Metabolism of Capsaicin by Cytochrome P450 Produces Novel

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Metabolism of Capsaicin by Cytochrome P450 Produces Novel Dehydrogenated Metabolites and Decreases Cytotoxicity to Lung and Liver Cells Christopher A. Reilly,† William J. Ehlhardt,‡ David A. Jackson,‡ Palaniappan Kulanthaivel,‡ Abdul E. Mutlib,§ Robert J. Espina,| David E. Moody,† Dennis J. Crouch,† and Garold S. Yost*,† Department of Pharmacology and Toxicology, Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84112, Drug Disposition, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana, 46285, Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, 2800 Plymouth Road, Ann Arbor, Michigan 48105, and Metabolism and Pharmacokinetics, Bristol Myers Squibb, Route 141, Wilmington, Delaware 19880 Received August 15, 2002

Capsaicin is a common dietary constituent and a popular homeopathic treatment for chronic pain. Exposure to capsaicin has been shown to cause various dose-dependent acute physiological responses including the sensation of burning and pain, respiratory depression, and death. In this study, the P450-dependent metabolism of capsaicin by recombinant P450 enzymes and hepatic and lung microsomes from various species, including humans, was determined. A combination of LC/MS, LC/MS/MS, and LC/NMR was used to identify several metabolites of capsaicin that were generated by aromatic (M5 and M7) and alkyl hydroxylation (M2 and M3), O-demethylation (M6), N- (M9) and alkyl dehydrogenation (M1 and M4), and an additional ring oxygenation of M9 (M8). Dehydrogenation of capsaicin was a novel metabolic pathway and produced unique macrocyclic, diene, and imide metabolites. Metabolism of capsaicin by microsomes was inhibited by the nonselective P450 inhibitor 1-aminobenzotriazole (1-ABT). Metabolism was catalyzed by CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Addition of GSH (2 mM) to microsomal incubations stimulated the metabolism of capsaicin and trapped several reactive electrophilic intermediates as their GSH adducts. These results suggested that reactive intermediates, which inactivated certain P450 enzymes, were produced during catalytic turnover. Comparison of the rate and types of metabolites produced from capsaicin and its analogue, nonivamide, demonstrated similar pathways in the P450-dependent metabolism of these two capsaicinoids. However, production of the dehydrogenated (M4), macrocyclic (M1), and ω-1-hydroxylated (M3) metabolites was not observed for nonivamide. These differences may be reflective of the mechanism of formation of these metabolites of capsaicin. The role of metabolism in the cytotoxicity of capsaicin and nonivamide was also assessed in cultured lung and liver cells. Lung cells were markedly more sensitive to cytotoxicity by capsaicin and nonivamide. Cytotoxicity was enhanced 5 and 40% for both compounds by 1-ABT in BEAS-2B and HepG2, respectively. These data suggested that metabolism of capsaicinoids by P450 in cells represented a detoxification mechanism (in contrast to bioactivation).

Introduction Capsaicinoids are the principal pungent substances found in “hot” peppers (Capsicum annum and Capsicum frutescens) (1-5). Six naturally occurring compounds have been characterized as follows: capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, homodihydrocapsaicin, and nonivamide (1-6). Nonivamide, commonly referred to as synthetic capsaicin, has recently been identified as a natural product (5, 6). Capsaicin is the most abundant of the capsaicinoids, constituting approximately 40-60% of the total capsaicinoid content * To whom correspondence should be addressed. Tel: 801-581-7956. Fax: 801-585-3945. E-mail: [email protected]. † University of Utah. ‡ Lilly Corporate Center. § Pfizer Global Research and Development. | Bristol Myers Squibb.

in hot pepper products; dihydrocapsaicin constitutes 2040%, with the remainder being the other capsaicinoids (1-5). The absolute and relative capsaicinoid content of capsaicin in hot peppers can vary, depending on the species and variety of pepper, growing conditions, and time of harvest (1, 4, 5). For centuries, humans have utilized oleoresin capsicum (the oily extract obtained from solvent extraction of dried hot peppers) for the preparation of spicy foods (1) and traditional medications (7). Recently, however, capsaicinoids have been used for a variety of other purposes including neurobiological research (8), weight loss (9, 10), local/topical analgesia (11, 12), antimicrobial defense (13), and the manufacture of self-defense pepper spray products (5, 14, 15). Use of capsaicin in pepper spray products stems from the property of the capsaicinoids to deter attack and incapacitate aggressive individuals through

10.1021/tx025599q CCC: $25.00 © 2003 American Chemical Society Published on Web 02/13/2003

Metabolism and Cytotoxicity of Capsaicin

the production of intense pain, temporary blindness, and uncontrollable coughing. The ability of the capsaicinoids to cause nociception results from their interaction with, and stimulation of, the TRPV1 (VR1, capsaicin receptor) (7, 16, 17).1 TRPV1 has been described as a molecular integrator of noxious chemical and physical stimuli (i.e., capsaicin, acidic pH, and temperature >42 °C), whose activation results in the perception of burning, intense pain, and discomfort (7, 16). The interaction between TRPV1 and capsaicin has been shown to exhibit stringent structural requirements with respect to the aromatic (4-hydroxy-3-methoxybenzylamide) and alkyl portions of the molecule (1822). Capsaicin and nonivamide were the most potent of the structural variants tested, exhibiting nearly identical potency (18-22). In addition to causing severe pain, capsaicin has been shown to elicit a variety of other physiological responses including coughing, respiratory depression, bronchospasm (8, 23), pulmonary edema (24), plasma extravasation (23, 24), altered cellular Ca2+ and K+ homeostasis (8, 25-27), altered cell membrane properties (28), perspiration (29), and neurogenic inflammation (7, 30, 31). Neurogenic inflammation results from the capsaicin-induced release of neuropeptides (i.e., substance P, neurokinin A, and calcitonin gene-related peptide) and subsequent recruitment and stimulation of inflammatory cells (8, 30, 31). Prolonged and/or repeated exposure to capsaicin has also been shown to cause a Ca2+-dependent desensitization in peripheral sensory neurons due, in part, to depletion of substance P from peripheral nerve termini, as well as peripheral neuropathy (8, 16, 23). Desensitization has been used as the basis for the many therapeutic applications of capsaicin, including the treatment of pain (11, 12). Capsaicin has been classified as a potent irritant with moderate toxicity (32). The LD50 for capsaicin in mice has been estimated at 0.56, 1.6, 7.7, 9.0, 190, and >510 mg/ kg for acute iv, intratracheal, ip, sc, po, and topical doses, respectively (32). In all instances, however, the cause of death was attributed to severe respiratory depression and cardiovascular dysfunction (32). The relative safety of oral and topical capsaicin was associated with low systemic bioavailability, presumably due to limited absorption and extensive hepatic metabolism (32-35). There have also been several reports of toxicity to humans after exposure to self-defense pepper sprays (37, 38), oral capsaicin (7), and topical creams containing capsaicin (39). In most instances, the exposed individuals exhibited mild to moderate respiratory distress. However, an infant accidentally exposed to pepper spray required manual ventilation and extensive treatment to overcome the severe respiratory impairment and tissue damage (38). Similarly, an infant treated with oral capsaicin died as a result of severe respiratory and cardiovascular complications (7). Of the 30 adult fatalities reported between August 1990 and December 1993 following pepper spray exposure, 22 were attributed to factors other than capsaicin exposure, including concomitant drug and alcohol use, existing disease (e.g., asthma), and positional asphyxiation (36, 40). 1 Abbreviations: P450, cytochrome P450; 1-ABT, 1-aminobenzotriazole; TRPV1 or VR1, vanilloid receptor type-1; MSD, mass selective detector; BEAS-2B, human bronchiolar epithelial cells; HepG2, human hepatoma cells; LHC, Lechner and LaVeck medium; EMEM, Eagle’s minimum essential medium; FBS, fetal bovine serum.

