Drug Metabolism and Disposition Fast Forward
First published on April 29, 2005; DOI: 10.1124/dmd.104.002634
0090-9556/05/3308-1115-1123$20.00
DMD 33:1115-1123, 2005
METABOLISM OF THE 
,ß-UNSATURATED KETONES, CHALCONE AND TRANS-4-PHENYL-3-BUTEN-2-ONE, BY RAT LIVER MICROSOMES AND ESTROGENIC ACTIVITY OF THE METABOLITES
Yoichi Kohno,
Shigeyuki Kitamura,
Seigo Sanoh,
Kazumi Sugihara,
Nariaki Fujimoto, and
Shigeru Ohta
Graduate School of Biomedical Sciences (Y.K., S.K., S.S., K.S., S.O.), and Research Institute for Radiation Biology and Medicine (N.F.), Hiroshima University, Hiroshima, Japan
(Received October 14, 2004;
accepted April 21, 2005)
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Abstract
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When chalcone and trans-4-phenyl-3-buten-2-one (PBO) were incubated with liver microsomes of untreated rats in the presence of NADPH, 4-hydroxychalcone and trans-4-(4-hydroxyphenyl)-3-buten-2-one (4-OH-PBO), respectively, were formed as major metabolites. Two minor metabolites of chalcone, 4'-hydroxychalcone and 2-hydroxychalcone, were also observed. The oxidase activity affording 4-hydroxychalcone was inhibited by SKF 525-A, disulfiram, ketoconazole, and 
-naphthoflavone. The oxidase activities leading to 4-hydroxychalcone and 4'-hydroxychalcone were enhanced in liver microsomes of 3-methylcholanthrene- and phenobarbital-treated rats, respectively. The activity generating 2-hydroxychalcone was enhanced in liver microsomes of 3-methylcholanthrene- and dexamethasone-treated rats. The oxidation of PBO to 4-OH-PBO was inhibited by SKF 525-A, ketoconazole, disulfiram, and sulfaphenazole. This activity was enhanced in liver microsomes of 3-methylcholanthrene-, acetone- and phenobarbital-treated rats. 4-Hydroxylation, 4'-hydroxylation, and 2-hydroxylation of chalcone were catalyzed by rat recombinant cytochrome P450 1A1, 1A2, and 2C6; by 1A1 and 2C6; and by 1A1 and 3A1, respectively. PBO was oxidized by cytochrome P450 1A1, 1A2, 2C6, and 2E1. Chalcone and PBO were negative in an estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, 4-hydroxychalcone, 2-hydroxychalcone, 4'-hydroxychalcone, and 4-OH-PBO exhibited estrogenic activity.
,ß-unsaturated ketones, which are structures in which the double bond is adjacent to the carbonyl group, have been used as starting materials for the synthesis of various chemicals, including plastics, resins, pesticides, dyes, and pharmaceuticals (Opdyke, 1973
). They are also found in naturally occurring compounds, such as plant allelochemicals, and insect hormones and pheromones (Wadleigh and Yu, 1987). In addition, these structures are formed during combustion of carbonaceous materials, and are found in automobile exhausts, tobacco smoke, and flue gases (Eder et al., 1991). ,ß-unsaturated ketones are reactive compounds, due to their electrophilic properties, and undergo nucleophilic attack, e.g., with SH groups in proteins. They exhibit genotoxicity and mutagenicity, and show an anticarcinogenic effect toward cultured tumor cells, such as human colon adenocarcinoma cells, B16 melanoma 4A5 cells, and K562 leukemia or melanoma cells (Eder et al., 1993; Prestera et al., 1993; Czerny et al., 1998; Sabzevari et al., 2004). They also showed inhibitory effects on glucose 6-phosphatase, glutathione S-transferase, catechol-O-methyltransferase, nitric oxide synthase, and quinone reductase (Jørgensen et al., 1992; Wikberg et al., 1993; Chien et al., 1994; Pan et al., 2000). Chalcone (trans-1,3-diphenyl-2-propen-1-one) is an ,ß-unsaturated ketone that has the skeletal makeup of so-called "chalcones." Chalcones are open-chain flavonoids in which two aromatic rings, jointed by a three carbon linker, are synthesized by chalcone synthetase from 3-malonyl-CoA and a starter CoA ester such as 4-coumaronyl-CoA in plants (Schröder and Schröder, 1990). Chalcone synthetase functions as a key enzyme of flavonoid biosynthesis, utilizing the same substrates as stilbene synthetase (Schröder et al., 1988). Chalcone and chalcones are called "anthochlor pigments." This term was coined to identify a group of yellow pigments which turn red in the presence of alkali. In some plants, chalcones contribute significantly to the corolla pigmentation.
trans-4-Phenyl-3-buten-2-one (PBO), also called trans-phenyl styryl ketone or benzalacetone, has a wide range of uses as an industrial material for synthesis of chemicals and drugs, and as a flavoring additive for cosmetics, soaps, detergents, cigarettes, and foods. It has mutagenic and antimutagenic effects (Prival et al., 1982; Motohashi et al., 1997). We have studied the metabolism of PBO in rats and dogs (Kitamura et al., 1999; Okamoto et al., 1999), and identified the double bond-reduced metabolite, 4-phenyl-2-butanone (PBA), and the carbonyl-reduced metabolite, trans-4-phenyl-3-buten-2-ol (PBOL), which lacks mutagenic activity, as in vivo metabolites of PBO in rats and dogs (Kitamura et al., 1999). Furthermore, it was demonstrated that the reductions of the double bond and carbonyl group were catalyzed in rats by ",ß-ketoalkene double bond reductase" in liver cytosol and by a novel microsomal carbonyl reductase, respectively (Kitamura and Tatsumi, 1990; Okamoto et al., 1999). We also showed that purified ,ß-ketoalkene double bond reductase exhibited significant reducing activity toward the double bonds of chalcone and some ,ß-unsaturated ketones (Kitamura and Tatsumi, 1990). In contrast, Sauer et al. (1997a,b) found some glutathione conjugates of PBO in the blood and urine of rats and mice dosed with PBO. However, the oxidative metabolism of PBO and chalcone in animals and humans has not been examined.
