Chem. Res. Toxicol. 1994, 7, 260-266
260
Contact Allergy to Resin Acid Hydroperoxides. Hapten Binding via Free Radicals and Epoxides Elisabeth Gafvert'st Li Ping ShaofJ Ann-Therese Karlberg,? Ulrika Nilsson,! and J. Lars G. Nilssont Department of Occupational Dermatology, National Institute of Occupational Health, Solna, and Karolinska Hospital, Stockholm, Sweden, Department of Pharmaceutical Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, Uppsala, Sweden, and Department of Analytical Chemistry, National Institute of Occupational Health, Solna, Sweden Received November 4,1993'
For a better understanding of the mechanisms of contact allergic reactions, the patterns of cross-reactivity between different resin acid oxidation products were studied. The 13,14(a)epoxide and the 13,14(P)-epoxide of abietic acid and 15-hydroperoxydehydroabieticacid (15HPDA) were shown in experimental sensitization studies to be contact allergens. Cross-reactivity was observed between the a-and P-epoxides and also between the epoxides and the previously identified rosin allergen 15-hydroperoxyabietic acid (15-HPA). This indicates that 15-HPA may form an epoxide which then reacts with skin protein to generate the complete antigen. 15-HPA and 15-HPDA cross-reacted as well. This can be explained by the formation of similar alkoxy radicals from both hydroperoxides which further react with skin protein. Cross-reactivity patterns of the resin acid oxidation products indicate that 15-HPA may react with skin proteins either as a radical or as an epoxide, thus generating different antigens. The presence in rosin of the epoxides of abietic acid was also studied. The p-epoxide was detected in gum rosin. Moreover, the epoxides elicited reactions in rosin-allergic individuals. Thus, the 13,14(@)epoxide of abietic acid was identified as a new, important rosin allergen.
Introduction
a:
16
Rosin (colophony) is a resin obtained from coniferous trees. Gum rosin, which is obtained from living trees, and tall oil rosin, which is obtained as a byproduct in the pulp industry, are the two dominating rosin types. Rosin is a complex mixture of diterpene resin acids (85-95 % ) and their oxidation products, diterpene alcohols, aldehydes, and hydrocarbons (1). Due to its stickiness, emulsifying, and insulating properties, this resin, in unmodified or modified form, is a common ingredient in many products such as adhesives, printing ink, paper size (used in a process to make paper hydrophobic), paint, varnishes, soldering fluxes, cutting fluids, dental products, and cosmetics (2, 3). The major resin acids are abietic acid and dehydroabietic acid (Figure 1). Rosin is well-known to cause contact allergy (3-6). In previous studies we have isolated and identified oxidation products of abietic acid and dehydroabietic acid as contact allergens (Figure 2a-e) (710). The isolation procedures involve methyl esterification of the carboxylic acid groups, and in the figures the isolated products are depicted as methyl esters. A hydroperoxide of abietic acid, isolated as methyl 15-hydroperoxyabietate (15-HPA;' Figure 2a), was identified as a major allergen in rosin (7).We have also synthesized2the corresponding hydroperoxide of methyl dehydroabietate, methyl 15hydroperoxydehydroabietate(15-HPDA,l Figure 20. Ox-
idation products of resin acids have also been identified as contact allergens by Hausen et al. (11, 12). Abietic acid is easily oxidized by atmospheric oxygen. A probable oxidation product could be an epoxide formed at the double bond in the 13,14-position, which is more susceptible to epoxidation than the double bond in 7,8position (13). Such an epoxide would be an electrophile and therefore would be likely to react with nucleophilic
'Address correspondence to this author a t the Department of Occupational Dermatology, National Institute of Occupational Health, 9-171 84 Solna, Sweden. t Department of Occupational Dermatology, National Institute of Occupational Health, Solna. Uppsala University. 1 Department of Analytical Chemistry, National Institute of Occupational Health, Solna. e Abstract published in Aduance ACS Abstracts, March 1, 1994.