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The purpose of this study was to investigate the metabolism of capsaicin and nonivamide by recombinant P450 enzymes and liver and lung microsomes in order to thoroughly identify the major human metabolites of capsaicinoids and to determine whether metabolism caused toxicity in respiratory tissues. The formation of potentially deleterious reactive intermediates produced by P450-dependent metabolism of capsaicin as a potential mechanism of toxicity was investigated. In addition, several unique metabolic pathways for the biotransformation, and presumably detoxification, of capsaicin and nonivamide were identified. The association of these data to previous studies on the toxicity of capsaicin, as well as the implications for safety of various pepper-based products, are discussed.

Materials and Methods Caution: Capsaicin, dihydrocapsaicin, and nonivamide are potent dermal, ocular, and respiratory irritants that cause reddening of the skin, the sensation of intense burning and pain, as well as uncontrollable coughing. Use gloves and a fume hood when handling concentrated solutions or the powdered forms of these chemicals. Chemicals. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), nonivamide, dihydrocapsaicin, 4-hydroxy-3-methoxybenzylamine hydrochloride, NADPH, GSH, EDTA, 1-ABT, D2O (99%), CD3CN (99%), and CH3OD (99%) were purchased from Sigma/ Aldrich Chemical Corp. (St. Louis, MO). HPLC grade methanol (MeOH), ethyl acetate, and n-butyl chloride were purchased from Burdick and Jackson (Muskegon, MI). Sodium phosphate monobasic, Tris-HCL, glycerol, sucrose, sodium dithionite, NaCl, and KCl were from Mallinckrodt (Paris, KY). Formic acid (88%) was purchased from J. T. Baker Chemical Corp. (Phillipsburg, NJ). All reagents were prepared in purified water (specific resistance >18.2 mΩ/cm) prepared using a Milli-Q Plus water purification system. The chemical names of the metabolites were obtained with AutoNom software from MDL Information Systems (San Leandro, CA). Microsome Preparation. Microsomal protein was prepared from rat, rabbit, mouse, human, and goat tissue as previously described (41). Briefly, the tissue was thawed, minced with scissors, and homogenized in 3 vol of 50 mM Tris buffer, pH 7.4, containing 1 mM EDTA, 10% (v/v) glycerol, 0.25 M sucrose, and 0.15 M KCl using a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 9000g for 30 min, and the supernatant was decanted through cheesecloth. The supernatant was then centrifuged at 105 000g for 90 min, and the pellets were washed once using the homogenization buffer. Microsomal pellets were reconstituted in 50 mM Tris, pH 7.4, containing 0.1 mM EDTA and 25% (v/v) glycerol and stored in aliquots at -80 °C. All procedures were performed at 4 °C. Protein content was determined using the Bicinchronic Acid Assay (Pierce, Rockford, IL) using bovine serum albumin as a standard. P450 content was calculated using the extinction coefficient 91 000 M-1 cm-1 from the difference spectrum of sodium dithionitereduced and CO-saturated P450 according to the method of Johannesen and DiPiere (42). Tissues were obtained from untreated animals or from organ transplantation donors in cooperation with the Intermountain Organ Recovery System (Salt Lake City, UT) and commercial human tissue sources (TCubed, Edison, NJ). The tissues were cut, frozen in liquid nitrogen, and stored at -80 °C until microsome preparation. Individual recombinant P450 enzymes, “supersomes”, (1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 4F3A) were purchased from Gentest Corporation (Woburn, MA). Recombinant 2F3 and goat 4B2 were expressed in Escherichia coli in our laboratory (43, unpublished studies). Purified rabbit 4B1 was generously provided by Dr. Alan Rettie (University of Washington, Seattle, WA) (44, 45). Metabolism of Capsaicin. Incubations (1 mL) consisted of either 100 µM capsaicin or nonivamide and 100 pmol recombi-

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nant P450 enzyme (250 pmol for 2F3 and goat 4B1), 0.25 mg/ mL liver (∼100 pmol/mL for human liver microsomes) or 1.25 mg/mL lung microsomal protein in PBS (25 mM sodium phosphate, pH 7.2, containing 150 mM NaCl). Incubations containing microsomes were standardized for protein content since spectroscopic determination of P450 content was not feasible for some microsomal samples, due to limited quantities. Stock solutions of capsaicin and nonivamide were prepared in 0.05 N NaOH and were stored at -20 °C, protected from light for up to 2 weeks. Reactions were initiated by the addition of 2 mM NADPH (prepared daily) and incubated at 37 °C in a shaking water bath; control samples did not contain NADPH. At specified time points, the reactions were terminated by addition of 5 mL of n-butyl chloride:ethyl acetate (3:2) and vortexed. Samples were extracted by shaking for 15 min at room temperature and centrifuged at 1000g for 10 min. The upper organic layer was collected and evaporated to dryness under a stream of air using a TurboVap LV evaporator maintained at 40 °C. Samples were reconstituted to a volume of 100 µL using 75% MeOH:25% H2O, transferred to autosampler vials, and analyzed by LC/MS and/or LC/MS/MS. Rates for the metabolism of capsaicin were obtained by quantifying the decrease in capsaicin concentration over time. The concentration of capsaicin was determined using a standard curve (0-200 µM) prepared in buffer using nonivamide as an internal standard. Calibration curves were constructed using the peak area ratio (capsaicin/nonivamide) (y-axis) and the fortified concentration (x-axis), and the data were fit with a weighted quadratic equation (1/Y2). Incubations were performed as described above, terminated at 0, 5, 10, 20, 40, and 60 min by addition of 5 mL of n-butyl chloride:ethyl acetate (3:2), fortified with internal standard (nonivamide; 50 µM), vortexed, extracted, and assayed by LC/MS. LC/MS and LC/MS/MS. LC/MS was performed using a Hewlett-Packard Series 1100 LC-MSD (Agilent Technologies, Palo Alto, CA). Samples (15 µL/injection) were chromatographed over a MetaSil Basic C2-C8 (150 mm × 3.0 mm, 3 µm) reversedphase HPLC column (MetaChem Technologies, Torrance, CA) eluted at a flow rate of 0.25 mL/min with a mobile phase consisting of 60% MeOH and 40% 0.1% (v/v) aqueous formic acid. The column temperature was maintained at 40 °C. The HPLC was interfaced with an HP Series 1100 diode array detector and MSD. The mass spectrometer was equipped with an electrospray ionization source and was set to scan for positive ions in the range of m/z 100-400. The capillary voltage was set at 3500 V, fragmenter at 45 V, drying gas temperature at 350 °C, gas flow (nitrogen) at 10 L/min, and nebulizer pressure at 25 psi. These instrument parameters were determined to be optimum for both capsaicin and nonivamide. Metabolite identity was confirmed by LC/MS/MS using a ThermoQuest/Finnigan TSQ 7000 triple-quadrapole mass spectrometer (ThermoQuest Instruments, San Jose, CA) equipped with Hewlett-Packard Series 1100 solvent delivery system. Product ions m/z 100-350 were scanned following collision-induced dissociation of the precursor ions m/z 292, 294, 304, 306, 320, 322, corresponding to the metabolites identified by LC/MS. These m/z values were 12 amu less for the analysis of nonivamide and its metabolites. The collision gas was argon at a pressure of 3.2 mT, and the collision offset voltage was set at -20 eV. The electron multiplier was set at 2000 V, API gas flow at 50 psi, and auxiliary gas flow at 10 U. LC conditions were as described above, and additional MS/MS parameters were optimized for detection of capsaicin and its fragment ions. For the hydrogen-deuterium exchange MS/MS studies, 60% CH3OD:40% D2O was used as the LC solvent. For detection of GSH adducts of the metabolites, the mass spectrometer was set to scan for ions m/z 250-750 using a linear gradient (20-80%; 40 min) of aqueous formic acid and methanol at 35 °C. MS/MS spectra (m/z 100-700) of the GSH adducts m/z 597 and 627 were also obtained using the conditions described above. Confirmation of Metabolites by LC/NMR. The identity of the unique dehydrogenated metabolites of capsaicin was