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FIG. 1. HPLC chromatograms of the extracts of incubation mixtures of chalcone and PBO with liver microsomes of 3-methylcholanthrene-treated rats. A, HPLC chromatogram of the metabolites of chalcone. B, HPLC chromatogram of the metabolites of PBO. The absorbance (full scale, 0.016 absorbance unit) was measured at 254 nm. An incubation mixture consisting of 0.1 µmol of chalcone or PBO, 0.5 µmol of NADPH, and 20 µl of liver microsomes was incubated for 20 min. The extract of the mixture with diethyl ether was analyzed by HPLC as described under Materials and Methods.
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Various chemicals produced by humans mimic the effects of ß-estradiol. These chemicals are called environmental estrogens and include agricultural pesticides, such as kepone, 1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane (o,p'-DDT), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDD), dieldrin and methoxychlor, and industrial chemicals such as bisphenol A, nonylphenol, and some polychlorinated biphenyl congeners (Andersen et al., 1999). The structural requirements for estrogenic activity include a hindered hydroxyl group on an aryl ring and a hydrophobic group attached para to the hydroxyl group (Blair et al., 2002; Hong et al., 2002). Environmental estrogens may be playing a role in the increasing incidence of breast cancer, testicular cancer, and other problems of the reproductive system. Naturally occurring phytoestrogens also show estrogenic activity in vitro in receptor binding assay, despite their beneficial effects, such as anticarcinogenicity (Branham et al., 2002). Some flavonoids are phytoestrogens. Chalcones are a source of phytoestrogens, acting as C15 precursors in plant flavonoid biosynthesis. PBO also has a flavonoid skeleton. Recently, we showed that trans-stilbene, the parent compound of diethylstilbestrol, was not itself estrogenic but exhibited a potent estrogenic activity after metabolic activation by a microsomal cytochrome P450 (P450) system (Sugihara et al., 2000). We suggested that the estrogenic activity of trans-stilbene was due to the hydroxylated metabolites, trans-4-hydroxystilbene and trans-4,4'-dihydroxystilbene. Despite the known estrogenic activity of stilbene derivatives such as resveratrol and trans-4-hydroxystilbene, the estrogenic action of chalcone and PBO has not been reported (Gehm et al., 1997; Sugihara et al., 2000).
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FIG. 2. Oxidase activities of rat liver microsomes toward chalcone and PBO. A, chalcone-oxidase activity to 4-OH chalcone; B, chalcone-oxidase activity to 4'-OH chalcone; C, chalcone-oxidase activity to 2-OH chalcone; D, PBO-oxidase activity to 4-OH-PBO. Each bar represents the mean ± S.D. of four rats. A mixture containing 0.1 µmol of chalcone or PBO, 0.5 µmol of NADPH, and 20 µl of liver microsomes in 0.1 M phosphate buffer (pH 7.4) was incubated at 37°C for 20 min. The 4-hydroxychalcone, 2-hydroxychalcone, 4'-hydroxychalcone, and 4-OH-PBO formed were determined using HPLC as described under Materials and Methods. N.D., not detected. *, p < 0.05, **, p < 0.01 compared with control. Control, 3-MC, Acetone, DEX, and PB represent liver microsomes of untreated, 3-methylcholanthrene-treated, acetone-treated, dexamethasone-treated, and phenobarbital-treated rats, respectively.
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Here, we describe a study of the metabolism of chalcone and PBO by rat liver microsomes. The estrogenic activities of the parent compounds and their metabolites were examined using an estrogen-responsive element (ERE)-luciferase reporter assay in MCF-7 cells. We identified the P450 isoforms functioning in the oxidation of chalcone and PBO and demonstrated that these hydroxylated ,ß-unsaturated ketones exhibit estrogenic activity.
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Materials and Methods
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Chemicals. Chalcone, 4-hydroxychalcone, 4'-hydroxychalcone, 2-hydroxychalcone, 2'-hydroxychalcone, PBO, PBA, and trans-4-(4-hydroxyphenyl)-3-buten-2-one (4-OH-PBO) were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). 17ß-Estradiol was obtained from Sigma-Aldrich (St. Louis, MO). Rat recombinant P450 isoforms expressed in a baculovirus system were obtained from BD Gentest (Woburn, MA). PBOL was synthesized by a reported method (Okamoto et al., 1999).
Animals. Male Sprague-Dawley rats (210–230 g) were obtained from Japan SLC, Inc. (Shizuoka, Japan). The animals were housed at 22°C with a 12-h light/dark cycle, with free access to tap water and a standard pellet diet MM-3 (Funabashi Farm, Funabashi, Japan). In some experiments, rats were given phenobarbital, 3-methylcholanthrene, or dexamethasone intraperitoneally once per day for 3 consecutive days at 80, 25, or 100 mg/kg, respectively, or acetone orally once at 3 g/kg at 24 h before sacrifice.
Preparation of Liver Microsomes. The livers were excised from exsanguinated rats and immediately perfused with 1.15% KCl. The livers were homogenized in 4 volumes of the KCl solution using a Potter-Elvehjem homogenizer. The microsomal fraction was obtained from the homogenate by successive centrifugation at 9000g for 20 min and 105,000g for 60 min. The fraction was washed by resuspension in the KCl solution and resedimentation. The pellets of microsomes were resuspended in the solution to make 1 ml equivalent to 1 g of liver.