Abbreviations: IbHPA, methyl 15-hydroperoxyabietate; 16-HPDA, methyl 15-hydroperoxydehydroabietate;FCA, Freund's complete adjuvant; EI, electron impact; n-CPBA, m-chloroperbenzoic acid; FCAT, Freund's complete adjuvant teet;CCET, cumulative contact enhancement test; pet., white petrolatum; 16-DAP,bis(methy1dehydroabietate-15-y1) peroxide. 2L.P. Shao, E. Gifvert, A.-T. Karlberg, and J. L. G. Nilsson. 15Hydroperoxydehydroabieticacid-analogue of potent contact allergen in Portuguese colophony. Unpublished results.
0893-228x/94/2707-0260$04.50/0
b:
)"/ COOH Figure 1. Major constituents in rosin. (a) Abietic acid; (b) dehydroabietic acid.
0 1994 American Chemical Society
Chem. Res. Toxicol., Vol. 7,No. 2, 1994 261
Contact Allergy to Resin Acid Hydroperoxides b:
was also studied in subjects with known contact allergy to rosin.
Experimental Procedures
C:
d:
f:
Figure 2. Methylated oxidation products of resin acids which are identified as contact allergens. (a) Methyl 15-hydroperoxyabietatq (b) methyl 7-oxodehydroabietate;(c)methyl 15-hydroxy7-oxodehydroabietate; (d) methyl 15-hydroxydehydroabietate; (e) bis(methy1 dehydroabietate-15.~1) peroxide; (f) methyl 15hydroperoxydehydroabietate;(9)methyl 13,14(a)-epoxyabietate; (h) methyl 13,14(P)-epoxyabietate. groups present in skin proteins and thereby induce contact allergy (14).In contact allergy, i.e., delayed type hypersensitivity, the allergens are low-molecular-weight compounds (haptens) bound covalently to endogenous proteins in the skin (15). The binding to protein is pivotal both at t h e first contact with the allergen (the sensitization phase) as well as at t h e subsequent contacts (the elicitation phase). Cross-reactivity between different haptens can take place when the haptens form complete antigens that have identical or very similar epitopes which make them react with t h e same T-lymphocytes (15). Epoxides of abietic acid are interesting from a mechanistic point of view since it is possible t h a t the hydroperoxide 15-HPA could yield a n epoxide in the 13,14-position before i t reacts to form t h e complete antigen (16-19). T h e aim of this study was t o identify the 13,14-epoxides of abietic acid in rosin (colophony) and to study their sensitizing potential. We wanted to study t h e crossreactivity pattern of t h e epoxides and the 15-hydroperoxides of abietic acid and dehydroabietic acid t o elucidate the mechanism involved when the hydroperoxides of t h e resin acids function as haptens. For reference purposes the 13,14-epoxides of methyl abietate were prepared according t o literature procedures (13). Sensitizing capacity and cross-reactivity were studied in animal experiments. T h e allergenic activity of the oxidation products
Chemicals and Instrumentation. Abieticacid was prepared according to the procedure described in the literature (20). m-Chloroperbenzoic acid was obtained from Janssen Chimica (Beerse, Belgium). 