ARTICLE pubs.acs.org/crt
Impact of a Heteroatom in a StructureActivity Relationship Study on Analogues of Phenyl Glycidyl Ether (PGE) from Epoxy Resin Systems Ida B. Niklasson,† Tamara Delaine,† Kristina Luthman,‡ and Ann-Therese Karlberg†,* † ‡
Department of Chemistry, Dermatochemistry and Skin Allergy, University of Gothenburg, SE-412 96 Gothenburg, Sweden Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-412 96 Gothenburg, Sweden
bS Supporting Information ABSTRACT: Epoxy resins are among the most common causes of occupational contact dermatitis. They are normally used in so-called epoxy resin systems (ERS). These commercial products are combinations of epoxy resins, curing agents, modifiers, and reactive diluents. The most frequently used resins are diglycidyl ethers based on bisphenol A (DGEBA) and bisphenol F (DGEBF). In this study, we have investigated the contact allergenic properties of a series of analogues to the reactive diluent phenyl glycidyl ether (PGE), all with similar basic structures but with varying heteroatoms or with no heteroatom present. The chemical reactivity of the compounds in the test series toward the hexapeptide H-Pro-His-Cys-Lys-Arg-MetOH was investigated. All epoxides were shown to bind covalently to both cysteine and proline residues. The percent depletion of nonreacted peptide was also studied resulting in ca. 60% depletion when using either PGE, phenyl 2,3-epoxypropyl sulfide (2), or N-(2,3-epoxypropyl)aniline (3), and only 15% when using 1,2-epoxy-4-phenylbutane (4) at the same time point. The skin sensitization potencies of the epoxides using the murine local lymph node assay (LLNA) were evaluated in relation to the observed physicochemical and reactivity properties. To enable determination of statistical significance between structurally closely related compounds, a nonpooled LLNA was performed. It was found that all investigated compounds containing a heteroatom in the R-position to the epoxide were strong sensitizers, congruent with the reactivity data, indicating that the impact of a heteroatom is crucial for the sensitizing capacity for this type of epoxides.
’ INTRODUCTION Allergic contact dermatitis (ACD) is a common occupational disease caused by chemicals (haptens) that come in close contact with the skin. A correlation between the reactivity of a compound and its ability to sensitize has been shown in both clinical experience and experimental studies. Most contact allergens contain electrophilic functional groups which are able to react with nucleophilic moieties in amino acid side chains in skin proteins to form immunogenic complexes.1 The details of this process are still largely unknown, even though extensive research has been carried out within the area. To prevent ACD, exposure to the allergenic compound must be avoided. Thus, it is important not only to identify new haptens, but also to search for nonallergenic substitutes. Using structureactivity relationship (SAR) data, skin sensitization potency related to physicochemical parameters of a hapten can be understood. Epoxy resins are among the most common causes of occupational contact dermatitis.2 They are normally used in so-called epoxy resin systems (ERS). These commercial products are combinations of epoxy resins, curing agents, modifiers, and reactive diluents used in applications where strong, flexible, and lightweight construction materials are required. Because of r 2011 American Chemical Society