Cell Culture. MCF-7 cell lines were maintained in minimal essential medium (Sigma-Aldrich), containing penicillin and streptomycin, with 5% fetal bovine serum (Invitrogen, Carlsbad, CA).
Microsomal Incubation Mixtures for Identification of Oxidative Metabolites of Chalcone and PBO. The metabolites of chalcone and PBO were isolated from an incubation mixture, which consisted of 0.2 µmol of chalcone or PBO, 1 µmol of NADPH, and 0.1 ml of microsomes in a total volume of 1 ml of 0.1 M K/Na phosphate buffer (pH 7.4). After incubation at 37°C for 20 min, the mixture was extracted with 5 ml of diethyl ether. The supernatant fraction was separated by centrifugation for 10 min at 1000g and evaporated to about 50 µl at 0°C, and 0.1 ml of methanol was added. The solution was injected into a high-performance liquid chromatography (HPLC) instrument equipped with a photodiode array UV detector (Beckman Coulter, Fullerton, CA) or with liquid chromatography-mass spectrometry operated in tandem (LC-MS/MS).
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FIG. 3. Effects of some chemicals on oxidase activities of rat liver microsomes toward chalcone and PBO. A, chalcone-oxidase activity to 4-OH chalcone; B, chalcone-oxidase activity to 4'-OH chalcone; C, chalcone-oxidase activity to 2-OH chalcone; D, PBO-oxidase activity to 4-OH-PBO. Each bar represents the mean ± S.D. of four rats. A mixture containing 0.1 µmol of chalcone or PBO, 0.5 µmol of NADPH, and 20 µl of liver microsomes in 0.1 M phosphate buffer (pH 7.4) was incubated at 37°C for 20 min. Inhibitors were added at the concentration of 10-4 M. The 4-hydroxychalcone, 2-hydroxychalcone, 4'-hydroxychalcone, and 4-OH-PBO formed were determined using HPLC as described under Materials and Methods. N.D., not detected. *, p < 0.05, **, p < 0.01 compared with control.
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Assay of Liver Microsomal Oxidase Activity. An incubation mixture consisted of 0.1 µmol of chalcone or PBO, 0.5 µmol of NADPH, and 0.2 ml of liver microsomes equivalent to 200 mg of liver wet weight (0.3–0.5 mg protein) or 90 µl of rat recombinant P450 isoforms (CYP 1A1, 1A2, 2B1, 2E1, 2D1, 2C6, and 3A1; about 0.05 nmol P450 equivalent) in a final volume of 1 ml of 0.1 M K/Na phosphate buffer (pH 7.4). The incubation was performed at 37°C for 20 min. After incubation, 0.1 µmol of methyl p-aminobenzoate was added as an internal standard, and the mixture was extracted with 5 ml of diethyl ether. The extract was evaporated to dryness, the residue was dissolved in 0.1 ml of methanol, and an aliquot (5 µl) was analyzed by HPLC or LC-MS/MS. The 4-hydroxylase activities toward chalcone and PBO were detected by HPLC. The 4'- and 2-hydroxylase activities toward chalcone were detected by LC-MS/MS. Rat recombinant P450 isoforms were expressed using a baculovirus expression system. Baculovirus-infected insect cells were used to prepare microsomes.
Assay of Estrogenic Activity. An ERE-luciferase reporter assay was performed to assess estrogenic activity (Sugihara et al., 2000). MCF-7 cells were plated at 2 x 104 cells/well in 48-well plates (Nalge Nunc International, Naperville, IL). After 24 h, cells in each well were transiently transfected with 0.3 µg of (ERE)3-SV40-luc and 0.01 µg of phRL-CMV (Promega, Madison, WI,) with 0.3 µg of TransFast (Promega), a transfection reagent containing a synthetic cationic lipid. After 24 h of incubation with chemicals, cells were harvested with 30 µl of cell lysis buffer (Promega). The firefly and Renilla luciferase activities were determined with the Dual Luciferase Assay Kit (Promega) by measuring luminescence with a Wallac Micro-Beta scintillation counter (PerkinElmer Life and Analytical Sciences, Boston, MA). Firefly luciferase reporter activity was normalized to Renilla luciferase activity from phRL-CMV. For the assay of the activated metabolites of chalcone in the liver microsomal system, chalcone (0.1 µmol) was incubated with 0.1 ml of rat liver microsomes in the presence of 1 µmol of NADPH for 30 min in a final volume of 1 ml of 0.1 M phosphate buffer. After the incubation, the mixture was extracted with 5 ml of ethyl acetate and evaporated to dryness. The residue was dissolved with 1 ml of ethanol, and an aliquot was used for the estrogenic activity assay. The total concentration of the substrate and its metabolites was calculated from the original amount of the substrate.
HPLC. HPLC was performed in a Hitachi L-6000 chromatograph (Hitachi, Tokyo, Japan) fitted with a 125 x 4 mm Inertsil ODS-3 column (GL Science, Tokyo, Japan). The mobile phase consisted of acetonitrile/water (2:3, v/v) for 40 min, then a linear gradient to acetonitrile/water (1:1, v/v) over 100 min, followed by 40 min of acetonitrile/water (1:1, v/v) for the separation of chalcone and the metabolites. The chromatograph was operated at a flow rate of 0.5 ml/min with detection at a wavelength of 280 nm. In the case of PBO, the mobile phase was acetonitrile/water (3:7, v/v). The chromatograph was operated at a flow rate of 0.5 ml/min at a wavelength of 254 nm. The elution times of 4-OH-PBO, methyl p-aminobenzoate (internal standard for PBO), PBO, methyl p-aminobenzoate (internal standard for chalcone), 4-hydroxychalcone, 4'-hydroxychalcone, 2-hydroxychalcone, chalcone, and 2'-hydroxychalcone were 13.3, 16.2, 45.2, 8.7, 33.0, 35.4, 46.4, 94.9, and 132.2 min, respectively.
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FIG. 4. Oxidase activities of rat recombinant P450 isoforms toward chalcone and PBO. A, chalcone-oxidase activity to 4-OH chalcone; B, chalcone-oxidase activity to 4'-OH chalcone; C, chalcone-oxidase activity to 2-OH chalcone; D, PBO-oxidase activity to 4-OH-PBO. Each bar represents the mean of duplicate experiments. A mixture containing 0.1 µmol of chalcone or PBO, 0.5 µmol of NADPH, and 90 µl of rat recombinant P450 isoform (CYP 1A1, 1A2, 2B1, 2E1, 2D1, 2C6, and 3A1; about 0.05 nmol P450 equivalent) in 0.1 M phosphate buffer (pH 7.4) was incubated at 37°C for 20 min. Hydroxylated metabolites formed were determined using HPLC as described under Materials and Methods. N.D., not detected.
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LC-MS/MS Analysis. LC-MS/MS was performed using an API 2000 (Applied Biosystems, Foster, CA) equipped with a triple-stage quadrupole mass spectrometer coupled to an Agilent 1100 HPLC system (Agilent Technologies, Palo Alto, CA). An electrospray ionization source was used for measurement. A reversed-phase Inertsil ODS-3 column (125 x 4 mm) was used as the separation column, and its temperature was maintained at 30°C. The mobile phase was 40% acetonitrile/water containing 0.1% acetic acid for 50 min, then a linear gradient to 80% acetonitrile/water containing 0.1% acetic acid over 55 min, followed by a hold for 10 min. The column effluent was monitored in the negative ion mode with multiple reaction monitoring. The temperature of the Turbo-Ionspray auxiliary gas was 500°C, and the ionization voltage was -4500 V. Nitrogen was used as nebulizer, auxiliary, curtain, and collision-activated dissociation gas, at 40, 80, and 30 psi, respectively, and a value of 4 (1 psi = 6894.76 Pa). Analytical conditions of the selected ion pair (m/z for Q1/Q3), dwell time, declustering potential, focusing potential, entrance potential, collision energy, and collision exit potential (4-hydroxychalcone: 222.7/144.7, 250 ms, -70 V, -260 V, -11 V, -36 V, and -20 V; 4'-hydroxychalcone: 222.7/120.7, 250 ms, -81 V, -300 V, -11 V, -40 V and -18 V; 2-hydroxychalcone: 222.7/194.8, 250 s, -90 V, -330 V, -10.5 V, -24 V, and -39 V; N-propylparaben (an internal standard): 178.7/91.7, 250 ms, -46 V, -320 V, -10.5 V, -30 V, and -18 V, respectively) were automatically determined by Analyst (the application software for quantitative determination with the API 2000). multiple reaction monitoring chromatograms of the metabolites were compared with those of authentic standards to confirm the identity of metabolites. The elution times of 4-hydroxychalcone, 4'-hydroxychalcone, 2-hydroxychalcone, and n-propylparaben (an internal standard) were 32.7 min, 34.9 min, 45.0 min, and 21.3 min, respectively. The detection limits (S/N value) of 4-hydroxychalcone, 4'-hydroxychalcone, and 2-hydroxychalcone were 3, 2, and 2 at concentrations of 100 nM, 500 nM, and 10 nM, respectively. Calibration plots were linear up to 10 µM for 4-hydroxychalcone, 4'-hydroxychalcone, and 2-hydroxychalcone. Recoveries of 4-hydroxychalcone, 4'-hydroxychalcone, and 2-hydroxychalcone were 94, 96, and 97%, respectively.
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Results
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Metabolism of Chalcone and PBO by Rat Liver Microsomes. Chalcone was incubated with liver microsomes of untreated rats in the presence of NADPH to obtain metabolites as described under Materials and Methods. Four peaks were detected in an HPLC chromatogram of the extract of the incubation mixtures. These peaks were not detected in the control, which was incubated without the substrate. Retention times of the chalcone and its metabolites detected at 33.0, 35.4, 46.4, and 94.9 min corresponded to those of 4-hydroxychalcone, 4'-hydroxychalcone, 2-hydroxychalcone, and chalcone, respectively (Fig. 1A). The peak corresponding to 4-hydroxychalcone was much higher than those of the 4'-hydroxyl and 2-hydroxyl derivatives. No peak corresponding to 2'-hydroxychalcone was detected. PBO was also incubated with liver microsomes of untreated rats in the presence of NADPH for the detection of the metabolites. Three peaks, which were not detected in the control, were detected in an HPLC chromatogram of the extract of the incubation mixture. The retention times of 13.3, 33.1, and 45.2 min corresponded to those of 4-OH-PBO, PBOL, and PBO, respectively (Fig. 1B). The identities of the hydroxylated metabolites were confirmed by mass and UV spectral comparison with authentic samples (data not shown).
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FIG. 5. Estrogenic activity of chalcone, PBO, and their hydroxylated metabolites using ERE-luciferase reporter assay in MCF-7 cells. A, estrogenic activity of chalcone and its hydroxylated metabolites; B, estrogenic activity of PBO and its metabolite. Each bar represents the mean ± S.D. of four experiments. Estrogenic activity was expressed as a relative activity with respect to the control using MCF-7 cells. *, p < 0.05, **, p < 0.01 compared with control.
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Oxidase Activity of Rat Liver Microsomes. When chalcone or PBO was incubated with liver microsomes of untreated rats, the amounts of hydroxyl derivatives formed increased linearly for 20 min (data not shown). The oxidase activities of rat liver microsomes toward chalcone and PBO were examined using liver microsomes of untreated and phenobarbital-, 3-methylcholanthrene-, dexamethasone-, or acetone-treated rats. When chalcone was incubated with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, significant oxidase activities affording 4- and 2-hydroxyl derivatives were observed. In contrast, when chalcone was incubated with liver microsomes of phenobarbital-treated rats, a significant oxidase activity affording 4'-hydroxychalcone was observed. The oxidase activity affording 4-hydroxychalcone was much higher than those leading to 4'-hydroxychalcone and 2-hydroxychalcone (Fig. 2, A–C). When oxidase activity transforming PBO to the 4-hydroxyl derivative was compared in liver microsomes of rats treated with various inducers in the presence of NADPH, the highest activity was observed in liver microsomes of 3-methylcholanthrene-treated rats. The oxidase activity was enhanced to a lesser extent when liver microsomes of acetoneor phenobarbital-treated rats were used instead of those of untreated rats (Fig. 2D). Only marginal oxidase activity toward chalcone and PBO was observed with NADH instead of NADPH (data not shown).
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FIG. 6. Estrogenic activity of chalcone with liver microsomes using ERE-luciferase reporter assay in MCF-7 cells. Each bar represents the mean ± S.D. of four experiments. Estrogenic activity was expressed as relative activity with respect to the control using MCF-7 cells. Chalcone was incubated with liver microsomes in the presence of NADPH, and the extract of the incubation mixture was subjected to the screening test. PB: phenobarbital, MC, 3-methylcholanthrene. *, p < 0.05, **, p < 0.01 compared with control.
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Inhibitory Effect of Chemicals on Microsomal Oxidase Activity. The NADPH-dependent activities of liver microsomes of untreated rats to generate 4-, 4'-, and 2-hydroxyl derivatives of chalcone were markedly inhibited by the addition of SKF 525-A and partly by -naphthoflavone and disulfiram, except for 2-hydroxylation, which was stimulated by -naphthoflavone. Ketoconazole also inhibited these oxidations, especially 2-hydroxylation. Secobarbital and sulfaphenazole did not show inhibitory effects on the oxidation of chalcone by rat liver microsomes (Fig. 3, A–C). In the case of 3-methylcholanthrene-treated rat liver microsomes, -naphthoflavone inhibited 4-, 4'-, and 2-hydroxylation of chalcone (data not shown). The microsomal oxidase activity with NADPH toward PBO was inhibited by the addition of ketoconazole, disulfiram, sulfophenazole, and SKF 525-A (Fig. 3D).
P450 Isoforms Catalyzing the Oxidation of Chalcone and PBO. Next, we attempted to identify P450 isoforms involved in the oxidation of chalcone and PBO by using various rat recombinant P450 isoforms. CYP 1A1 and 1A2 mainly catalyzed the oxidation of chalcone to 4-hydroxychalcone, and CYP 2B1 and 2C6 also catalyzed the oxidation. However, CYP 2E1, 2D1, and 3A1 lacked this activity (Fig. 4A). The oxidation to 4'-hydroxychalcone was catalyzed by CYP 1A1 and 2C6 (Fig. 4B). The oxidation to 2-hydroxychalcone was catalyzed by CYP 1A1 and 3A1 (Fig. 4C). CYP 1A2 and 2C6 mainly catalyzed the oxidation of PBO to 4-OH-PBO. CYP 1A1 and 2E1 also catalyzed this reaction (Fig. 4D). The highest oxidase activity was observed in chalcone 4-hydroxylation, compared with other oxidations of chalcone and PBO, similar to the case of liver microsomes.
Estrogenic Activities of Chalcone, PBO, and their Hydroxylated Metabolites. The estrogenic activities of chalcone, PBO, and their hydroxylated metabolites were examined using ERE-luciferase reporter assay in MCF-7 cells. Chalcone did not show estrogenic activity in this assay. However, 4-hydroxychalcone, 2-hydroxychalcone, and 4'-hydroxychalcone showed estrogenic activity. The 2'-hydroxychalcone metabolite was inactive. 4-Hydroxychalcone exhibited the highest activity at 1 x 10-4 M. These compounds were cytotoxic to MCF-7 cells at concentrations above 1 x 10-4 M, except for 2-hydroxychalcone, so the apparent estrogenic activities decreased at higher concentrations (Fig. 5A).
The estrogenic activities of PBO and its metabolites were also examined. PBO was negative in the assay with MCF-7 cells. However, the hydroxylated metabolite, 4-OH-PBO, showed estrogenic activity at 1 x 10-5 to 1 x 10-4 M (Fig. 5B). In contrast, PBA, the double bond-reduced metabolite of PBO, and its hydroxylated metabolite, 4-(4-hydroxyphenyl)-2-butanone, did not show estrogenic activity at 1 x 10-4 M (data not shown). These results suggest that chalcone and PBO were converted to active metabolites by rat liver microsomes.
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FIG. 7. Metabolic activation pathway of chalcone and PBO to estrogens by rat liver microsomal enzyme system.
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Estrogenic Activity of Chalcone with a Microsomal Oxidation System. The estrogenic activity of chalcone in the presence of a rat liver microsomal oxidation system was examined. When chalcone was incubated with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, the extract of the incubation mixture exhibited an estrogenic activity in the range of 1 x 10-6 to 1 x 10-5 M. In contrast, a smaller effect was obtained when liver microsomes of phenobarbital-treated rats were used (Fig. 6). These results suggest that chalcone is mainly metabolically activated to 4- and 2-hydroxychalcones by liver microsomes from 3-methylcholanthrene-treated rats and to 4'-hydroxychalcone by microsomes from phenobarbital-treated rats.