15-HPA was isolated from gum rosin as previously described (7,9). Unmodified gum rosin was a gift from Socer-Comercio et Industria de Resinas, SA (Pombal, Portugal). Tall oil rosin was a gift from Bergvik Kemi (Sandarne, Sweden). White petrolatum (pet.)' was obtained from Witco Corp. (New York). Freund's complete adjuvant (FCA)' was obtained from Difco (Detroit, MI). Standard chemicals used were of analytical or pharmaceutical grade. IRspectra were obtained using a Perkin-Elmer 298 instrument. NMR spectra were performed on a JEOL EX 270 instrument using deuteriochloroform (CDCla) solutions. TMS was used as reference. GC/MS was performed using a Finnigan Incos 50 quadropole instrument equipped with a Varian 3400 gas chromatograph. The GC column used was a DB-5 (30 m, stationary phase 0.1 hm, i.d. 0.25 mm) from J&W Scientific (Folsom, CA) and the temperature programming of the GC was as follows: 50 OC for 0.5 min followed by a temperature increase of 15 OC/min up to 295 OC. The transfer line between the GC and the MS was held at 310 OC. The MS analyses were performed in electron impact (EI)' mode. The scan range was mlz 50-500, scan cycle time 0.6 s. The temperature of the ion source was 150 "C, and the electron energy was set to 70 eV. The samples were dissolved in equal amounts of distilled cyclohexane and acetone prior to the GC/MS analysis. The injections were made using the oncolumn technique. TLC was performed using precoated TLC plates, silica gel 60 F-254,layer thickness 0.2 mm, and aluminum oxide F-254,layer thickness0.25mm, from E. Merck (Darmstadt, Germany). Synthesis of Methyl 13,14(ar)-Epoxyabietate and Methyl 13,14(@)-Epoxyabietate(a-a n d 8-Epoxyabietate). The synthesis was a modification of the procedure described by Valverde etal. (13). Activatedpotassiumfluoride(KF)l(258mg,4.4mmol) and m-chloroperbenzoic acid (m-CPBA)' (348 mg, 2.2 mmol) in 8.4 mL of dry dichloromethane (CHZC12) were stirred under nitrogen at room temperature for 0.5 h. Methyl abietate (540 mg, 1.7 mmol) in 9 mL of dry CHzClz was added. The reaction mixture was stirred under nitrogen at -11 "C for 24 h. The precipitate was filtered off and discarded, the solvent was evaporated, and the crude product was dried in a vacuum. The mixture of products was chromatographed on neutral alumina which was pretreated with water-saturated ethyl acetate and eluted with hexane/ethyl acetate (water-saturated) (982). Fractions with similar polarity, when checked with TLC, were pooled. Three compoundswere isolatedwith the following approximate Rfvalues on TLC (silica gel eluted with hexane/diethyl ether, 91): 0.86 (unreacted methyl abietate), 0.19, and 0.10. The two new products were obtained in yields of 23% and 14%,respectively, and were identified with IR, NMR, and MS as follows: (A)Compound with RfO.19. IR: 2940 cm-l (aliphatic CHI, 1730 cm-l (ester carbonyl). 'H-NMR: 5.82 ppm (8, lH, vinylic at C7), 3.65 ppm (8, 3H at C21), 3.13 ppm (s, 1H at C14), 1.23 ppm (s, 3H at C19), 0.98 and 0.94 ppm (2 doublets, 6H at C16 and C17), 0.80 ppm (s,3H at C20). See Figure 1for numbering of atoms. W-NMR: 178.94 ppm (ClS),132.85 ppm (C8), 129.68 64.26ppm (C13),60.95 ppm (C14). MS: mlz 332 (M+, ppm (C7), 35), mlz 317 (M - CHs, 8), mlz 290 (38), mlz 247 (base peak), mlz 121 (84). This is in accordance with the spectral data of methyl 13,14(@)-epoxyabietatein the literature (Figure 2h) (13). (B)Compound with RfO.10. I R 2940 cm-1 (aliphatic CH), 1730 cm-1 (ester carbonyl) [the spectrum was identical to that of methyl-l3,14(8)-epoxyabietateabove]. 'H-NMR. 5.91 ppm ( 8 , lH, vinylic at C7), 3.64 ppm (8, 3H at C21), 3.12 ppm ( 8 , 1H at C14), 1.24 ppm (s,3H at C19), 0.97 and 0.93 ppm (2 doublets, 6H at C16 and C17), 0.76 ppm (s,3H at C20). laC-NMR 178.94
Gafvert et al.
