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Cutaneous Metabolic Activation of Carvoxime, a Self-Activating, Skin-Sensitizing Prohapten Hagen Ott,*,†,‡ Moa Andresen Bergstro¨m,†,§ Ruth Heise,‡ Claudia Skazik,‡ Gabriele Zwadlo-Klarwasser,‡,| Hans F. Merk,‡ Jens M. Baron,‡ and Ann-Therese Karlberg§ Department of Dermatology and Allergology and Interdisciplinary Centre for Clinical Research (IZKF) BIOMAT, RWTH Aachen UniVersity, D-52074 Aachen, Germany, and Dermatochemistry and Skin Allergy, Department of Chemistry, Go¨teborg UniVersity, SE-412 96, Go¨teborg, Sweden ReceiVed October 2, 2008
Bioactivation of low molecular weight compounds in the skin can cause contact sensitization. We have previously shown that the R,β-unsaturated oxime R-carvoxime [1, (R)-2-methyl-5-isopropenylcyclohex-2-enone oxime] is bioactivated to two diastereomeric highly reactive and strongly sensitizing R,β-epoxy oxime metabolites. To investigate if this metabolic activation is catalyzed by the major cytochrome P450 (P450) enzymes found in human skin, incubations of 1 with a skinlike P450 cocktail in the presence of glutathione were carried out. We identified three glutathione conjugates in the incubation mixture arising from two diasteomeric R,β-epoxy oxime metabolites of 1, thus showing that the metabolic activation of 1 is P450-mediated. A P450 identification study using the individual P450 enzymes present in the skinlike P450 cocktail showed the involvement of P450 1A1 and 1B1 and also to some extent 2B6. P450 1B1 metabolism of 1 was found to be stereoselective as glutathione conjugates from only one of the R,β-epoxyoxime metabolites were identified (metabolite 2). Additionally, 1 was found to be an inducer of P450 1B1 (but not 1A1) in human monocyte-derived dendritic cells (moDCs) and to some extent in normal human epidermal keratinocytes. A further transcriptional gene expression change observed in moDCs was a 44-fold upregulation of IL-8, a marker often used for assessment of sensitizing potential of contact allergens. The autoinduction of P450 1B1 by 1 may be a key event in the development of contact allergy to 1 and may also explain why only metabolite 2, and not 3, was found to elicit an allergic response in mice sensitized to 1. Our data show that the R,β-unsaturated oxime 1 is bioactivated by human cutaneous P450, thus forming highly allergenic metabolites, and has the potential to induce its own bioactivation pathway, particularly in antigen-presenting cells. Introduction 1
Allergic contact dermatitis (ACD) is the clinical manifestation of contact allergy, a chronic skin disease caused by skin contact with protein-reactive chemicals in the environment (1, 2). ACD is proposed as the most frequent manifestation of immunotoxicity in humans (3), and it is estimated that 15-20% of the adult European population is sensitized to one or more chemicals (4, 5). To prevent ACD, exposure to the allergenic compound must be avoided (6). Thus, it is important to identify contact allergens and evaluate their sensitizing potency to make proper risk assessments. Currently, much effort is directed toward the development of in vitro assays for the prediction of skin sensitization capacity (7, 8). Key challenges in the development of such assays are, among others, the identification and consequent potency assessment of prohaptens, that is, contact allergens that need prior activation to become skin * To whom correspondence should be addressed. Tel: +49-241-8088330. Fax: +49-241-8082413. E-mail:
[email protected]. † Both authors contributed equally. ‡ Department of Dermatology and Allergology, RWTH Aachen University. § Dermatochemistry and Skin Allergy, Go¨teborg University. | Interdisciplinary Centre for Clinical Research (IZKF) BIOMAT, RWTH Aachen University. 1 Abbreviations: ACD, allergic contact dermatitis; AhR, aryl hydrocarbon receptor; IL, interleukin; LCs, Langerhans cells; moDCs, monocyte-derived dendritic cells; NHEKs, normal human epidermal keratinocytes; PAHs, polycyclic aromatic hydrocarbons; PBMCs, human peripheral blood monocytes; rh, recombinant human; RT-PCR, reverse transcription polymerase chain reaction; TNBS, 2,4,6-trinitrobenzenesulfonic acid.