Reilly et al. confirmed by LC/NMR, using instrumentation and conditions previously described (46). Briefly, 1H NMR spectra of the metabolite were obtained using a Bruker Advance 500 MHz spectrometer equipped with a 2.5 mm 1H/13C inverse flow-probe and interfaced with a Hewlett-Packard Series 1100 solvent delivery system. Metabolite samples were chromatographed on a Waters Symmetry 150 mm × 3.9 mm C18 reversed-phase HPLC column and eluted at 0.8 mL/min using a linear gradient (20-80% over 20 min) of CD3CN and D2O containing 0.05% trifluoroacetate at room temperature. Suppression of residual water and acetonitrile signals was carried out using the WET solvent suppression method. Additional 1D and 2D 13C and 1H NMR experiments were carried out on the dehydrogenated metabolites (M1 and M4) using a column trapping technique in conjunction with LC/NMR. Samples of the metabolites were first isolated by HPLC using the conditions described for Figure 2. The analyte of interest was then isolated and transferred to a 2 mL HPLC injector loop connected to the LC/NMR system using a flow splitter to dilute the sample with D2O (2 mL/min) and direct the sample to a secondary HPLC column (Keystone Aquasil 30 mm × 3 mm C18). After transfer was complete (as monitored by UV detection at 225 nm) and protonated solvents were removed, the flow was reversed and the analyte was transferred to the LC/NMR flow cell by eluting with 50% CD3CN:D2O at 1 mL/min. 1D and 2D spectra were recorded with standard Varian pulse sequences on a 600 MHz instrument equipped with a Varian triple resonance flow probe containing a 60 µL active volume flow cell. WET solvent suppression was used to reduce the signals from residual water and acetonitrile. Cell Culture and Cytotoxicity Assays. Immortalized human bronchiolar epithelial (BEAS-2B) and human hepatoma (HepG2) cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). BEAS-2B cells were cultured in LHC-9 media (Biofluids, Inc., Rockville, MD) containing retinoic acid (33 nM) and epinephrine (2.75 µM) using plastic cell culture dishes coated with LHC basal media containing BSA (100 µg/mL), collagen (30 µg/mL), and fibronectin (10 µg/mL) (4 h at 37°C). HepG2 cells were cultured in EMEM (Gibco BRL, Grand Island, NY) supplemented with 1 mM sodium pyruvate, 24 mM sodium bicarbonate, and 10% FBS. All cells were maintained in 75 cm2 flasks at 37 °C in an air-ventilated and humidified incubator maintained at 5% CO2. Culture media was renewed every 2-3 days, and cells were subcultured every 5-6 days using 0.25% trypsin. For cytotoxicity assays, cells were subcultured into 96 well cell culture plates at ∼75% confluency (∼2.0-2.5 × 104 cells/well) and permitted to adhere for 12-24 h at 37 °C. Cells were washed 30 min with serum-free cell culture medium, with and without 1-ABT (500 µM) at 37 °C. Cells were then treated with increasing concentrations of capsaicin (0-200 µM) in the presence and absence of 1-ABT (500 µM) in serum-free cell culture medium (minus FBS) for 20 h at 37 °C. Cell viability was assessed using the Dojindo Cell Counting Kit-8 (Dojindo Laboratories, Gaithersburg, MD), according to the supplier recommendations. Cell viability was expressed as percentage of viable cells relative to untreated controls. All experiments were performed in triplicate on three separate occasions.

Results Metabolism of Capsaicin. A proposed scheme for the microsomal metabolism of capsaicin is presented in Figure 1. Incubation of capsaicin with various recombinant P450 enzymes, liver or lung microsomes, and NADPH resulted in the detection of a variety of analytes exhibiting increased polarity, as well as a single metabolite with decreased polarity. A mass chromatogram obtained from the analysis of capsaicin and its metabolites (generated using human liver microsomes) by scanmode LC/MS is shown in Figure 2. Analytes exhibiting a base peak m/z value of 292, 304, 306, 320, and 322 were



Figure 1. Schematic representation for the metabolism of capsaicin by P450. The structure for capsaicin has been numbered according to Figure 6 to aid in the discussion of the results presented throughout the study. Table 1. Rates of Capsaicin Metabolism by Microsomes Isolated from the Lung and Liver of Various Species species/tissue

specific activity (nmol/min × mg)

activity/nmol P450 × min

mouse liver mouse lung

6.0 ( 0.9a 0.23 ( 0.02

7.0 ( 1.0 NDb

human liver human lung

11.0 ( 1.0a 0.6 ( 0.3

11.0 ( 1.0 NDb

rat liver rat lung

4.2 ( 0.2a 0.10 ( 0.02

7.4 ( 0.2 NDb

goat liver goat lung

4.7 ( 0.3a 0.22 ( 0.01

14.5 ( 0.8b 1.35 ( 0.05

3.9 ( 0.4a 0.9 ( 0.2

5.4 ( 0.5b 2.5 ( 0.4

rabbit liver rabbit lung

Figure 2. Representative scan mode LC/MS chromatogram of capsaicin and metabolites produced by human liver microsomes. Samples contained 100 µM capsaicin in PBS buffer, pH 7.2, and 0.25 mg/mL human liver microsomal protein in the presence (s) or absence (- - -) of 2 mM NADPH. Samples were incubated at 37 °C for 60 min and analyzed by LC/MS as described under Materials and Methods.

observed and were consistent with capsaicin (m/z 306), O-demethylation (m/z 292), dehydrogenation (m/z 304), dehydrogenation and hydroxylation (m/z 320), or hydroxylation (m/z 322) of capsaicin. The m/z values for capsaicin and each metabolite obtained by LC/MS were used for additional MS/MS studies designed to identify the sites of biotransformation. No metabolites were observed when human liver cytosol (1 mg/mL) was used in place of the microsomal protein (data not shown). Identical metabolites (i.e., same retention times and m/z values) were observed when human liver microsomes were replaced with liver or lung microsomes from various species, albeit the relative proportions of each metabolite varied (data not shown). The rate for metabolism of capsaicin by lung microsomes was 2-200-fold lower than for liver microsomes and was dependent upon species and tissue source (Table 1).

a Represents significant difference between liver and lung samples from the same species. Statistical significance was determined by analysis of three individual samples consisting of the same microsomal preparations and using the two sample t-test at a 95% confidence level (P < 0.025). b No P450 spectrum was detected in the sample.

Identification of Metabolites by LC/MS, LC/MS/ MS, and LC/NMR. Tandem mass spectrometry of capsaicin (m/z 306) resulted in the formation of four prominent product ions at m/z 182, 170, 153, and 137 (Figure 3). The product ion at m/z 137 was assigned to the vanillyl portion of capsaicin resulting from cleavage of the C7-N8 bond and subsequent rearrangement of the double bonds of the aromatic ring structure (Figure 3). The identity of this product ion was further confirmed by comparison to the MS and MS/MS spectra for authentic 4-hydroxy-3-methoxybenzylamine (data not shown) and to published MS/MS data for structurally related analytes (47). The product ions m/z 182, 170, and 153 were attributed to fragmentation of the alkyl chain at various positions (Figure 3). In some spectra, a fragment ion at m/z 135 was observed and probably formed by the loss of H2O from m/z 153. The proposed fragmentation of capsaicin was verified using deuterium-hydrogen

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Figure 3. Mass chromatogram of the product ions generated from collision-induced dissociation of capsaicin. Data were obtained by infusing capsaicin (10 ng/mL) at 10 µL/min into an HPLC flow consisting of 60% MeOH:40% 0.1% (v/v) formic acid (0.25 mL/min) and introducing the LC flow into the mass spectrometer. Tandem MS was performed as described under Materials and Methods. The inset is a schematic representation of the major product ions produced from collision-induced dissociation of capsaicin.

exchange LC/MS/MS and was consistent with previous MS/MS and MS3 studies of capsaicin (15, 48). Identical fragmentation patterns were observed for nonivamide and dihydrocapsaicin (the saturated analogue of capsaicin); however, the m/z values for the product ions, derived from the alkyl portion of the molecules, were 12 amu less or 2 amu greater than capsaicin, respectively (data not shown). These data further support the proposed fragmentation pathway presented in Figure 3. Changes in the MS/MS spectra of the metabolites were used to determine the portion of capsaicin that was modified by metabolism. Identification of aromatic-hydroxylated metabolites (M5 and M7) was achieved by MS/ MS analysis of precursor ions at m/z 322 and observing a shift in the product ion at m/z 137 to m/z 153, corresponding to the addition of oxygen (16 amu) to the 4-hydroxy-3-methoxybenzylamide ring moiety (Figure 4). The retention times for these metabolites were 12.3 (M5) and 14.3 (M7). Identification of the precise location of the hydroxyl group on the aromatic ring for M5 and M7 was not possible by MS/MS (Figure 4). An additional unique ring oxygenated metabolite was also observed with a m/z value of 320 (M8). This metabolite has been tentatively identified as a ring oxygenated metabolite (8-methyl-non-6-enoic acid 4-hydroxy-3-methoxy-5-oxo-cyclohexa-1,3-dienylmethyleneamide) of the N-dehydrogenated product (M9, 8-methylnon-6-enoic acid 4-hydroxy-3-methoxy-benzylideneamide), formed after tautomerization of the ketone arising from an epoxide intermediate (Figures 5 and 9). The retention time for this analyte was 15.5 min. The UV/vis absorbance spectrum for M8 was red-shifted relative to capsaicin with maxima at 260 and 370 nm (Figure 5), consistent with a quinoid structure with extended conjugation. Additional evidence for the structure of this metabolite was achieved by MS/MS. The MS/MS spec-

trum of this metabolite showed product ions at m/z 168, 153, and 135. These product ions were consistent with fragmentation at the N8-C9 bond to yield m/z 153 (an alkyl fragment) and 168 (an oxygenated ring structure with the nitrogen retained). A requirement for this fragmentation pathway was the presence of the double bond between C7-N8 prior to collision-induced dissociation, as well as the additional oxygen on the ring structure. The existence of a double bond between C7 and N8 was supported by MS analysis in D2O:CH3OD, where an M + D ion at 322 was observed. Observation of the ion at m/z 322 showed that there was one exchangeable proton, rather than a predicted m/z of 323 if there would have been two exchangeable protons. The location of the oxygen at the C5 position of the ring was essential to produce the extended conjugation shown in Figures 5 and 9. Addition of oxygen at other positions would not be favorable since the resulting metabolite would not be stabilized by extended conjugation into the imide oxygen, as indicated for the proposed structure of M8 (Figure 9). Identification of O-demethylated capsaicin (M6) (m/z 292) by MS/MS was straightforward. O-demethylated capsaicin exhibited a retention time of 13.2 min and an MS/MS spectrum that demonstrated a loss in the product ion m/z 137 and the appearance of m/z 123, indicative of a net loss of 14 amu from the ring moiety. The characteristic alkyl fragments were unchanged. These data are also presented in Figure 4. Two alkyl hydroxylated metabolites were differentiated from aromatic hydroxylated metabolites by detecting a shift (16 amu) in the product ions that corresponded to the characteristic alkyl fragment ions of capsaicin (i.e., 153 f 169, 170 f 186, and 182 f 198) and the presence of m/z 137 corresponding to an intact and unmodified aromatic ring. The retention times for the alkyl hydroxylated metabolites were 5.4 (M2) and 6.3 (M3) min. The



Figure 4. Representative mass spectra of various metabolites of capsaicin produced from incubation with human liver microsomes. Incubation mixtures contained 0.25 mg/mL human liver microsomal protein and 100 µM capsaicin in PBS buffer, pH 7.2. Reactions were initiated by addition of 2 mM NADPH and conducted for 60 min at 37 °C. The identities of the metabolites from upper left to lower left are O-demethylated (M6), macrocyclic and alkyl dehydrogenated (M1 and M4), and N-dehydrogenated (M9) capsaicin. The identities of the metabolites from upper right to lower right are ω-hydroxylated (M2), ω-1-hydroxylated (M3), and aromatic hydroxylated (M5 and M7) metabolites of capsaicin. The structures and fragmentation patterns of key diagnostic ions are presented within the figure. Samples were analyzed by LC/MS/MS as described under Materials and Methods.

location of the hydroxyl group on the alkyl chain was also determined by MS/MS studies. ω-Hydroxylation (M2) (m/z 322) (49) was identified by observing a characteristic product ion [M - CH2O]+ at m/z 292, corresponding to the loss of CH2O (-30 amu) from m/z 322. In addition, a series of hydroxylated alkyl fragment ions, with and without the CH2O portion (i.e., m/z 198 and 168, 186 and 156, and 169 and 139) were observed and provided additional support for the proposed metabolite structure (Figure 4). Hydroxylation of the alkyl chain at the ω-1 position (M3) was a unique metabolite for capsaicin and was also confirmed by MS/MS. This metabolite was identified by the appearance of product ions corresponding to the net neutral loss of C3H4O (-56 amu), presumably arising via fragmentation of the alkyl chain between C15 and C16 and concomitant saturation of the alkyl chain to produce an ion (m/z 266) that was similar in structure to a fragment produced from nonivamide (see Figure 10 inset), except that the alkyl chain was seven carbon atoms in length. The proposed fragmentation pathway to generate m/z 266 was supported by the presence of the corresponding alkyl fragment ion at m/z 142. These data are also presented in Figure 4. An additional unique metabolite was identified that appeared to be formed by N-dehydrogenation (M9) (18.6

min; m/z 304). LC/MS analysis of this metabolite using CH3OD:D2O as the mobile phase produced an ion at m/z 306, indicating the presence of only a single exchangeable proton, vs two exchangeable protons for capsaicin (m/z 309) (data not shown). The presence of an intense product ion in the MS/MS spectrum of M9 at m/z 152 (using CH3OH:H2O as the mobile phase) corresponded to a fragment with the nitrogen retained on the aromatic ring. This ion was likely produced from an alternate fragmentation pathway than that which was observed for capsaicin (Figure 4). In this spectrum, the characteristic aromatic ring fragment ion of capsaicin (m/z 137) corresponded to the loss of CH3 (15 amu) from the product ion m/z 152 or from loss of H2O from m/z 153 (if formed). The preexisting double bond between the C7 and the N8 of this metabolite (M9) resulted in predominant fragmentation at the amide bond between C9 and N8, rather than between C7 and N8. This fragmentation pathway was also observed for M8. Because the basic nitrogen was primarily retained on the ring structure of M9, the alkyl fragment ions were a very minor component of the MS/MS spectrum. The alkyl product ions that were present, however, were also shifted to lower m/z values by 2 amu (m/z 180 and 168 vs 182 and 170) providing additional support for the presence of a double bond between C7 and N8. These