262 Chem. Res. Toxicol., Vol. 7, No. 2, 1994
ppm (C18), 134.10 ppm $81,127.67 ppm (C7), 66.76 ppm (C13), 62.91 ppm ((214). MS: mlz 332 (M+,37), mlz 317 (M - CHs, lo), mlz 290 (46), mlz 247 (base peak), mlz 121 (98). This is in accordance with the spectral data of methyl 13,14(a)-epoxyabietate in the literature (Figure 2g) (13). Isolation and Identification of a-and 8-Epoxyabietates in Rosin. Gum rosin and tall oil rosin were fractionated with preparative TLC on aluminum oxide plates which were pretreated with water-saturated ethyl acetate. On each plate 50 mg of rosin was applied. Synthetic methyl 13,14(@)-epoxyabietatewas applied as a reference. The plates were developed in hexane/ ethyl acetate (water-saturated) (96:4). An area of the plate, around the same Rf value as the epoxide reference, was scraped off and eluted with ethyl acetate. The components in the extract were separated and analyzed with GC/MS. Sensitization Experiments. Female, outbred DunkinHartley albino guinea pigs weighing 300-350 g, supplied by AB Sahlins FBrsiiksdjursfarm (Malm& Sweden), were used for the animal experiments. The animals were kept on a standard diet obtained from EWOS AB (SGdedje, Sweden). Sensitizing Potential of a-and j3-Epoxyabietate According to Freund's Complete Adjuvant Test (FCAT).' The experiment was performed as described by Klecak but slightly modified according to earlier experience (21, 22). Forty-five animals were divided into three groups of 15 animals: group I was treated with a-epoxyabietate, group I1 was treated with @-epoxyabietate,and the third group was the control group (group 111). (A) Induction. On days 0,6, and 10 the animals were given one intradermal injection (0.1 mL) on the upper back. Group I was injected with 5 % (w/w) a-epoxyabietate in FCA/H20 emulsion (1:l) (corresponding to 0.15 mmol/g), group I1 was injected with 5 % j3-epoxyabietate in FCA/H20 emulsion (l:l), and the control animals were given the FCA/H20 emulsion only. The concentrations of the epoxides were chosen to correspond to the concentration earlier used for induction of sensitivity to 15-HPA (7). (B)Challenge. Closed challenge testing was performed on day 21. The test material was applied on the shaved flanks of theanimalsfor 24husingaluminatestchambers(FinnChambers, i.d. 7 mm). The reactions were assessed at 48 and 72 h after application. The minimum criterion for a positive reaction was a confluent erythema. The animals were challenged with a-epoxyabietate in two concentrations; 5 % and 1% in petrolatum (pet.) (corresponding to 0.15 and 0.03 mmol/g), and with j3-epoxyabietate in three concentrations, 5 % , 1% , and 0.2 % in pet. (corresponding to 0.15, 0.03, and 0.006 mmol/g). The 5 % test concentrations were chosen to correspond to the concentration used for 15-HPA. The animals were further tested with 15-HPA, 5 % in pet. (corresponding to 0.14 mmol/g), and with gum rosin, 10% in pet. The concentrations were chosen according to previous experience (7, 22). The test concentrations of the epoxides were shown in a pretest on FCA-treated animals to be nonirritating. Sensitizing Potential of j3-Epoxyabietate According to t h e Cumulative Contact Enhancement Test (CCET).' The test was performed according to the test procedure described by Tsuchiya et al. (23). However, the controls were sham treated, and the challenge testing was performed according to previous experience (24). A group of 15 animals (group IV)was induced with &epoxyabietate, and another group of 15 animals was the control group (group V). (A) Induction. The exposed group was treated with four closed epidermal applications of j3-epoxyabietate, 5 % in pet., on the upper back (days 0,2,7,9). The applications were removed after 24 h. On day 7 also two injections of 0.1 mL of FCA/H20 emulsion (1:l)were given intradermallyet the epidermalexposure site. The control group was treated in the same way but with neat petrolatum in the epidermal applications. (B)Challenge. Closed challenge testing was performed on day 21 as described above. The test material applied was a-epoxyabietate, 5 % in pet.; &epoxyabietate, 5 % ,1% ,and 0.2 %