sensitizing (9). This activation process can occur either before skin contact [e.g., by autoxidation (10-12)] or in the skin [e.g., by cytochrome P450 (P450)-mediated oxidation (13-15)]. P450s are an important xenobiotic-metabolizing enzyme group involved in the oxidative metabolism of numerous drugs, naturally occurring substances, and environmental pollutants (16). They are highly expressed in the liver and gut but can be found in virtually every tissue, including the skin (17). To provide a tool for the study of cutaneous P450-mediated metabolism, we have previously described the development of an enriched skinlike P450 cocktail (14). Even though the purpose of P450-mediated biotransformation is detoxification, the same mechanisms can lead to the conversion of inherently harmless compounds into reactive and toxic species (2, 14-16, 18-20). Furthermore, many P450 enzymes can be induced by chemicals. For example, polycyclic aromatic hydrocarbons (PAHs) are known to induce P450 1A1 by interaction with the aryl hydrocarbon receptor (AhR), thereby inducing their own metabolism, which leads to increased formation of reactive metabolites after repeated exposure to the toxic compound (21). We have previously shown that the R,β-unsaturated oxime R-carvoxime [1, (R)-2-methyl-5-isopropenyl-cyclohex-2-enone oxime (Scheme 1)] is a skin sensitizer of strong potency (22). As the chemical reactivity of 1 toward nucleophiles is too low to explain its high sensitizing potency, 1 was proposed to be a prohapten, bioactivated by skin metabolism (22). Three glutathione (GSH) conjugates formed from two reactive mono-
10.1021/tx8003642 CCC: $40.75 2009 American Chemical Society Published on Web 01/07/2009
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Scheme 1. Proposed Bioactivation Pathways of Carvoxime (1)
Scheme 2. Cytochrome P450-Mediated Epoxidation of 1 and Subsequent Conjugation with GSH
oxygenated metabolites of 1 were identified in reactive metabolitetrapping experiments with GSH and liver microsomes (15). The conjugates formed were derived from two diasteomeric R,βepoxy oxime metabolites (2 and 3), formed by enzymatic epoxidation of the endocyclic carbon-carbon double bond of 1 (Scheme 2). Metabolites 2 and 3 were sensitizers of extreme potency in the murine local lymph node assay (LLNA) and highly chemically reactive toward the nucleophilic model peptide Pro-His-Cys-Lys-Arg-Met. The reason for the high reactivity and sensitizing capacity of metabolites 2 and 3 is probably due to their ability to form nitroso intermediates (Scheme 1) (15, 23). However, despite the fact that epoxy oximes 2 and 3 display the same sensitizing and electrophilic characteristics, only 2 was able to elicit a response in mice sensitized to 1. The reason for this finding may be that 2, but not 3, is formed from 1 by stereoselective cutaneous metabolism. The aim of this study was to further investigate the mechanisms of cutaneous metabolic activation of 1. For this reason, metabolite-trapping experiments with 1, GSH, and the skinlike P450 cocktail were carried out. The metabolic activation of 1 by the individual P450 enzymes present in the cocktail was also studied. To study the ability of 1 to induce P450 and to stimulate an allergenic response in vitro, incubations of 1 with human monocyte-derived dendritic cells (moDCs) and keratinocytes were performed, and the expression of interleukin (IL)-8, P450 1A1, and P450 1B1 was measured using real-time reverse transcriptase polymerase chain reaction (RT-PCR).
Experimental Procedures Caution: Skin contact with 1 must be aVoided. As a skinsensitizing substance, this compound must be handled with care. Chemicals and Biochemicals. Compound 1 was synthesized as previously described (22). Recombinantly expressed P450 bactosomes (coexpressed with P450-reductase in Escherichia coli) were purchased from tebu-bio (Roskilde, Denmark). Instrumental. LC-MS analyses of the GSH conjugates formed in metabolite-trapping experiments were performed using negative
Ott et al. electrospray ionization 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 quadrupole mass spectrometer. The analyses were performed as previously described (15), with the following modification: Negative ionization and SIM detection of the m/z 487 ion were used. This modification was performed to increase the sensitivity of the analysis. Metabolite-Trapping Experiments with 1, GSH, and the Skinlike P450 Cocktail. Each incubation contained substrate (200 µM), the skinlike rh (recombinant human) P450 cocktail (22.2 pmol) (14), GSH (5.0 mM), HEPES buffer (50 mM, pH 7.4), MgCl2 (30 mM), and NADPH (1 mM) in a total volume of 500 µL. The rhP450 cocktail was prepared by premixing recombinantly expressed P450 bactosomes P450 1A1 (3.6 pmol), 1B1 (2.0 pmol), 2B6 (0.035 pmol), 2E1 (11 pmol), and 3A5 (5.6 pmol). The incubations were performed in duplicate and were initialized by addition of NADPH after 3 min of preincubation at 37 °C and terminated after 2 h by addition of acetonitrile (500 µL). The incubation mixtures were, after centrifugation (3000 rpm, 4 °C, 10 min), analyzed using LCMS. Control experiments were run in the absence of NADPH. Metabolite-Trapping Experiments with 1, GSH, and Single Skin P450 Enzymes. The incubations were performed, workedup, and analyzed as