their great technical advantages, ERS will always find new usages. Thus, allergic contact dermatitis caused by ERS in products different from those where this contact is known to occur can take place. Today, many cases of epoxy allergy are caused by ERS present in cement for repair work. The most frequently used resins are diglycidyl ethers based on bisphenol A (DGEBA) and bisphenol F (DGEBF). Alternatives to DGEBA and DGEBF have presently received commercial interest, as epoxy resins with various properties are desired. ERS are often modified by the addition of reactive diluents, which are used mainly to reduce the viscosity and improve polymerization. The most important allergens are the epoxy resin monomers but also ERS components other than the resin monomers have been shown to be potent sensitizers.3 Aromatic, aliphatic, or allylic glycidyl ethers are commonly used as reactive diluents.4,5 Phenyl glycidyl ether (PGE) (Figure 1) has been demonstrated to be a strong sensitizer, and most patients sensitized to reactive diluents reacted to PGE.6,7 We have previously shown that the sensitizing capacity of PGE analogues can be reduced by making small changes in the Received: December 3, 2010 Published: March 03, 2011 542
dx.doi.org/10.1021/tx100417r | Chem. Res. Toxicol. 2011, 24, 542–548
Chemical Research in Toxicology
ARTICLE
and the flow rate was 1.2 mL/min. The temperature program started at 35 °C for 1 min, increased by 10 °C/min, and ended at 250 °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 50 to 1500. LC/MS analyses were performed using electrospray ionization (EIS) on a Hewlett-Packard 1100 HPLC/MS. The system included a vacuum degasser, a binary pump, an autoinjector, a column thermostat, a diode array detector, and a single quadropole mass spectrometer. The HPLC was equipped with a HyPURITY C18 column (150 3 mm i. d., particle size 3 μm, Thermo Hypersil-Keystone, Thermo Electron Corp., Bellafonte, PA). The mobile phase consisted of 0.005% pentafluoropropanoic acid, 0.1% acetic acid, and 5% acetonitrile in water (solvent A), and 0.005% pentafluoropropanoic acid, 0.1% acetic acid, and 50% water in acetonitrile (solvent B). A linear gradient from 0% to 100% B in 30 min, followed by 10 min of isocratic elution was used. The flow rate was 0.40 mL/min and the column temperature was set to 40 °C. The electrospray interface was used with the following spray chamber settings: nebulizer pressure, 40 psgi; capillary voltage, 3500 V; drying gas temperature, 350 °C; and drying gas flow rate, 10 L/min. Fragmentor voltage was set to 120 V to produce molecular fragments. The mass spectrometer was used in scan mode detecting molecular ions with m/z values ranging from 50 to 1600. Column chromatography was performed on Merck silica gel 60 (230400 mesh ASTM); in one purification activated neutral aluminum oxide, Brockmann scale 4, (150 mesh) was used. Short path distillation was performed using a Buchi GKR-50 Kugelrohr distiller. Chemistry. 2,3-Epoxypropyl phenylether was purchased from Aldrich Chemicals (Stockholm, Sweden). H-Pro-His-Cys-Lys-Arg-MetOH (98.5%) was purchased from Bachem (Bubendorf, Switzerland). Acetone was purchased from Merck (Darmstadt, Germany) and olive oil from Apoteket AB (Goteborg, Sweden). Unless otherwise indicated, reagents were obtained from commercial suppliers and used without further purification. TLC was performed using silica gel coated aluminum plates. The purity of both synthesized and purchased test compounds was >98% (GC/MS) before testing of sensitizing capacity. Phenyl 2,3-epoxypropyl Sulfide (2). The synthesis was performed as previously described11 producing 2 in 64% yield. Compound 2 was isolated by distillation at reduced pressure as a colorless liquid. On vacuum distillation at 7 mbar, the product was collected at 7678 °C. 1H NMR δ 2.47 (1H, dd, J = 4.9, 2.2 Hz), 2.702.73 (1H, m), 2.882.94 (1H, m) 3.093.15 (2H, m), 7.167.20 (1H, m), 7.237.28 (2H, m), 7.377.40 (2H, m). 13C NMR (CDCl3): δ 36.7 (C1), 47.5 (C3), 51.1 (C2), 126.8 (C7), 129.2 (C5, C9), 130.3 (C6, C8), 135.5 (C4). EI-MS (70 eV), m/z (%) 166 (72) (Mþ), 123 (56), 110 (100), 91 (10), 77 (18), 65 (21), 51 (14). N-(2,3-Epoxypropyl)aniline (3). The synthesis was performed as previously described12 producing 3 in 20% yield. The product was purified with column chromatography on aluminum oxide, Brockmann scale 4 (3% ethyl acetate in hexanes), affording a mixture of 3 and (2,3epoxipropyl)aniline. The two components were separated with short path distillation. On vacuum distillation at 0.5 mbar, a fraction was collected at 6264 °C, which was proved to be 3. The product was a light lemon-colored liquid. 