in pet.; gum rosin, 10% in pet.; methyl-esterified gum rosin, 10% in pet.; and tall oil rosin, 10% in pet. The challenge testing and assessment of the reactions were performed as described above. Elicitation Reactions to a-a n d j3-Epoxyabietate in Guinea Pigs Sensitiaed with Gum Rosin According to CCET. Fourteen animals (group VI) sensitized to gum rosin, 20% in pet., according to the CCET procedure were rechallenged with a-epoxyabietate, 5 % in pet.; 8-epoxyabietate, 5 % in pet.; and gum rosin, 10% in pet., 6 weeks after the last induction application. The test material at the first challenge included modified rosin components not cross-reacting with gum rosin (25). The challenge testing and assessment of the reactions were performed as described above. Cross-Reactivity Study on 15-HPDA and 15-HPA According to FCAT. The procedure followed the FCAT method as described above. Forty-five animals were divided into three equal groups: group VI1 was induced with 5% 15-HPDA (corresponding to 0.14 mmol/g) in FCA/H20 emulsion, group VI11 with 5 % 15-HPA (corresponding to 0.14 mmol/g) in FCA/ H2O emulsion, and group IX was the control group. The concentration of 15-HPDAwas chosento correspondto the molar concentration of 15-HPA according to previous experience (7). Challenge testing was performed with 5 % and 1% 15-HPDA in pet. (corresponding to 0.14 and 0.03 mmol/g) and 5% 15-HPA in pet. (corresponding to 0.14 mmollg). The test concentrations of 15-HPDA were shown in a pretest on FCA-treated animals to be nonirritating. The animals were rechallenged with gum rosin, lo%,and tall oil rosin, 10% in pet., 5 weeks after the first challenge. A control group of 10 FCA-treated guinea pigs (group X)was challenge tested in the same way as the two exposed groups. Statistical Methods. Exposed animals were compared to control animals using Fisher's exact test. Patch Testing i n Rosin-Allergic Subjects. Patch testing (26)was performed usingFinn Chambers on Scanpor tape. White petrolatum (pet.) was used as the vehicle. Twelve subjects with known contact allergy to gum rosin were tested with a-epoxyabietate, 5 % and 1% ,fi-epoxyabietate, 5 % and 1% , 15-HPA, 55% , and tall oil rosin, 10%. The rosin allergy was confirmed with a patch of gum rosin, 5 % . The material was applied and kept on for 48 h, and the reactions were assessed 72 h after application. The minimum criterion for a positive reaction was erythema with infiltration. Eight control subjects were tested in the same way. Another 7 rosin-allergic subjects were tested with 15-HPDAand 15-HPA in concentrations of 5 % , 2.5%, and 1% in pet. Gum rosin, lo%, was tested for a confirmation of the rosin allergy. The hydroperoxides were also tested in 10 healthy control subjecta. The study was approved by the local ethical committee.
Results Isolation and Identification of a- and 8-Epoxyabietate6 in Rosin. The synthesized reference compounds a-and 8-epoxyabietatewere completely separated using the DB-5GC column. The 8-epoxyabietate eluted before the a-epoxyabietate did. Ingum rosin the fraction analyzed contained 8-epoxyabietate (MW 332)according to GCIMS. The retention and the mass fragmentation were identical to those of the synthetic reference compound. The amount of 8-epoxyabietate in gum rosin was roughly estimated to be 1% . The a-epoxide could not be detected. In tall oil rosin neither the a- nor the 8-epoxyabietate could be identified. However, other compounds of molecular weight 332 were detected. Sensitization Experiments. Results from the animal experiments are presented in Tables 1-5. The readings at 72 h are presented. The readings from 48 h did not differ from those at 72 h and are thus not presented.
Contact Allergy to Resin Acid Hydroperoxides
Chem. Res. Toxicol., Vol. 7,No.2, 1994 263
Table 1. Sensitizing Potential of Methyl 13,14(a)-Epoxyabietateand Methyl 13,14(@)-Epoxyabietate(a-and @-Epoxyabietate) According to Freund’s Complete Adjuvant Test (FCAT) group P (n = 15) group IIb(n = 15) group IIIc(n = 15) challenge material (in pet.)
concn (% )
positive reactions
p(exp/co)
positive reactions
p(exp/co)
positive reactions
a-epoxyabietate a-epoxyabietate 8-epoxyabietate 8-epoxyabietate 8-epoxyabietate 15-HPA gum rosin vehicle control (pet.)
5 (0.15)d l(0.03) 5 (0.15) l(0.03) 0.2 (0.006) 5 (0.14) 10
15 13 11 5 1 10 2 0
co.001