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Figure 5. LC/MS/MS (upper panel) and UV/vis absorbance (lower panel) spectra for the N-dehydrogenated and ring oxygenated metabolite (M8) produced by human liver microsomes. Data were collected using an in-line diode array detector during LC/MS analysis. The UV/vis absorbance spectra for M8 is represented by the solid line, and capsaicin is represented by the dashed line. LC/MS/MS was performed as described under Materials and Methods.

fragment ions originated from the minor fragmentation pathway involving cleavage between C7 and N8 (as observed for capsaicin). The mass spectrum of this metabolite is presented in Figure 4. Unique alkyl dehydrogenated metabolites of capsaicin (m/z 304) eluting at 5.4 (M1) and 11.6 min (M4) were also identified by LC/MS/MS. The MS/MS spectra of the two dehydrogenated metabolites were characterized by a 2 amu shift in the fragment ions that corresponded to the alkyl portion of capsaicin (i.e., 182 f 180, 170 f 168, and 153 f 151) and the presence of the product ion m/z 137. Although these metabolites were not identical, their MS/MS spectra were essentially identical. The fragmentation of M1 to produce ions at m/z 168 and 151 is illustrated in Figure 4. This mechanism is proposed to involve loss of the benzylic portion of this metabolite to form the ion at m/z 168 and loss of water from the macrocycle to form the ion at m/z 151. The dehydrogenation of capsaicin by certain P450 enzymes was a novel metabolic pathway. Because of the unique nature of this metabolic pathway, we performed extensive additional spectroscopic analyses to fully identify these products. LC/NMR was used to characterize the structure of the dehydrogenated metabolites (M1, 1-(4-hydroxy-3-methoxy-benzyl)-9,9-dimethyl-1,3,4,5,6,9-hexahydro-azonin-2one, and M4, 8-methyl-nona-6,8-dienoic acid 4-hydroxy3-methoxy-benzylamide) since preparative HPLC and standard 1H NMR were not successful (i.e., much of the material was lost during sample preparation due to the apparent high volatility of M1 and M4). The 1H NMR (LC/NMR) spectrum for capsaicin (50) and the dehydrogenated metabolites eluting at 5.4 (M1) and 11.6 min (M4) are presented in Figure 6. The spectrum for M1

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exhibited no change in the three aromatic protons (designated as numbers 2, 5, and 6), a loss in the coupling of the terminal isopropyl methyl groups (number 17), a downfield shift in the six methyl protons to 1.2 ppm, a loss of coupling for the olefinic protons (numbers 14 and 15) with a concomitant shift of these signals to ∼5.5 ppm. These data were consistent with a structure of a macrocyclic ring that was formed between the tertiary carbon and the amide nitrogen (M1), rather than the simple introduction of a double bond into the alkyl chain, as observed for M4. The 1H NMR spectrum for M4 exhibited characteristic aromatic resonances at 6.7-6.9 ppm, new olefinic resonances at 6.15, 5.7, and 4.7 (numbers 15, 14, and 18), a loss of coupling for the methyl groups (number 17), and a shift in the remaining methyl proton resonances from 0.8 to 1.8 ppm (number 17) (Figure 6). These data for M4 were consistent with the formation of a diene, with the second double bond located between the tertiary carbon and the terminal methyl group. Predicted 1H NMR spectra for both of these metabolites closely resembled the experimental spectra (data not shown). Additional confirmation of the cyclic structure of M1 was achieved using deuterium-hydrogen exchange and LC/MS/MS. Scan-mode LC/MS analysis of capsaicin demonstrated the presence of two exchangeable hydrogens, attributable to the hydrogens on the amide nitrogen and phenol group. Deuterium exchange at these positions resulted in the detection of an M + D ion at m/z 309 (Figure 7) and an [M + Na]+ adduct of deuteriocapsaicin at m/z 330 (data not shown). Analysis of the macrocyclic metabolite (M1) demonstrated the presence of a single exchangeable proton (M + D ) 306), while the other dehydrogenated metabolite (M4) (eluting at 11.6 min) exhibited two exchangeable protons (M + D ) 307). Deuterium-hydrogen exchange MS/MS analysis of these metabolites confirmed that the single exchangeable proton for the macrocyclic metabolite was attributable to the phenol group, based on the presence of a characteristic product ion at m/z 138 and unique cyclic alkyl fragments m/z 180, 170, and 151 (Figure 7). In contrast, deuterium-hydrogen exchange MS/MS analysis of M4 (M + D ) 307) was consistent with a linear structure with dehydrogenation of the alkyl chain at the terminal methyl position, as demonstrated using 1H NMR (Figure 6). The presence of an intact phenolic group on both of the dehydrogenated metabolites was also confirmed by UV/vis diode array spectroscopy under neutral and basic conditions (spectra were collected with and without the post-HPLC column addition of 10 N NaOH at 2.5 µL/min (final [NaOH] ) 0.1 N) (data not shown). These studies confirmed that the phenolic chromophore was retained in these metabolites by demonstrating identical bathochromic shifts in the absorbance maxima for the metabolites and capsaicin when the pH was changed from ∼7 to ∼10. These data confirmed that the macrocycle was not formed through an ether bond with the phenol group; rather, it was formed through the amide nitrogen via carbon-nitrogen bond formation. Final elucidation of the precise structure of M1 was achieved using 2D LC/NMR and heteronuclear multiple bond correlation (HMBC) techniques (Figure 8 and Table 2). The chemical shifts for the carbon atoms and protons in Table 2 were achieved using DQCOSY and TOCSY, while the carbon shifts were obtained by HSQC and HMBC data. In the HMBC spectrum (Figure 8), the six



Figure 6. 1H NMR (LC/NMR) spectra for capsaicin (upper panel), the macrocyclic metabolite (M1) (middle panel), and the terminal diene (M4). Proton assignments for the spectra are represented by number and correspond to the chemical structures within each panel. Residual CH3CN and H2O resonances and resonances due to contaminants are noted with an “X”. Data were collected as described under Materials and Methods. Table 2. 1H and

13C

NMR Chemical Shift Assignmentsa of Capsaicin and M1

capsaicin

M1

CD3CN position 1 2 3 4 5 6 7 8 (NH) 9 10 11 12 13 14 15 16 17, 18 3-OMe 4-OH

1H

δ, m (J Hz)

6.84 d (2) 6.73 d (9) 6.69 dd (9,2) 4.21 d (7) 6.06 br 2.12 obsc 1.55 m 1.32 m 1.95 m 5.33 dt (18, 7) 5.38 dd (18, 6) 2.21 m 0.93 d (8) 3.80 s 6.42 s

CD3CN:D2O, 1:1 13C

δ

132.6 112.3 148.3 146.2 115.7 121.2 43.5 173.7 37.0 26.3 30.2 33.2 128.1 139.0 32.0 23.2 56.8

1H

δ, m (J Hz)

6.84 d (2.4) 6.76 d (8.4) 6.70 dd (8.4, 2.4) 4.18 s 2.16 m 1.50 m 1.26 m obsc 5.48 overlapped 5.48 overlapped 1.16 s 3.74 s

CD3OD 13C

1H

δ

131.2 112.1 147.9 145.5 115.8 120.7 42.9 176.5 36.1 25.4 28.5 NO 127.5 137.8 71.2 28.9 56.2

δ

6.92 6.83 6.78 4.27 2.25 1.60 1.35 obsc 5.56 overlapped 5.56 overlapped 1.25 3.83

a Proton chemical shift assignments were made by the interpretation of DQCOSY and TOCSY data. Proton-bearing and quaternary carbon chemical shift assignments were made by the interpretation of HSQC and HMBC data, respectively. S, singlet; br, broad; d, doublet; dd, double doublet; dt, double triplet; m, multiplet; obsc, obscured by solvent resonance; NO, not observed.