described above with the following modification: Individual rhP450 enzymes (20 pmol; P450 1A1, 1B1, 2B6, 2E1, or 3A5) were used instead of the rhP450 cocktail. Control experiments were run in the absence of NADPH. Enzyme-Free Incubations with 1 and GSH. The incubations were performed as described above with the following modifications: No P450 enzymes, MgCl2, or NADPH was added, and HEPES (50 mM, pH 7.4) or potassium phosphate buffer (100 mM, pH 7.4) was used as the reaction medium. The incubation mixtures were analyzed without prior sample workup using LC-MS as previously described (15). Cell Culture and Incubation. P450 1A1 and 1B1 Induction in Keratinocytes. Normal human epidermal keratinocytes (NHEKs) were obtained from foreskin specimens by dispase separation (BD Biosciences, Bedford, MA) of the epidermal sheet from the dermis and subsequent trypsin digestion (Cambrex, Walkersville, MD) of the epidermis (24). Trypsin was neutralized with trypsin neutralization solution (TNS, Cambrex). Cells were fed with keratinocyte growth medium (KGM bullet kit C3111, Cambrex) three times a week. For this study, proliferating keratinocytes in the first and second passage were used. Compound 1 was added to NHEKs in culture at 37 °C and 5% CO2 to a final concentration of 200 µM. RNA isolation (RNA Isolation Kit, Roche, Penzberg, Germany) of the treated cells was performed after 24 h of incubation. The RNA samples were harvested (see below) and stored at -80 °C prior to quantitative RT-PCR analysis. P450 1A1 and 1B1 Induction and IL-8 Upregulation in Dendritic Cells. Human peripheral blood monocytes (PBMCs) from a total of 13 healthy donors were separated from purchased single buffy coats (Institut fu¨r Transfusionsmedizin, Universita¨tsklinikum Aachen, Germany) over a Ficoll-Paque gradient (Amersham Pharmacia Biotech, Uppsala, Sweden), and CD2, CD7, CD19, CD56, CD16, and CD235a positive leukocytes were depleted using a negative monocyte isolation kit (Dynal Monocyte Negative Isolation Kit, Invitrogen, Oslo, Norway). The remaining monocytes were suspended in complete medium consisting of RPMI-1640 (PAA Laboratories, Pasching, Germany) enriched with 1.5% heatinactivated autologous plasma and 1.1 mL of L-glutamine/100 mL. The cell suspension was plated at a density of 3 × 106 cells per reaction well. To induce DC differentiation, the culture medium was supplemented with 800 U/mL GM-CSF and 1000 U/mL IL-4 (R&D-System, Bu¨hlmann, Basel, Switzerland), and the cells were kept at 37 °C and 5% CO2 for 3 days. 1600 U/mL GM-CSF and 1000 U/mL IL-4 were added to the culture medium on day 4. The DC phenotype was determined on day 5 by flow cytometric analysis using saturating concentrations of fluorescein isothiocyanate (FITC)or phycoerythrine (PE)-conjugated monoclonal antibodies (BD Pharmingen, Erembodegem, Belgium). The investigated dendritic cells displayed a phenotype characteristic of immature DCs, that
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Figure 1. LC-MS chromatograms (SIM at m/z 487) of mono-oxygenated GSH conjugates formed from 1 in metabolite-trapping incubations with the skinlike cytochrome P450 cocktail in the presence of NADPH (A) and without NADPH (control experiment, B). The incubations were performed as described in the Experimental Procedures.
is, CD1ahigh, HLA-DRintermediate, CD80intermediate, CD86low, CD83negative, and CD14negative, and were incubated with either 1, DMSO, TNBS (2,4,6-trinitrobenzenesulfonic acid), or SDS. The test compounds were individually prepared in complete medium (200 µM 1, 200 µg/mL TNBS, 5 µg/mL SDS, and 0.1% DMSO), and each test solution was incubated with at least 6 × 106 DCs for 24 or 30 h. RNA isolation (RNA Isolation Kit, Roche) of the treated cells was performed after 24 h (for P450 1A1 and P450 1B1 detection) or 30 h (for measurement of IL-8 mRNA expression) of incubation. The RNA samples were harvested (see below) and stored at -80 °C prior to quantitative RT-PCR analysis. RNA Isolation and RT-PCR. Total RNA from NHEKs or DCs was isolated using the high-pure RNA isolation kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions, including efficient on-column digestion of DNA with RNase-free DNase I. The RNA concentration of each sample was measured using the Nanodrop system (NanoDrop Technologies, Rockland, DE), and equal amounts of RNA were used for quantitative RTPCR. Purified polyA+ RNA was reverse transcribed with the TaqMan Reverse Transcription Reagents kit (Applied Biosystems, Weiterstadt, Germany) with random hexamers as primers. TaqMan experiments were carried out on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) using Assay-on-Demand gene expression products for P450 1A1 (Hs00153120), P450 1B1 (Hs00164383), and IL-8 (Hs00174103) according to the manufacturer’s recommendations. An Assay-on-Demand product for 18S rRNA (Hs99999901) was used as an internal reference to normalize the target transcripts of NHEKs, while cyclophilin A (Hs99999904_m1) and GADPH (Hs99999905_m1) were used as housekeeping genes in DCs experiments. P450, IL-8, 18S, cyclophilin A, and GADPH rRNA sequences were amplified independently in separate reaction wells in triplicate. The RT-PCR efficiencies were determined for each primer/probe set from standard curves generated from serial dilutions of polyA+ RNA (14).