1H NMR δ 2.632.65 (1H, m), 2.77 (1H, app. t), 3.14 (1H, m), 3.443.48 (1H, m), 3.683.75 (1H, m), 4.14 (1H, s), 6.68 (2H, d), 6.796.83 (1H, m), 7.217.28 (2H, m). 13C NMR δ 44.8 (C9), 50.9 (C8), 45.2 (C7), 112.8 (C4, C5), 117.7 (C1), 129.2 (C2, C3), 147.7 (C6). EI-MS (70 eV), m/z (%) 149 (39) (Mþ), 129 (34), 106 (100), 93 (30), 51 (16). 1,2-Epoxy-4-phenylbutane (4). The synthesis was performed as previously described13 producing 4 in 92% yield. 1H NMR δ 1.811.96 (2H, m), 2.49 (1H, dd, J = 5.0, 2.8 Hz), 2.742.89 (3H, m), 2.942.98 (1H, m), 7.227.35 (5H, m). 13C NMR δ 32.4 (C5), 34.5 (C6), 47.3 (C8), 51.8 (C7), 126.2 (C1), 128.5 (C3, C9), 128.6 (C2, C10), 141.4
Figure 1. Structures of compounds studied.
chemical structure.6 It was demonstrated that the aromatic ring was important for the sensitizing capacity of the glycidyl ethers. Furthermore, investigations of analogues with a core structure identical to that of PGE but with varying lengths of the carbon chains between the ether oxygen and the aromatic system, alternatively an extended carbon chain between the ether oxygen and the epoxide moiety, were performed. In this case, it was observed that an extended distance between the ether oxygen and the epoxide moiety decreased the sensitizing potency more than a similar distance alteration between the ether oxygen and the aromatic system. The chemical reactivity correlated well to the sensitizing capacity in this series of structurally modified PGE analogues.6 In a study of the acute and chronic bacterial toxicity of 34 organic compounds, Blaschke et al. observed an increased toxicity of activated epoxides such as PGE compared to that of other nonaliphatic and aliphatic epoxides.8 As the strong sensitizer, PGE is a commonly used reactive diluent in ERS, and the structure of DGEBF consists of two identical PGE moieties. We found it adequate to use PGE as a starting point also for further SAR studies of ERS, with the ultimate goal to try to reduce the sensitizing effects of ERS used in the future. In order to analyze how the chemical reactivity and sensitizing capacity depend on whether a heteroatom is present in the structure and if so which heteroatom, a series of structural analogues of PGE (24) (Figure 1) was synthesized. Their reactivity was evaluated through experiments with a model peptide, and data from the depletion of nonreacted peptide and the formed reaction products were analyzed. The sensitizing potency of the epoxides within the series was determined using the murine local lymph node assay (LLNA).9 To investigate if there was a statistically significant difference in sensitizing potency between the structurally closely related compounds, a modified LLNA with nonpooled lymph nodes was performed.6,10 To the best of our knowledge, no studies of the allergenic potency of modified epoxy products used in the present study have been published to date. Increased knowledge of their overall properties will provide the basis for future investigations toward the prevention of epoxy resin allergy.
’ EXPERIMENTAL PROCEDURES Caution: This study involves skin sensitizing compounds which must be handled with care. Instrumentation and Mode of Analysis. 1H and 13C NMR spectroscopy was performed on a Jeol Eclipse 400 spectrometer at 400 and 100 MHz, respectively, using CDCl3 solutions (residual CHCl3 δ 7.26 and CDCl3 δ 77.0 as internal standards). Electron-ionization mass spectral analysis (70 eV) was performed on a Hewlett-Packard 5973 mass spectrometer connected to a gas chromatograph (Hewlett-Packard 6890). The GC was equipped with a cool on-column capillary inlet and an HP-5MSi fused silica capillary column (30 m 0.25 mm, 0.25 μm, Agilent Technologies, Palo Alto, CA). Helium was used as carrier gas, 543
dx.doi.org/10.1021/tx100417r |Chem. Res. Toxicol. 2011, 24, 542–548
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(C4). EI-MS (70 eV), m/z (%) 148 (7) (Mþ), 117 (100), 104 (37), 91 (92), 51 (9).