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Reilly et al. Table 3. Enzyme Selective Pathways for the Metabolism of Capsaicin

metabolite/reaction N-macrocycle (M1) ω-hydroxylation (M2) ω-1-hydroxylation (M3) alkyl dehydrogenation (M4) ring hydroxylation (M5) O-demethylation (M6) ring hydroxylation (M7) N-dehydrogenation and ring oxygenation (M8) N-dehydrogenation (M9)

mass and retention time (m/z and min) 304, 5.4 322, 5.5 322, 6.3 304, 11.6 322, 12.5 292, 13.4 322, 14.3 320, 15.5 304, 18.6

P450 enzymea 2C9, 2C19, 2E1 (2C8) 2E1, 2C9 3A4, 2C8 2C9, 2C19, 2E1 (2C8) 1A2, 2C19 1A2, 2C19 2B6, 2C8, 2E1 3A4, 1A1, 2E1, 2C8, 2D6, and 2B6 3A4 (1A1 and 2B6)

a

Metabolism of capsaicin was considered positive when the approximate amount of a metabolite was equal to or greater than the amount of metabolite produced by human liver microsomes. Enzymes in parentheses represent metabolite production at rates just below that observed for human liver microsomes. Table 4. Effects of GSH (2 mM) on the P450-Dependent Metabolism of Capsaicin by Human Liver Microsomesa metabolite/reaction

Figure 7. Representative deuterium-hydrogen exchange LC/ MS/MS spectra for capsaicin (upper panel), the macrocyclic metabolite (M1) (middle panel), and alkyl dehydrogenated metabolite (M4) (bottom panel). LC/MS/MS was performed as described under Materials and Methods using 60% MeOD:40% D2O as the mobile phase. Data were collected as described under Materials and Methods.

methyl protons on C17 exhibited coupling to itself (gemdimethyl; 28.9 ppm), the quaternary carbon at C16 (71.2 ppm), as well as coupling to C15 (137.8 ppm). Similarly, the olefinic protons (14 and 15) exhibited correlations with C16 and C17, while C14 (127.5 ppm) was correlated to the number 12 protons. These data, along with the 1H NMR and LC/MS/MS data presented in Figures 6 and 7, respectively, confirmed the structure of M1. During the manipulations of M1, we observed that this metabolite decomposed slowly to form M4 in the presence of trifluoracetic acid, which was added to acidify the HPLC solvents. A proposed mechanism for the formation of the dehydrogenated metabolites is presented in Figure 9. Enzyme Selective Metabolism of Capsaicin. The metabolism of capsaicin by recombinant P450 enzymes was mediated by 1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 but not by 2A6, 2C18, 4F3A, 2F3, 4B2, or 4B1. The metabolites produced by the P450 enzymes are presented in Table 3. It is noteworthy to point out that ω-hydroxylation was selectively catalyzed by 2E1 and 2C9, N-dehydrogenation by 3A4 (and to a lesser extent 1A1 and 2B6), and alkyl dehydrogenation by 2C9, 2C19, and 2E1 (and to a lesser extent 2C8). Interestingly, 4F3A, 2F3, 4B2, and 4B1 were not capable of metabolizing capsaicin, even though these enzymes have been shown to catalyze the oxygenation and/or dehydrogenation of certain highly lipophilic compounds (43-45, 51). Modulation of Capsaicin Metabolism. Metabolism of capsaicin by microsomes was inhibited ∼80% relative to controls when the nonselective P450 inhibitor 1-ABT (1 mM) was included in the incubation mixtures (52).

N-macrocycle (M1) ω-hydroxylation (M2) ω-1-hydroxylation (M3) alkyl dehydrogenation (M4) ring hydroxylated (M5) O-demethylation (M6) ring hydroxylation (M7) N-dehydrogenation and ring oxygenation (M8) N-dehydrogenation (M9)

relative abundance (% control)

GSH adduct (m/z)b

172 ( 12 71 ( 8 139 ( 9 104 ( 9 73 ( 3 2(1 22 ( 6 2(1

ND ND ND ND ND 597 627 627

103 ( 5

ND

a

Incubations were performed with 2 mM GSH in the reaction mixture using human liver microsomes and analyzed by LC/MS and LC/MS/MS, as described under the Materials and Methods section. b ND, none detected.

Addition of GSH (2 mM) to incubations stimulated the overall metabolism of capsaicin by ∼2.5-fold with marked increases in the production of the macrocyclic and ω-1hydroxylated metabolites. These data suggested that metabolism of capsaicin by selective P450 enzymes produced reactive intermediates that were capable of inhibiting P450 turnover. Preliminary studies in our laboratory have shown that CYP2E1 is inactivated by capsaicin in a pseudo mechanism-based manner (unpublished data). Because the inactivation was ameliorated by inclusion of GSH, it is possible that inactivation was caused by release of electrophilic metabolites (intermediates) such as M8 or M9. GSH also prevented the formation/detection of the O-demethylated (M6), N-dehydrogenated and ring oxygenated (M8), and the aromatic hydroxylated (M5) metabolites, as well as decreased the production/detection of the aromatic hydroxylated products (M5 and M7) and the ω-hydroxylated metabolite (M3). Data showing the effects of GSH on the metabolism of capsaicin are presented in Table 4. LC/MS and MS/MS analysis of incubations containing GSH demonstrated the formation of several GSH adducts (dependent upon both NADPH and GSH) consistent with addition of GSH to M6 (m/z 597), M7 (m/z 627), and M8 (m/z 627) (Table 4). The conclusion that the adducts observed at m/z 627 were the result of trapping M7 and M8 was based on the GSH inhibition data presented in Table 4. A proposed structure for the GSH adduct of M8 is shown in Figure 9.



Figure 8. HMBC NMR spectrum for M1. Key diagnostic elements in the spectrum, as discussed in the Results section, are represented in bold text within the figure. Data were collected as described under Materials and Methods.

Metabolism of Nonivamide. To our knowledge, the metabolism of nonivamide by P450 has not been described in previous research. Representative LC/MS profiles for metabolites produced from capsaicin and nonivamide are shown in Figure 10. The primary differences in the metabolism of nonivamide by P450 were an absence in the formation of the corresponding macrocyclic (M1*), alkyl dehydrogenated (M4*), and ω-1-hydroxylated (M3*) metabolites. The inability of P450 enzymes to produce these metabolites from nonivamide provides strong evidence that the macrocycle (M1) from capsaicin is formed through oxidation of the tertiary, allylic carbon to its stabilized free radical, which is then intercepted by the amide nitrogen to form the macrocycle (Figure 9). The absence of a tertiary, allylic carbon at the alkyl terminus of nonivamide probably prevented the formation of the corresponding macrocyclic, dehydrogenated, or ω-1-hydroxylated metabolites from this capsaicinoid. A proposed mechanism for the formation of these metabolites from capsaicin is presented in Figure 9 and was based, in part, on the differences in metabolite formation between these structural analogues. Despite these differences, however, the relative rates of substrate disappearance for nonivamide and capsaicin were nearly identical (data not shown). Cell Culture and Cytotoxicity Assays. Capsaicin and nonivamide were cytotoxic to both BEAS-2B (human bronchiolar epithelial) and HepG2 (hepatoma) cells. The LC50 values for capsaicin and nonivamide are presented

in Table 5. Briefly, capsaicin was slightly more cytotoxic than nonivamide to both BEAS-2B and HepG2 cells. Addition of 1-ABT (500 µM) to the incubations caused a slight (5-10%) increase in cell killing in BEAS-2B cells for both nonivamide and capsaicin, while an approximate 30-40% increase in cell death was observed for HepG2 cells. Although statistical significance was not observed in these studies, due in part to limits on the solubility of capsaicin in culture media for HepG2 cells without 1-ABT, the overall trends provided valuable insight into the role of metabolism in the cytotoxicity of capsaicin. These data implied that lung cells probably possessed less metabolic capacity to detoxify capsaicin than HepG2 cells (∼5-10% vs 30-40% differences between samples with and without 1-ABT) and that the metabolism of capsaicin and nonivamide by P450 enzymes in these cells protected the cells from the cytotoxic effects of these compounds (1-ABT increased cytotoxicity).

Discussion In this research, we characterized the P450-dependent metabolism of capsaicin and its analogue, nonivamide, and assessed the importance of metabolism in the cytotoxicity of these compounds in vitro. Using a combination of analytical techniques, we have confirmed the formation of O-demethylated (M6), aromatic (M5 and M7) (53-55) and ω-hydroxylated (M2) (49) metabolites of capsaicin (proposed and/or identified in previous studies), as well

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Figure 9. Hypothetical mechanisms for the formation of M1, M2, M3, M4, M8 (and its GSH adduct), and M9 by P450 enzymes. Hypothetical intermediates for several of the metabolic steps are shown in brackets. Table 5. Cytotoxicity of Capsaicin and Nonivamide in BEAS-2B (Bronchiolar Epithelial) and HepG2 (Hepatoma) Cells in the Presence and Absence of the P450 Inhibitor 1-ABT approximate LC50 value (µM)a cell type and treatment

no additions

+1-ABT (500 µM)

BEAS-2B, capsaicin BEAS-2B, nonivamide HepG2, capsaicin HepG2, nonivamide

100 ( 8 115 ( 12 >200b ∼200b

90 ( 6 105 ( 10 180 ( 8 130 ( 12

a Approximate LC 50 values were obtained from dose-response curves generated from three separate experiments. Data were corrected for toxicity observed with 1-ABT (500 µM) only. b The 200 µM amount was the maximum concentration of capsaicin used in the cytotoxicity assays. Capsaicin was not soluble in cell culture media at concentrations >200 µM.

Figure 10. Representative scan mode LC/MS chromatogram of capsaicin (s) and nonivamide (- - -) and their metabolites, as produced by human liver microsomes. The metabolites named in the figure are those of capsaicin that have been characterized in this study, as well as the ω-hydroxylated metabolite of nonivamide (M2*). Samples contained 100 µM capsaicin or nonivamide in PBS buffer, pH 7.2, 0.25 mg/mL human liver microsomal protein, and 2 mM NADPH. Samples were incubated at 37 °C for 60 min and analyzed by LC/MS as described under Materials and Methods.

as identified several new metabolites involving ω-1hydroxylation (M3) (CYP3A4 and 2C8), N-dehydrogenation (M9) (CYP3A4 and to a lesser extent 1A1 and 2B6), N-dehydrogenation and ring oxygenation (M8) (CYP3A4, 1A1, 2E1, 2C8, and 2D6), and alkyl dehydrogenation (M1 and M4) (CYP2C9, 2C19, 2E1 and to a lesser extent 2C8), including formation of an interesting and unique macrocyclic product (M1) (Figure 1). The metabolism of nonivamide by P450 has, to our knowledge, not been

previously investigated. We have also obtained evidence for the production of reactive metabolites capable of inhibiting P450 turnover (Table 4). Although significant research on the metabolism of capsaicin by P450 enzymes has been performed, limitations in sensitivity and selectivity of the analytical techniques used to assess metabolism (i.e., HPLC/UV, TLC, and GC/MS) prevented the identification of many of the metabolites characterized in this study (49, 53). Similarly, comprehensive studies designed to assess the importance of metabolism in the cytotoxicity of capsaicinoids have not been previously performed. The data presented in this study focus on these objectives and resolve several important issues surrounding the role of metabolism in the toxicity of capsaicinoids. Previous studies on the structure-activity relationships among the capsaicinoids and synthetic variants


have demonstrated the necessity of a 4-methoxy-3hydroxy ring structure for pharmacological activity at TRPV1 (18, 49). In addition, the presence of hydrogen atoms at positions 2, 5, and 6 of the ring moiety and an unmodified, hydrophobic alkyl chain between 8 and 12 carbon atoms in length were determined to be essential for maximum potency (18, 49). It has also been previously demonstrated that concomitant exposure to phenobarbital and capsaicin increased the duration of phenobarbital-induced anesthesia, presumably due to a decreased rate of metabolism of phenobarbital caused by capsaicin (49). Coadministration of either the ring or the alkyl hydroxylated metabolites (presumably mixtures of M2, M5, and M7) of capsaicin, however, had no effect on the duration of anesthesia (49). We have characterized several metabolites of capsaicin that would be predicted by structure-activity relationship studies to ameliorate the pharmacological (TRPV1 binding), and possibly toxicological, properties of capsaicin. Using in vitro cytotoxicity assays, we demonstrated a protective effect for P450dependent metabolism of capsaicin (Table 5). Collectively, these data imply that the metabolism of capsaicin by P450 represents a mechanism for inactivation of the pharmacological and toxicological properties of capsaicin. Metabolism can frequently increase toxicity through the activation of xenobiotics to reactive, cytotoxic intermediates. Previous research on the metabolism of capsaicin has proposed the formation of potentially deleterious reactive intermediates including electrophilic quinones, a quinone methide, a ring epoxide, and a phenoxyl radical produced by CYP2E1-mediated one electron oxidation of the phenol group (2, 54, 55). One could also propose the P450-, peroxidase-, or transition metal-mediated oxidation of O-demethylated (M6) or catechol metabolites of capsaicin to produce electrophilic quinones or semiquinone radicals capable of redox cycling and the production of reactive oxygen species (ROS). We have detected the production of a quinoid type metabolite (M8), presumably arising from ring oxygenation of M9 via an epoxide intermediate (Figures 5 and 9), as well as O-demethylated (M6) and aromatic hydroxylated products (M5 and M7). GSH adducts of M6, M7, and M8 were detected by LC/MS analysis. The formation of these adducts confirmed the presence of these electrophilic metabolites (intermediates) (Table 4). Although we have not characterized these adducts, a hypothetical structure from GSH addition to M8 is shown in Figure 9. Previous studies have used 3H-labeled dihydrocapsaicin to demonstrate the formation of protein adducts and the concomitant inactivation of P450 enzymes. As such, metabolic activation to reactive intermediates was proposed as a mechanism of toxicity (48, 55). We have found that addition of GSH stimulated the overall metabolism of capsaicin ∼2.5-fold and decreased the detection of the O-demethylated (M6), N-dehydrogenated and ring oxygenated metabolite (M8), and an uncharacterized aromatic hydroxylated metabolite (M7). In particular, we observed marked (40-70%) increases in the production of the macrocyclic (M1) and ω-1-hydroxylated (M3) metabolites (Table 4). These data may imply that the P450 enzymes that catalyzed ω-1-hydroxylation and macrocycle formation (i.e., 2C8, 2C9, 2E1, 2C19, and 3A4) are particularly susceptible to inactivation during the metabolism of capsaicin. Interestingly, many of these same P450 enzymes catalyzed the formation of the N-dehydrogenated and ring oxygenated metabolite that