Results and Discussion Metabolite Trapping Experiments. We have previously shown that 1 is metabolically activated to R,β-epoxy oximes 2 and 3 in incubations with human and mouse liver microsomes (15). To investigate if this metabolic activation is catalyzed by the major P450 enzymes found in human skin, incubations of 1 with GSH and the skinlike P450 cocktail were carried out. The three previously (15) identified GSH conjugates formed from 2 (conjugates i and ii) and 3 (conjugate iii) were detected in the incubations (Scheme 2 and Figure 1A). Thus, these results show that the metabolic activation of 1 is P450-mediated and that P450 enzymes expressed in human skin are able to convert 1 into both 2 and 3. Three additional isobaric GSH conjugates (m/z 488) were identified in the incubation mixtures (tR ) 16.3, 17.7, and 18.9 min, Figure 1A). These conjugates were not observed in previous incubations with liver microsomes (15). However, the conjugates at tR ) 16-19 min (and an additional conjugate at tR ) 14.8 min) were also identified in the control experiments containing no NADPH (Figure 1B). Therefore, as P450-mediated oxidation is NADPH-dependent (16), these conjugates did not arise from a metabolite of 1 formed by P450mediated oxidation. When incubating 1 and GSH in the same concentrations as used in the metabolism experiments in HEPES buffer (pH 7.4) or potassium phosphate buffer (used previously in liver microsomal incubations, pH 7.4), the conjugates at tR ) 16-19 min were formed exclusively in the HEPES incubations (data not shown). Consequently, these conjugates are probably nonenzymatically formed by a direct reaction addition of GSH to 1 in HEPES buffer, followed by autoxidation of the sulfide bond. We have previously observed the nonenzymatic
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Figure 2. LC-MS chromatograms (SIM at m/z 487) of mono-oxygenated GSH conjugates formed from 1 in metabolite-trapping incubations with the following cytochrome P450 enzymes: 1A1 (A), 1B1 (B), 2B6 (C), 2E1 (D), and 3A5 (E). The incubations were performed as described in the Experimental Procedures.
formation of mono-oxidized GSH conjugates from similar R,βunsaturated oximes (23). No nonoxidized conjugates originating from a direct reaction between 1 and GSH were identified in any of the incubations. Incubations with the individual P450 enzymes present in the skinlike P450 cocktail were performed to determine which specific enzymes are able to convert 1 into 2 and 3 (Figure 2A-E). The incubations were performed with equal amounts of P450 enzyme (20 pmol/incubation). The incubations of 1 with P450 1A1 contained the highest amounts of GSH conjugates formed from 2 and 3 (i-iii, Figure 2A). P450 1B1 and
2B6 were also able to convert 1 into epoxy oxime metabolites but did so diastereoselectively as only i and ii, the GSH conjugates formed from 2 (Scheme 2), were identified (Figure 2B and C, respectively). Only traces of i-iii were identified in the incubations with P450 2E1 and 3A5 (Figure 2D and E, respectively). P450 2B6 has previously been shown to be highly active in the metabolic activation of a prohaptenic conjugated diene [(5R)-5-isopropenyl-2-methyl-1-methylene-2-cyclohexene] into a strongly sensitizing epoxide (14). However, this enzyme is only found in small amounts in the skin relative to the other skin P450 enzymes (such as P450 1A1 and 1B1) and
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Figure 3. In vitro assessment of the sensitizing capacity of compound 1. TaqMan RT-PCR analysis of IL-8 mRNA expression by DCs stimulated for 30 h with 1 (200 µM), the positive control TNBS (200 µg/mL), the irritant SDS (5 µg/mL), and the vehicle DMSO (0.1%). The relative RNA levels were normalized to cyclophilin and GADPH, and results are displayed as x-fold regulation as compared to mRNA expression of mediumtreated controls (NK). The data shown are mean values ( SD (bars) of 13 single donor experiments.
is therefore present at
