to cinnamal (9). Concomitant reactions to cinnamyl alcohol and cinnamal are frequently seen, and can be explained by co-exposure and cinnamyl alcohol being a prohapten causing allergy, owing to its bioactivation to cinnamal (3). However, among consecutively tested patients, 5% of those with positive reactions to either cinnamyl alcohol or cinnamal reacted to only one of the compounds (31). An explanation for this could be that cinnamyl alcohol acts as a sensitizer independently of its bioactivation to cinnamal, as the allergenic epoxy cinnamyl alcohol is formed outside the skin by autoxidation. This is in accordance with the results obtained with oxidized limonene and oxidized linalool (3 36), where the autoxidized fragrance compounds were found to be more frequent sensitizers in consecutive dermatitis patients than the pure compounds, and it should be investigated whether autoxidized cinnamyl alcohol clinically detects more cases of contact allergy than the cinnamyl alcohol as presently tested. Alternatively, testing with epoxy cinnamyl alcohol should be performed. Conclusions We have shown that cinnamyl alcohol readily oxidizes when exposed to air, forming a highly sensitizing epoxide and cinnamal, thus acting as a prehapten. We have also shown that there is no difference in the degradation of cinnamyl alcohol and the formation of oxidation products whether the air exposure is performed with or without stirring and a daylight lamp. nce the oxidation has started, these factors are not important. The present study confirms the importance of thorough experimental investigations to understand the phenomena underlying clinical results. To obtain a complete picture, the bioactivation of cinnamyl alcohol and cinnamal should also be thoroughly investigated. Further mechanistic studies are necessary to fully understand the complex prehapten/prohapten cinnamyl alcohol, and such studies are in progress in our group. With regard to contact allergy to cinnamyl alcohol and cinnamal, additional testing with epoxy cinnamyl alcohol should be performed to investigate whether new cases of fragrance allergy can be detected. Acknowledgements Financial support by AFA Insurance and the Swedish Council for Working Life and Social Research (FAS) is acknowledged. The skilful technical assistance of Susanne Exing, Gabriella Wendt and Anders Eliasson with the LLNA is gratefully acknowledged. We also thank Ulrika Nilsson for valuable discussions. The work was performed within the Centre for Skin Research (SkinResQU) at the University of Gothenburg, Sweden. Contact Dermatitis, 68,

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