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Discussion
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Previously, we demonstrated that the double bond and carbonyl group of ,ß-unsaturated ketones were reduced by liver microsomes and cytosol of experimental animals (Kitamura and Tatsumi, 1990; Kitamura et al., 1999; Okamoto et al., 1999). Here, we describe the oxidative metabolism of ,ß-unsaturated ketones, chalcone and PBO, by rat liver microsomes. These compounds were activated to estrogens by oxidative conversion to their hydroxylated metabolites. ,ß-unsaturated ketones are easily detoxified to reduced metabolites by double bond reductase, a cytosolic enzyme, triple bond reductase, a microsomal enzyme, or microsomal and cytosolic carbonyl reductases. In contrast, when ,ß-unsaturated ketones are oxidized at the phenyl ring by P450, they are converted to active estrogenic compounds. Inasmuch as ,ß-unsaturated ketones are taken in through foods, food additives, and cigarette smoke, it is necessary to consider the possible endocrine-disrupting activity of these compounds, including their metabolites. In this study, we obtained evidence that chalcone is oxidized to a major metabolite, 4-hydroxychalcone, by CYP 1A1/2 isoforms. PBO was oxidized to 4-OH-PBO by CYP 1A1/2; CYP 2C6 and 2E1 also contributed to this oxidation. Furthermore, CYP 2C6 and 1A1 were major isoforms involved in the 4'-hydroxylation of chalcone. The oxidation of chalcone to 2-hydroxychalcone was catalyzed by CYP 1A1 and 3A1. The CYP 1A subfamily commonly catalyzes the oxidation of chalcone and PBO.
It is interesting to note that CYP 2C6 contributes to the oxidation of chalcone and PBO. ,ß-unsaturated ketone structure of chalcone and PBO may be important for metabolism by this P450, as reported in the oxidation of ketones such as tolbutamide, phenytoin, and warfarin (Lewis, 1998). Disulfiram is a well known potent inhibitor of CYP 2E1. In this study, the chemical inhibited the 4- and 4'-hydroxylations of chalcone, whereas CYP 2E1 did not catalyze these oxidations. Martini et al. (1997) reported that disulfiram inhibits not only CYP 2E1-, but also CYP 2C- and 3A-mediated metabolism in rat liver microsomes. Therefore, the decreased 4- and 4'-hydroxylating activities toward chalcone may be due to the inhibitory effect of disulfiram on CYP 2C6 and 3A1. Ketoconazole commonly inhibited the 4-, 4'-, and 2-hydroxylations of chalcone, presumably due to its inhibitory effects on rat CYP 1A2, 2C6, and 3A1/2 (Kobayashi et al., 2003). In contrast, 4'-hydroxylation of chalcone was catalyzed by rat CYP 2C6 and 1A1. However, no inhibitory effect of sulfaphenazole, a human CYP 2C9 inhibitor, on 4'-hydroxychalcone formation was observed. This may be due to differential sensitivity to sulfaphenazole in humans and rats, i.e., the involvement of CYP 2C9 and CYP 2C6. Otherwise, the inhibitory effect may be masked by the activity of CYP 1A1. In this study, 2-hydroxylation of chalcone by untreated rat liver microsomes was stimulated by the addition of -naphthoflavone, an inhibitor of CYP 1A. There are other reports that -naphthoflavone stimulated various CYP 3A-dependent reactions (Ueng et al., 1995; Harlow and Halpert, 1998). Indeed, the oxidase activity of 3-methylcholanthrene-treated rat liver microsomes for conversion of chalcone to 2-hydroxychalcone was markedly inhibited by -naphthoflavone.
In this study, we obtained evidence that chalcone is converted to estrogenically active hydroxylated derivatives by rat liver microsomes. 4'-Hydroxychalcone and 2-hydroxychalcone were minor metabolites of chalcone, and 2'-hydroxychalcone was not formed. Their estrogenic activities were lower than that of 4-hydroxychalcone. In the microsomal system used in this study, the estrogenic activity of chalcone is thought to be mainly due to 4-hydroxychalcone, which is a major metabolite. PBO was also metabolically activated to an estrogen by a microsomal enzyme system, and 4-OH-PBO, which is a major metabolite in the microsomal system, exhibited estrogenic activity. These facts would suggest that chalcone and PBO are metabolically activated to estrogenic hydroxylated derivatives in rats. We reported that PBO was reduced to PBA by liver cytosolic double bond reductase (Kitamura and Tatsumi, 1990). 4-Hydroxyphenyl-2-butanone, an oxidized metabolite of PBA, was not estrogenic (data not shown). A 4-hydroxyl group on the phenyl moiety is an essential factor for the estrogenic activity, and a double bond conjugated with the phenyl ring may be also necessary (Fig. 7). The balance between oxidative and reductive metabolism of chalcone and PBO is an important problem. The level of microsomal carbonyl reductase activity toward PBO in rat livers found in our previous paper is almost equal to that of oxidase activity shown in this study (Okamoto et al., 1999). In our preliminary study, cytosolic double bond reductase activities toward chalcone and PBO were 14 and 5 nmol/min/mg protein, respectively. These specific activities are higher than those of the microsomal oxidase activities observed in this study. Thus, the activation step of chalcone and PBO is not a major reaction. These compounds may pose a relatively low risk to humans and other animals in terms of estrogenic activity, despite the in vitro activity shown in this study. Further study in other animals and organs will be necessary to determine the relevance of these reactions in the body. It is worthwhile to investigate further the estrogenic activity of hydroxylated chalcones, because these compounds occur naturally.
Chalcone and PBO were shown to be proestrogens in this study. There are other examples of metabolic activation to estrogens. Methoxychlor requires demethylation by liver microsomal mixed function oxidase-involved CYP 1A2 and 2C19, to elicit estrogenic activity (Stresser and Kupfer, 1998). Elsby et al. (2001) also reported that methoxybisphenol A was activated through demethylation by human liver microsomes. trans-Stilbene is also a proestrogen, which is metabolically activated to trans-4-hydroxystilbene, a potent estrogen, by CYP 1A1/2 (Sugihara et al., 2000; Sanoh et al., 2002). Furthermore, we reported activation of styrene oligomers and 2-nitrofluorene to estrogenic metabolites by CYP 2B1 and 1A1, respectively (Kitamura et al., 2003; Fujimoto et al., 2003). It is known that polychlorinated biphenyls are converted to the hydroxylated metabolites in the animal body, and some hydroxylated polychlorinated biphenyls show estrogenic activity (Korach et al., 1988; Connor et al., 1997). Hydroxylated metabolites of benzo[a]pyrene also exhibit estrogenic activity (Charles et al., 2000). It is necessary to consider the activity of metabolites produced from the parent compounds for the proper assessment of estrogenic toxicity of many compounds, including ,ß-unsaturated ketones.
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Footnotes
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This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Japanese Ministry of Education, Science, Sports and Culture, and a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.104.002634.
ABBREVIATIONS: PBO, trans-4-phenyl-3-buten-2-one; PBA, 4-phenyl-2-butanone; PBOL, trans-4-phenyl-3-buten-2-ol; P450, cytochrome P450; ERE, estrogen-responsive element; 4-OH-PBO, trans-4-(4-hydroxyphenyl-3-buten-2-one; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography-mass spectrometry operated in tandem; SKF 525-A, N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride.
Address correspondence to: Dr. Shigeyuki Kitamura, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan. E-mail: skitamu{at}hiroshima-u.ac.jp
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References
|
|---|
Andersen HR, Andersson A-M, Arnold SF, Autrup H, Barfoed M, Beresford NA, Bjerregaard P, Christiansen LB, Gissel B, Hummel R, et al. (1999) Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals. Environ Health Perspect 107 (Suppl 1): 89-108.[CrossRef]
Blair RM, Fang H, Branham WS, Hass BS, Dial SL, Moland CL, Tong W, Shi L, Perkins R, and Sheehan DM (2002) The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. Toxicol Sci 54: 138-153.
Branham WS, Dial SL, Moland CL, Bruce LM, Hass BS, Blair RM, Fang H, Shi L, Tong W, Perkins RG, et al. (2002) Phytoestrogens and mycoestrogens bind to the rat uterine estrogen receptor. J Nutr 132: 658-664.[Abstract/Free Full Text]
Charles GD, Bartels MJ, Zacharewski TR, Gollapudi BB, Freshour NL, and Carney EW (2000) Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor- reporter gene assay. Toxicol Sci 55: 320-326.[Abstract/Free Full Text]
Chien C, Kirollos KS, Linderman RJ, and Dauterman WC (1994) ,ß-unsaturated carbonyl compounds: inhibition of rat liver glutathione S-transferase isozymes and chemical reaction with reduced glutathione. Biochim Biophys Acta 1204: 175-180.[CrossRef][Medline]
Connor K, Ramamoorthy M, Moore M, Mustain M, Chen I, Safe S, Zacharewski T, Gillesby B, Joyeux A, and Balague P (1997) Hydroxylated polychlorinated biphenyls (PCBs) as estrogens and antiestrogens: structure-activity relationships. Toxicol Appl Pharmacol 145: 111-123.[CrossRef][Medline]
Czerny C, Eder E, and Rünger TM (1998) Genotoxicity and mutagenicity of the ,ß-unsaturated carbonyl compound crotonaldehyde (butenal) on a plasmid shuttle vector. Mutat Res 407: 125-134.[Medline]
Eder E, Hoffman C, Bastian H, Deininger C, and Scheckenbach S (1991) Molecular mechanisms of DNA damage initiated by ,ß-unsaturated carbonyl compounds as criteria for genotoxicity and mutagenicity. Environ Health Perspect 88: 99-106.
Eder E, Scheckenbach S, Deininger C, and Hoffman C (1993) The possible role of ,ß-unsaturated carbonyl compounds in mutagenesis and carcinogenesis. Toxicol Lett 67: 87-103.[CrossRef][Medline]
Elsby R, Maggs JL, Ashby J, Paton D, Sumpter JP, and Park BK (2001) Assessment of the effects of metabolism on the estrogenic activity of xenoestrogens: a two-stage approach coupling human liver microsomes and a yeast estrogenicity assay. J Pharmacol Exp Ther 296: 329-337.[Abstract/Free Full Text]
Fujimoto T, Kitamura S, Sanoh S, Sugihara K, Yoshihara S, Fujimoto N, and Ohta S (2003) Estrogenic activity of an environmental pollutant, 2-nitrofluorene, after metabolic activation by rat liver microsomes. Biochem Biophys Res Commun 303: 419-426.[Medline]
Gehm D, McAndrews JM, Chien P-Y, and Jameson JL (1997) Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor. Proc Natl Acad Sci USA 94: 14138-14143.[Abstract/Free Full Text]
Harlow GR and Halpert JR (1998) Analysis of human cytochrome P450 3A4 cooperativity: construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics. Proc Natl Acad Sci USA 95: 6636-6641.[Abstract/Free Full Text]
Hong H, Tong W, Fang H, Shi L, Xie Q, Wu J, Perkins R, Walker JD, Branham W, and Sheehan DM (2002) Prediction of estrogen receptor binding for 58,000 chemicals using an integrated system of a tree-based model with structural alerts. Environ Health Perspect 110: 29-36.[Medline]
Jørgensen BM, Agerbo P, Jensen B, Børresen T, and Hølmer G (1992) Inhibition of microsomal glucose 6-phosphatase by unsaturated aliphatic aldehydes and ketones. Chem-Biol Interact 81: 209-218.[CrossRef][Medline]
Kitamura S, Ohmegi M, Sanoh S, Sugihara K, Yoshihara S, Fujimoto N, and Ohta S (2003) Estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes. Environ Health Perspect 111: 329-334.[Medline]
Kitamura S, Okamoto Y, Takeshita M, and Ohta S (1999) Reductive metabolism in vivo of trans-4-phenyl-3-buten-2-one in rats and dogs. Drug Metab Dispos 27: 767-769.[Abstract/Free Full Text]
Kitamura S and Tatsumi K (1990) Purification of NADPH-linked ,ß-ketoalkene double bond reductase from rat liver. Arch Biochem Biophys 282: 183-187.[CrossRef][Medline]
Kobayashi K, Urashima K, Shimada N, and Chiba K (2003) Selectivities of human cytochrome P450 inhibitors toward rat P450 isoforms: study with cDNA-expressed systems of the rat. Drug Metab Dispos 31: 833-836.[Abstract/Free Full Text]
Korach KS, Sarver P, Chae K, McLachlan JA, and McKinney JD (1988) Estrogen receptorbinding activity of polychlorinated hydroxybiphenyls: conformationally restricted structural probes. Mol Pharmacol 33: 120-126.[Abstract]
Lewis DFV (1998) The CYP2 family: models, mutants and interactions. Xenobiotica 28: 617-661.[CrossRef][Medline]
Martini R, Ingelman-Sundberg M, and Murray M (1997) Pretranslational and posttranslational regulation of rat hepatic CYPs 3A2 and 2E1 by disulfiram. Biochem Pharmacol 54: 1323-1329.[Medline]
Motohashi N, Ashihara Y, Yamagami C, and Saito Y (1997) Antimutagenic effects of dehydrozingerone and its analogs on UV-induced mutagenesis in Escherichia coli. Mutat Res 377: 17-25.[Medline]
Okamoto Y, Kitamura S, Takeshita M, and Ohta S (1999) Microsomal carbonyl reductase responsible for reduction of 4-phenyl-3-buten-2-one in rats. IUBMB Life 48: 543-547.[CrossRef][Medline]
Opdyke DLJ (1973) Monographs on fragrance raw materials. Food Cosmet Toxicol 11: 1011-1081.[Medline]
Pan M-H, Lin-Shiau S-Y, and Lin J-K (2000) Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IkB kinase and NFkB activation in macrophages. Biochem Pharmacol 60: 1665-1676.[CrossRef][Medline]
Prestera T, Holtzclaw WD, Zhang Y, and Talalay P (1993) Chemical and molecular regulation of enzymes that detoxify carcinogens. Proc Natl Acad Sci USA 90: 2965-2969.[Abstract/Free Full Text]
Prival MJ, Sheldon AT Jr, and Popkin D (1982) Evaluation, using Salmonella typhimurium, of the mutagenicity of seven chemicals found in cosmetics. Food Chem Toxicol 20: 427-432.[CrossRef][Medline]
Sabzevari O, Galati G, Moridani MY, Siraki A, and O'Brien PJ (2004) Molecular cytotoxic mechanisms of anticancer hydroxychalcones. Chem-Biol Interact 148: 57-67.[Medline]
Sanoh S, Kitamura S, Sugihara K, and Ohta S (2002) Cytochrome P450 1A1/2 mediated metabolism of trans-stilbene in rats and humans. Biol Pharm Bull 25: 397-400.[Medline]
Sauer J-M, Bao J, Smith RL, Kuester RK, Mayersohn M, and Sipes IG (1997a) Absorption, disposition and metabolism of trans-methyl styryl ketone in female B6C3F mice. Drug Metab Dispos 25: 1184-1190.[Abstract/Free Full Text]
Sauer JM, Smith RL, Bao J, Kattnig MJ, Kuester RK, McClure TD, Mayersohn M, and Sipes IG (1997b) Oral and topical absorption, disposition kinetics and the metabolic fate of trans-methyl styryl ketone in the male Fischer 344 rat. Drug Metab Dispos 25: 732-739.[Abstract/Free Full Text]
Schröder G, Brown JWS, and Schröder J (1988) Molecular analysis of resveratrol synthase. cDNA, genomic clones and relationship with chalcone synthase. Eur J Biochem 172: 101-109.[Medline]
Schröder G and Schröder J (1990) Stilbene and chalcone synthases: related enzymes with key functions in plant-specific pathways. Z Naturforsch Sect C Biosci 45: 1-8.
Stresser DM and Kupfer D (1998) Human cytochrome P450-catalyzed conversion of the proestrogenic pesticide methoxychlor into an estrogen. Role of CYP2C19 and CYP1A2 in O-demethylation. Drug Metab Dispos 26: 868-874.[Abstract/Free Full Text]
Sugihara K, Kitamura S, Sanoh S, Ohta S, Fujimoto N, Maruyama S, and Ito A (2000) Metabolic activation of the proestrogens trans-stilbene and trans-stilbene oxide by rat liver microsomes. Toxicol Appl Pharmacol 167: 46-54.[CrossRef][Medline]
Ueng Y, Shimada T, Yamazaki H, and Guengerich FP (1995) Oxidation of aflatoxin B1 by bacterial recombinant human cytochrome P450 enzymes. Chem Res Toxicol 8: 218-225.[CrossRef][Medline]
Wadleigh RW and Yu SJ (1987) Glutathione transferase activity of fall armyworm larvae toward ,ß-unsaturated carbonyl allelochemicals and its induction by allelochemicals. Insect Biochem 17: 759-764.[CrossRef]
Wikberg T, Vuorela A, Ottoila P, and Taskinen J (1993) Identification of major metabolites of the catechol-O-methyltransferase inhibitor entacapone in rats and humans. Drug Metab Dispos 21: 81-92.[Abstract]
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Abstract
Materials and Methods