appears to be formed via an epoxide intermediate (Table 3 and Figure 9). These data agree, in part, with previous reports on the inactivation of 2E1 by dihydrocapsaicin (2, 54, 55). However, the precise nature of the P450inactivating intermediate has not been elucidated. Regardless, P450-dependent activation of capsaicin to reactive metabolites capable of altering P450 activity, as well as altering the properties of other critical cellular macromolecules, may represent a mechanism of toxicity (2, 54, 55). However, as mentioned above, our cell culture results showed a protective role for P450 in cells treated with capsaicin, suggesting that bioactivation of capsaicin to potentially electrophilic intermediates, although present, does not appear to be involved in the cytotoxicity of capsaicinoids. As mentioned in the Introduction, capsaicin has been described as a potent irritant and moderately toxic substance, exhibiting LD50 values in mice that ranged from 0.56 to 510 mg/kg for iv and topical doses, respectively (32). Interestingly, the mechanism of death was attributed to severe cardiovascular and respiratory dysfunction and respiratory depression and failure (32). Given the apparent tissue selective toxicity of capsaicinoids to respiratory tissues, one could hypothesize that differences in metabolism of capsaicinoids in these tissues may be an important contributing factor. For example, the production of unique cytotoxic metabolites in respiratory tissues may predispose these cells to the toxic effects of capsaicin. In this study, we did not observe the production of unique metabolites by lung microsomes. However, the overall rate of capsaicin metabolism was markedly less for lung microsomes than liver microsomes (Table 1). Hence, an alternate hypothesis for the tissue selective nature of capsaicin toxicity may be that respiratory tissues are deficient in many of the P450 enzymes capable of metabolizing capsaicin (See Table 3). Such deficiencies could increase the half-life of capsaicin in lung tissues, such that the activity and toxicity of capsaicin in respiratory tissues are more pronounced. The metabolism (Table 1) and cytotoxicity data (Table 5) presented in this study support this hypothesis. Conversely, all tissues deficient in adequate P450 activity would be expected to be susceptible to the toxic effects of capsaicin, but such widespread toxicities have not been observed. One explanation for this phenomenon may be that most tissues are not exposed to high (cytotoxic) doses of capsaicin since the primary routes of exposure to capsaicin are oral, dermal, and inhalation. An alternate mechanism of capsaicin-mediated cellular damage that is consistent with the data presented here focuses on TRPV1 receptor activation. We have recently demonstrated that overexpression of TRPV1 in BEAS2B cells resulted in a 100-fold increase in cellular susceptibility to cytotoxicity by capsaicin, nonivamide, and other TRPV1 agonists (56). We also demonstrated that the relative susceptibility of BEAS-2B, A549, and HepG2 cells to cytotoxicity (BEAS-2B > A549 > HepG2) by capsaicin was correlated with the levels of TRPV1 expression in each cell type (56). These data suggested that TRPV1 binding regulated the cytotoxic effects of capsaicin. The role of TRPV1 in mediating the cytotoxic effects of capsaicin has been well-documented in peripheral sensory neurons and has been used as the basis for treatment of chronic pain with capsaicin. Collectively, these data support the hypothesis that metabolism of capsaicin represents a detoxification mechanism by

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transforming capsaicin to metabolites that are less potent agonists of TRPV1, a vital cellular receptor that controls the cytotoxicity of capsaicin. Previous research has also demonstrated variability in the pungency of the naturally occurring capsaicinoids, with capsaicin and nonivamide being the most pungent (1, 3, 18-22). Because of the similarity in pharmacological activity between capsaicin and nonivamide, the two chemicals are commonly used interchangeably or in combination in various product formulations where capsaicin is an active component. Recently, nonivamide has been identified as a natural product that is found in extracts of peppers, and thus, represents a small proportion of total capsaicinoids (1-4%) in all pepper-based products (5). Our data demonstrated that human liver microsomes were capable of metabolizing both capsaicin and nonivamide in a similar manner, with the exception that the formation of the corresponding dehydrogenated (M4*), macrocyclic (M1*), and ω-1-hydroxylated (M3*) metabolites was absent for nonivamide (Figure 10). These data are significant since they imply that differences in the structures of the alkyl chain portions of capsaicin and nonivamide affect the ability of P450 enzymes to metabolize, and presumably, detoxify capsaicin and nonivamide via alkyl dehydrogenation and oxygenation pathways. The cell culture data presented in Table 5 appear to contradict the hypothesis that nonivamide should be more cytotoxic due to decreased metabolism, since capsaicin was slightly more cytotoxic than nonivamide to lung and liver cells. These data support the hypothesis that metabolism of capsaicin to cytotoxic metabolites may be important for bioactivation and cytotoxicity. However, the overall rate for metabolism of nonivamide was similar to capsaicin. Therefore, an alternate explanation might be that the data exemplify the inherent differences in the binding affinities and agonist properties of capsaicin and nonivamide at TRPV1 or other unidentified mediators of cytotoxicity. Collectively, our data have provided new insight into the role of metabolism in the pharmacology/toxicology of capsaicin and nonivamide. We have demonstrated that liver and lung microsomes were capable of metabolizing capsaicin and nonivamide and that this trend was consistent among all species tested, albeit the rates and relative proportions of each metabolite were variable and depended upon the chemistry of the alkyl side chain. The combination of our data with previous research relating the pharmacological activity of capsaicinoids to molecular structure suggests that P450-mediated metabolism decreases the pharmacological and/or toxicological activity of capsaicin and may serve to ameliorate the effects of capsaicin by preventing key biochemical interactions with molecular determinants of toxicity (i.e., TRPV1). In general, this hypothesis was supported by our cell culture experiments that demonstrated an overall protective effect of P450 in the cytotoxicity of capsaicinoids in lung and liver cells. These data may also suggest that the route of exposure and resulting bioavailability of capsaicin may predispose certain tissues (e.g., lung or nerve cells) that are deficient in the appropriate P450 enzymes and/or rich in the molecular targets (i.e., TRPV1) that mediate cytotoxicity to the pharmacological/toxicological effects of capsaicin. Consequently, the relative safety of various capsaicin-based products (i.e., topical creams, oral supplements, or aerosolized pepper sprays) may be highly dependent on formulation and the method of use.

Reilly et al.

Acknowledgment. We thank Dr. Matthew Slawson for technical assistance with the LC/MS/MS experiments. This research was supported, in part, by the National Institute of Standards and Technology (Contract No. 60NANBOD0006) through the National Institute of Justice and by the Colgate-Palmolive Post-Doctoral Fellowship in In Vitro Toxicology through the Society of Toxicology.

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