and measurements were performed according to the ordinary LLNA method, but the single cell suspensions from the lymph nodes of each mouse were treated separately. Results are expressed as simulation index (SI), i.e., the ratio of individual test animal/mean of the control group. Statistical analysis. The nonparametric MannWhitney U test was used as a statistical method. To obtain global significance at a level of P < 0.05, the significance levels were corrected for multiple comparisons using the Bonferroni correction. For each comparison, a value of P < 0.017 was considered statistically significant.
Reactions of PGE and Epoxides 24 with the Model Peptide H-Pro-His-Cys-Lys-Arg-Met-OH (PHCKRM). All solvents were degassed with argon prior to use. Solutions of PGE in dimethyl sulfoxide (DMSO) (40 mM, 100 μL) together with potassium phosphate buffer (100 mM, pH 7.4), (200 μL) were added to a vial purged with argon containing PHCKRM in DMSO (40 mM, 100 μL). Accordingly, final concentrations of PGE and the model peptide in the reaction mixture were 10 mM and 1 mM, respectively. The reaction was kept under argon at room temperature and was monitored with HPLC/ ESI-MS for 20 h. Samples were collected from two sets of reactions, to be able to analyze every 20 min. Epoxides 24 were treated identically to PGE's treatment, and their reactions were investigated using the method described above. 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. Sensitizing Capacity of PGE and 24 in Mice. The local lymph node assay (LLNA)9 was used to assess the sensitizing capacity. Mice in six groups of three animals in each were treated by topical application on the dorsum of both ears with the test compound (25 μL) dissolved in acetone/olive oil (AOO) (4:1 v/v) or with the vehicle control. All solutions were freshly prepared for every application. Each compound was tested in five different concentrations. The test concentrations used were as follows: PGE and 24, 0.010, 0.10, 1.0, 10, and 20% (w/v); corresponding molar concentrations for PGE, 0.67 mM, 6.7 mM, 67 mM, 0.67 M, and 1.3 M; for epoxide 2, 0.60 mM, 6.0 mM, 60 mM, 0.60 M, and 1.2 M; for epoxide 3, 0.67 mM, 6.7 mM, 67 mM, 0.67 M, and 1.3 M; and for epoxide 4, 0.68 mM, 6.8 mM, 68 mM, 0.68 M, and 1.3 M. Treatments were performed daily for three consecutive days (day 0, 1, and 2). Sham treated control animals received vehicle alone. On day 5, all mice were injected intravenously via the tail vein with [methyl-3H]thymidine (2.0 Ci/mmol, Amersham Biosciences, UK) (20 μCi) in phosphatebuffered saline (PBS, containing 137 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer, pH 7.4) (250 μL). After 5 h, the mice were subjected to euthanasia, the draining lymph nodes were excised and pooled for each group, and single cell suspensions of lymph-node cells in PBS were prepared using cell strainers (Falcon, BD labware, 70 μm pore size). Cell suspensions were washed twice with PBS, precipitated with TCA (5%), and left in the refrigerator overnight. The samples were then centrifuged, resuspended in TCA (5%) (1 mL), and transferred to the scintillation cocktail (10 mL) (EcoLume, INC Radiochemicals, USA). The [methyl3 H]thymidine incorporation into DNA was measured by β-scintillation counting on Beckman LS 6000TA Instruments. Results are expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI), i.e., test group/control group ratio. Test materials that at one or more concentrations caused an SI greater than 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 sensitizing capacity was classified as the following:
