Galloylated Catechins as Potent Inhibitors of Hypochlorous Acid

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Chem. Res. Toxicol. 2008, 21, 1407–1414

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Galloylated Catechins as Potent Inhibitors of Hypochlorous Acid-induced DNA Damage Yoshichika Kawai,*,†,‡ Yuri Matsui,† Hajime Kondo,† Hiroshi Morinaga,† Koji Uchida,† Noriyuki Miyoshi,†,§ Yoshimasa Nakamura,†,| and Toshihiko Osawa† Laboratory of Food Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya UniVersity, Nagoya 464-8601, Japan, Department of Food Science, Graduate School of Nutrition and Biosciences, The UniVersity of Tokushima, Tokushima 770-8503, Japan, Department of Food and Nutritional Sciences, Graduate School of Nutritional and EnVironmental Sciences, UniVersity of Shizuoka, Shizuoka 422-8526, Japan, and Department of Biofunctional Chemistry, DiVision of Bioscience, Graduate School of Natural Science and Technology, Okayama UniVersity, Okayama 700-8530, Japan ReceiVed February 21, 2008

Hypochlorous acid (HOCl), a strong oxidant derived from myeloperoxidase in neutrophils and macrophages, can chlorinate DNA bases at the site of inflammation. Because little is known about the protective role of natural antioxidants, such as polyphenols, for the myeloperoxidase-derived DNA damage, we screened the inhibitory effects of various phenolic antioxidants on the chlorination of the 2′-deoxycytidine residue by HOCl in vitro and found that green tea catechins, especially (-)-epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg), significantly inhibited the chlorination. These catechins also reduced nucleoside- and taurine-chloramines, which can induce secondary oxidative damage, into their native forms. Mass spectrometric and nuclear magnetic resonance analyses showed that ECg and EGCg can effectively scavenge HOCl and/or chloramine species resulting in the formation of monoand dichlorinated ECg and EGCg. Using the HL-60 human leukemia cell line, it was found that ECg could efficiently accumulate in the cells. Immunocytometric analyses using antihalogenated 2′deoxycytidine antibody showed that pretreatment of cells with ECg inhibited the HOCl-induced immunofluorescence. In addition, the chlorinated ECg derivatives were detected in the HOCl-treated HL-60 cells. These results showed that green tea catechins, especially 3-galloylated catechins, may be the plausible candidate for the prevention of inflammation-derived DNA damage and perhaps carcinogenesis. Introduction Chronic inflammation increases the risk of cancer, raising the possibility that reactive intermediates generated by neutrophils, eosinophils, monocytes, and macrophages might damage nucleic acids and compromise the integrity of the genome (1–3). It has been suggested that reactive intermediates generated by phagocytes might damage nucleic acids in cells at the site of inflammation. In vitro studies have demonstrated that various modified nucleic acids and/or DNA strand breaks were formed during the reaction with reactive oxygen/nitrogen species (4–7). The heme enzyme myeloperoxidase (MPO),1 synthesized and secreted by neutrophils and monocytic/macrophage cells, is an important endogenous source of oxidants. It uses H2O2 generated by the phagocyte NADPH oxidase and chloride ion to produce * To whom correspondence should be addressed. Tel: 81-88-633-9592. Fax: 81-88-633-7089. E-mail: [email protected]. † Nagoya University. ‡ The University of Tokushima. § University of Shizuoka. | Okayama University. 1 Abbreviations: MPO, myeloperoxidase; 5-CldC, 5-chloro-2′-deoxycytidine; N-CldC, N4-chloro-2′-deoxycytidine; EC, (-)-epicatechin; EGC, (-)epigallocatechin; ECg, (-)-epicatechin gallate; EGCg, (-)-epigallocatechin gallate; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; Cl2dC, N4,5-dichloro-2′-deoxycytidine; N-CldT, N3chloro-thymidine; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; LC-MS, liquid chromatography-mass spectrometry; NMR, nucler magnetic resonance; mAb, monoclonal antibody; HMBC, heteronuclear multiple-bond connectivity.

the potent cytotoxin, hypochlorous acid (HOCl). HOCl can chlorinate/oxidize pyrimidines and purines. Henderson et al. (8) reported that the HOCl-derived Cl2 chlorinates 2′-deoxycytidine (dC) to form 5-chloro-2′-deoxycytidine (5-CldC) as the major product. Alternatively, in contrast to these stable carbonchlorinated products, the formation of semistable or unstable nitrogen-chlorinated products, that is, chloramines, was also observed. Unstable nucleoside chloramines, such as thymidine chloramine, are suggested to initiate DNA single and double strand breaks via nitrogen-centered radicals and to transfer their chlorine atoms to other nucleosides (9, 10). We have demonstrated that both the carbon- and the nitrogen-chlorination of dC residues could occur in tissues at the site of inflammation (11). Epidemiological studies have shown that the intake of polyphenols, such as flavonoids, potentially decreases in the risk of coronary heart diseases and cancer (12, 13). The beneficial health effects of natural compounds, especially phenolic antioxidants, on their antioxidative/anti-inflammatory activities during the development of several chronic diseases have been discussed. Although little attention has been paid the activity of natural products for scavenging MPO-derived oxidants such as HOCl, it has been reported that isoflavones can scavenge HOCl generated from neutrophils (14, 15). The reaction of quercetin, a major flavonoid in the human diet, with HOCl has been reported (16). These observations raise the possibility that phenolic antioxidants may prevent MPO-derived DNA damage in vivo. In this study, we screened various phenolic compounds

10.1021/tx800069e CCC: $40.75  2008 American Chemical Society Published on Web 05/31/2008

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for their scavenging activity for chlorinating intermediates including HOCl and chloramines and found that tea catechins, especially galloylated catechins, might be the good inhibitor of DNA base chlorination. We now demonstrate the chemical reaction mechanisms of the potent inhibitors on HOCl-induced DNA chlorination.

Experimental Procedures Materials. dC, sodium hypochlorite, (-)-epicatechin (EC), (-)epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), quercetin, ascorbic acid, R-tocopherol, mannitol, and methionine were purchased from Wako Pure Chemicals (Osaka, Japan). Ferulic acid and calf thymus DNA were purchased from the Sigma-Aldrich Co. (St. Louis, MO). Caffeic acid and chlorogenic acid were pbtained from Nakarai Tesuque, Inc. (Kyoto, Japan). Sesaminol was kindly provided from Takemoto Oil & Fat Co., Ltd. (Aichi, Japan). Cell Culture. HL-60 cells were obtained from the American Type Culture Collection and were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum, 100 µg/mL penicillin, and 100 units/mL streptomycin in a 5% CO2-containing atmosphere. Inhibition Assay for the Formation of Chlorinated dC Residues. For the N4-chloro-dC (N-CldC) formation assay, 2 mM dC was mixed with different concentrations of antioxidants (stock solution, H2O or methanol) and then incubated with 1 mM HOCl in phosphate buffer (pH 7.4) at 37 °C for 1 h. The concentrations of HOCl were spectrophotometrically determined using 292 ) 350 M-1 cm-1 (17). After incubation, the reaction mixtures were analyzed by reverse phase high-performance liquid chromatography (HPLC) using a Develosil ODS HG-5 column (4.6 mm × 250 mm, Nomura Chemicals, Aichi, Japan) with a linear gradient elution from 0.05% acetic acid to acetonitrile containing 0.05% acetic acid in 30 min with UV (260 nm) detection (condition I). For the N-CldC reduction assay, the N-CldC was semipurified from the reaction mixture of dC (40 mM) and HOCl (40 mM) by HPLC using a Develosil ODS HG-5 column (20 mm × 250 mm) equilibrated with 20% aqueous methanol containing 0.05% acetic acid at the flow rate of 6 mL/min with UV (270 nm) detection. The concentration of the purified N-CldC was determined by the reaction with 5-thio-2-nitrobenzoic acid as previously reported (9). The N-CldC (0.5 mM) was mixed with different concentrations of catechins in phosphate buffer (pH 7.4) at 37 °C for 1 h and then analyzed by HPLC as described above (condition I). For the 5-CldC formation assay, calf thymus DNA (1 mg/ml) was mixed with different concentrations of antioxidants (stock solution, H2O, or methanol) and then incubated with 1 mM HOCl in phosphate buffer (pH 7.4) at 37 °C for 1 h. After incubation, the reaction was terminated by adding 4 mM methionine. The enzymatic digestion of the DNA with DNase I, nuclease P1, alkaline phosphatase, and phosphodiesterase I/II was carried out as previously reported (18). After digestion, the reaction mixtures were filtered through an Ultrafree-MC membrane (nominal molecular weight limit 5000; Millipore) by centrifugation (10000 rpm, HIMAC centrifuge, Hitachi, Japan) to remove the enzymes; 10 µL of the filtrates was injected for the HPLC-tandem mass spectrometry (MS/MS) as described below. The amount of the nonmodified dC residues was determined by an HPLC-UV (260 nm) analysis. Chromatography was carried out using a Develosil ODS-HG-5 column (4.6 mm × 250 mm) with a gradient elution using solvent A (40 mM ammonium formate) and solvent B (acetonitrile containing 0.05% acetic acid): 0-5 (5% B), 5-20 (5-35% B), 20-25 (35-100% B), and 25-35 min (100% B); flow rate, 0.8 mL/min. For the N4,5-dichloro-dC (Cl2dC) assay by enzyme-linked immunosorbent assay (ELISA), calf thymus DNA (0.5 mg/ml) was mixed with different concentrations of antioxidants (stock solution, H2O, or methanol) and then incubated with 0.2 mM HOCl in phosphate buffer (pH 7.4) at 37 °C for 1 h. After termination of

Kawai et al. the reaction with 4 mM methionine, 0.5 µg of the DNA in phosphate-buffered saline (PBS) was coated onto an immunoplate at 4 °C overnight. The ELISA procedure using mAb2D3 was previously reported (11). Analysis of Chlorinated Catechin Derivatives. The chlorination products of the catechins formed during the reaction with HOCl or chloramine species were analyzed as follows. Each catechin (1 mM) was incubated with 0.5 mM HOCl or each chloramine in phosphate buffer (pH 7.4) at 37 °C for 1 h and then analyzed by HPLC (condition I) with UV (280 nm) detection. The chloramine of thymidine (N3-chloro-thymidine, N-CldT) was prepared by the reaction of 10 mM thymidine (dT) with 2 mM HOCl at 4 °C for 5 min. The reaction solution used 2 mM N-CldT, because HOCl can stoichiometrically generate chloramines on dT (9). The taurine chloramine (SO3H-CH2-CH2-NHCl) was prepared by incubating 100 mM taurine with 10 mM HOCl at 4 °C for 5 min. The concentration of the taurine chloramine in the solution was spectrophotometrically determined using 252 ) 415 M-1 cm-1. Thereactionproductswerealsoanalyzedbyanliquidchromatographymass spectrometry (LC-MS) (Micromass VG Platform II, Micromass, Manchester, United Kingdom) in the electrospray ionizationnegative mode using an HPLC gradient elution (condition I). For the structural identification of the chlorinated ECg and EGCg, the products formed in the reaction with N-CldC were purified by HPLC. Chromatography was carried out using a Develosil C30UG-5 column (8 mm × 250 mm) equilibrated with 22 (for ECg) or 15% (for EGCg) acetonitrile containing 0.05% acetic acid with UV (280 nm) detection at the flow rate of 2 mL/min. The products were analyzed by nuclear magnetic resonance (NMR) using a Bruker AMX400 (400 MHz; Bruker, Karlsruhe, Germany). Analysis of ECg and Chlorinated ECg in HL-60 Cells. The accumulation of ECg in HL-60 cells was determined as follows. Cells (8 × 106 cells) were treated with different concentrations of ECg dissolved in the medium for 0-24 h. After incubation, the cells were washed three times with PBS and then lysed in 200 µL of 10 mM Tris-HCl (pH 7.4) containing 10 mM ethylene diamine tetraacetic acid and 0.5% (v/v) Triton X-100 at 4 °C for 20 min. After centrifugation for 10 min (10000 rpm, HIMAC centrifuge, Hitachi, Japan), the supernatants were mixed with an equal volume of CH2Cl2 and then centrifuged again. The supernatants were filtered through an Ultrafree-MC membrane (nominal molecular weight limit 5000; Millipore) by centrifugation; 10 µL of the filtrates was injected for the HPLC-MS/MS as described below. The accumulation of the chlorinated ECg in HL-60 cells was determined as follows. Cells (8 × 106 cells) were treated with 1 mM ECg for 2 h. After incubation, the cells were washed three times with PBS and then treated with 1 mM HOCl for 15 min. After the addition of 4 mM methionine to terminate the reaction, the cells were lysed as described above, and the chlorinated ECg derivatives were analyzed by HPLC-MS/MS. HPLC-MS/MS Analysis. The HPLC-MS/MS analyses were carried out by an API 2000 triple quadrupole mass spectrometer (Applied Biosystems) through a TurboIonSpray source with an Agilent 1100 HPLC system. For analysis of the 5-CldC in the enzymatic hydrolysates of the DNA, chromatography was carried out using a Develosil ODS-HG-3 column (2.0 mm × 100 mm) with a gradient elution as follows: 0-15 min, 0.5% aqueous acetonitrile containing 0.01% formic acid; 15-20 min, linear gradient from 0.5% aqueous acetonitrile containing 0.01% formic acid to 20% aqueous acetonitrile containing 0.01% formic acid; and 20-20.1 min, linear gradient to 0.5% aqueous acetonitrile containing 0.01% formic acid; flow rate, 0.2 mL/min. The instrument response was optimized by infusion experiments of the standard compounds using a syringe pump at the flow rate of 5 µL/min. Halogenated nucleosides were detected using electrospray ionization MS/MS in the multiple reaction monitoring mode. Specific transitions used to detect products in the positive ionization mode were those between the molecular cation of the products and the characteristic daughter ion formed from the loss of the 2′-deoxyribose moiety.

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For analysis of ECg and the chlorinated derivatives, chromatography was carried out using a Develosil ODS-HG-3 column (2.0 mm × 100 mm) with a gradient elution from 0.1% formic acid to acetonitrile containing 0.1% formic acid in 30 (for ECg) or 20 min (for chlorinated ECgs) at a flow rate of 0.2 mL/min in the negative ionization mode. The specific fragmentations at m/z 440.9/168.7 (ECg), 474.9/168.7 (monochloro-ECg), and 508.9/356.8 (dichlorinated ECg) were used for the detection. Immunocytometric Analysis of HOCl-Modified DNA. The HL-60 cells in PBS were treated with HOCl at 37 °C for 15 min. Residual HOCl was inactivated by adding 4 mM methionine. The cells were washed three times with PBS and then fixed in phosphate buffer (pH 7.4) containing 4% paraformaldehyde at 4 °C for 1 h. To prevent nonspecific antibody binding, the cells were blocked for 1 h at room temperature with 2% BSA in TPBS. Membranes were permeabilized by exposing the fixed cells to PBS containing 0.3% Triton X-100. The cells were then incubated in the primary antibody (mAb2D3) for 1 h at room temperature. The cells were then incubated for 1 h in the presence of fluorescein isothiocyanatelabeled antimouse IgG (Dako Japan Co., Ltd., Kyoto, Japan) and rinsed with PBS containing 0.3% Triton X-100. The fluorescence intensity of cells was analyzed by a flow cytometer (Epics XL, Beckman Coulter).

Results Screening of Natural Antioxidants for the Inhibitory Effect on HOCl-Induced DNA Damage. It has been reported that HOCl can chlorinate DNA bases in vitro and in vivo, resulting in the formation of N- an/or C-chlorinated nucleobases (8–11, 19). The possible pathways for the formation of the chlorinated dC residues are illustrated in Figure 1A. The N4and/or C5-positions of dC could be chlorinated during the reaction with HOCl or molecular chlorine (Cl2), resulting in the formation of three different chlorinated dC adducts (8, 11, 20). HPLC analysis showed that the reaction of HOCl and dC resulted in the formation of a major product (Figure 1B, upper). The product exhibited the characteristic of chloramines, which can be reduced by methionine (Figure 1B, lower). Monochlorination of the product was confirmed by LC-MS characterization showing the molecular ion at m/z 262 with a deoxyribose fragmentation ion at m/z 146 (Figure 1C). It has been reported that the formation of another chlorinated dC, 5-CldC, requires acidic pH conditions (8). In addition, 5-CldC could not be reduced by methionine (8, 11). Furthermore, 1H NMR measurements showed that a signal for C-5 proton could be detected in the HOCl-treated dC (Supporting Information, Figure S1). On the basis of the observations, the product was finally identified to be N4-chloro-dC (N-CldC). Using this N-CldC generation system, we then screened the activities of natural phenolic antioxidants for scavenging and/ or reducing chlorinating intermediates in vitro. Methionine and mannitol were used as the control compounds for scavenging HOCl and hydroxyl radical, respectively. As shown in Figure 1D, among 11 phenolic compounds, flavonoids including tea catechins and quercetin exhibited a stronger inhibitory effect at a concentration of 100 µM. Especially, the inhibitory effect of two 3-galloylated catechins, ECg and EGCg, was quite significant (≈80% inhibition). We then focused on the four tea catechin molecules (see Figure 2A), especially ECg and EGCg, for their scavenging actions of chlorinating intermediates. Similar inhibitory effects were also obtained in the reaction system of calf thymus DNA and HOCl. It has been reported that 5-CldC is a major stable chlorinated nucleoside in HOCl-exposed DNA (21). The HPLCMS/MS analysis of 5-CldC in the enzymatic hydrolysates of DNA showed that the four catehicns inhibited the formation of

Figure 1. Inhibitory effects of various antioxidants on HOCl-induced dC chlorination. (A) Scheme for the chlorination of dC residues upon reaction with HOCl or molecular chlorine (Cl2). The chloramines can be reduced (red) in the presence of thiols or thioethers such as glutathione and methionine. (B) Upper, HPLC profile for the reaction mixture of HOCl (1 mM) and dC (2 mM) at 37 °C for 1 h; lower, HPLC profile for the reaction mixture after treatment with 4 mM methionine. (C) Electrospray ionization mass spectrometry (in positive mode) of the product obtained in panel B. Monochlorinated product, m/z 262 [M + H]+; a fragmentation ion, m/z 146 [M + H - dR]+; dR, deoxyrobose. (D) Inhibition assay of various antioxidants for N-CldC formation upon reaction of HOCl (1 mM) and dC (2 mM) in the presence of 100 µM each antioxidant. Methionine (gray bar) and mannitol were used as controls for scavenging HOCl and hydroxyl radical, respectively. Each point represents the mean of triplicate determinations.

5-CldC in DNA in a dose-dependent manner (Figure 2B). The inhibitory effect of ECg and EGCg was much more stronger than that of the nongalloylated catechins. We have previously reported the production of a monoclonal antibody (mAb2D3) to the HOCl-modified DNA and found that N4,5-dichlorinated dC (Cl2dC) is the major epitope (11). An ELISA experiment with mAb2D3 also showed that ECg and EGCg more efficiently inhibited the formation of the Cl2dC residues in DNA (Figure

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Figure 3. Reduction of chloramines by tea catechins. (A) Two possible pathways for the inhibitory actions of catechins during the dC chlorination reactions. (B) Reduction of N-CldC to the native dC form by catechins. The N-CldC (0.5 mM) was incubated with different concentrations of catechins at 37 °C for 1 h and then analyzed by HPLC-UV (280 nm) detection. Each point represents the mean of duplicate determinations.

Figure 2. Inhibitory effect of tea catechins on HOCl-induced dC chlorination in DNA. (A) Chemical structure of four major tea catechins. (B) Inhibitory effect of the catechins on the formation of 5-CldC in the reaction of calf thymus DNA (1 mg/mL) with HOCl (1 mM). The formation of 5-CldC was analyzed by HPLC-MS/MS. Each point represents the mean of duplicate determinations. (C) Inhibitory effect of the catechins on the formation of N4,5-Cl2dC in calf thymus DNA (0.5 mg/mL) during incubation with HOCl (0.2 mM). The formation of N4,5-Cl2dC was determined by ELISA using mAb2D3. Each point represents the mean of triplicate determinations.

2C). These results showed that tea catechins, especially ECg and EGCg, are efficient inhibitors for the formation of three types of chlorinated dC residues during the reaction with HOCl. Reaction Mechanism between Catechins and Chlorinating Intermediates. For inhibition of the chlorinated dC formation, two possible reactions were proposed as follows: (a) direct scavenging HOCl and (b) reduction of N-CldC to dC (Figure 3A). We first focused on the reduction activity of catechins for N-CldC. The reduction activity of catechins was determined by HPLC upon reaction of N-CldC and the catechins. As shown in Figure 3B, the catechins dose dependently reduced N-CldC

with the appearance of unmodified dC. The more significant reducing activity of the galloylated catechins (ECg and EGCg) than nongalloylated catechins (EC and EGC) was also observed, reflecting the inhibitory effect of these catechins shown in Figure 1. We confirmed that propyl gallate did not reduce N-CldC (Supporting Information, Figure S2), suggesting that the galloyl moiety itself could not be the scavenger for the chloramines. To examine the reaction mechanism for the reducing activity, the reaction of the catechins (ECg or EGCg) with chloramine compounds (N-CldC, N-CldT, or taurine chloramines) was performed and then, the products were analyzed by HPLC. As shown in Figure 4A, three major products were detected from the reaction of N-CldC with each catechin: peaks 1a-3a, with ECg; peaks 1b-3b, with EGCg. Similar HPLC profiles were obtained upon reaction with taurine chloramine. N-CldT generated 3a and 3b as the major products. Profiles similar to N-CldT were observed in the reaction with HOCl (data not shown). As shown in Figure 4B, these peaks time dependently increased during the reaction with N-CldC. Peaks 1 and 2 (1a, 2a, 1b, and 2b) significantly increased within 30 min, and peak 3 (3a and 3b) increased after 30 min, suggesting that peaks 3a and 3b might be the secondary products in this reaction. We could not detect similar oxidation products of EC and ECG in the reaction with HOCl or the chloramines (data not shown). To identify the products, the reaction mixtures were analyzed by LC-MS with selected ion monitoring in the negative ionization mode. Figure 5 showed that peaks 1 and 2 were detected at m/z 475 (1a and 2a, ECg + 35Cl) and m/z 491 (1b and 2b, EGCg + 35Cl), indicating the monochlorinated catechins, and peaks 3a,b were at m/z 509 (3a, ECg + 235Cl) and m/z 525 (3b, EGCg + 235Cl), indicating dichlorinated catechins. These chlorinated catechins were also detected from the reaction

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Figure 6. HMBC spectrum for peak 1a for identifying the chlorinated position. Upper, a magnified two-dimensional spectrum; lower, chemical structure of peak 1a (8-chloro-ECg). Arrows indicate the key correlations for identifying peak 1a. 37

Figure 4. Formation of chlorinated catechin derivatives upon reaction with chloramines. (A) HPLC profiles for the reaction mixtures of catechins (ECg or EGCg, 1 mM) and chloramines (N-CldC, N-CldT, or taurine chloramine, 0.5 mM) at 37 °C for 1 h. The products from ECg (left) and EGCg (right) are shown as 1a-3a and 1b-3b, respectively. (B) Time-dependent formation of the reaction products between catechins (1 mM) with N-CldC (0.5 mM). Each point represents the mean of duplicate determinations.

Figure 5. LC-MS analysis of the chlorinated derivatives of ECg and EGCg. The reaction mixtures shown in Figure 4 were analyzed by LCMS in the negative ionization mode with selected ion monitoring: (A) m/z 441 (ECg), m/z 475 (mono-Cl35 ECg), and m/z 509 (di-Cl35 ECg); (B) m/z 457 (EGCg), m/z 491 (mono-Cl35 EGCg), and m/z 525(diCl35 EGCg).

with HOCl (Supporting Information, Figure S3), which generated dichlorinated peaks as the major products. The isotopic peaks for 37Cl were also detected upon reaction with N-CldC at the same retention times: m/z 477 (1a and 2a, ECg + 37Cl) and m/z 493 (1b and 2b, EGCg + 37Cl), indicating the monochlorinated catechins, and m/z 511, 513 (3a, ECg + 35Cl/ 37 Cl, 37Cl/37Cl) and m/z 527, 529 (3b, EGCg + 35Cl/37Cl, 37Cl/

Cl), indicating the dichlorinated catehins (Supporting Information, Figure S4). The intensity of the peaks approximately satisfied the presumed isotopic ratio (monochloro, 35Cl:37Cl ) 3:1; dichloro, 35Cl/35Cl:35Cl/37Cl:37Cl/37Cl ) 9:6:1). The preferential formation of dichlorinated products upon reaction with N-CldT or HOCl may be due to their stronger reactivity. Monochlorinated catechins, if formed, could immediately be chlorinated by N-CldT or HOCl. To further identify the chemical structures, these six peaks (1a-3a and 1b-3b) were purified and then analyzed by NMR. The 1H NMR spectra (Supporting Information, Figure S5) showed that the 2H signal for H6 and H8 of ECg changed into the 1H signal in 1a and 2a and disappeared in 3a. No other significant changes were observed between the authentic ECg and 1a-3a. On the basis of the LC-MS and 1H NMR analyses, 1a and 2a were presumed to be the 6- or 8-chlorinated ECg, and 3a was the 6,8-dichlorinated ECg. To identify the chlorination positions of 1a and 2a, a heteronuclear multiple-bond connectivity (HMBC) experiment was performed for 1a. As shown in Figure 6, the singlet 1H signal at 6.12 ppm correlated with the C5 and C7 carbon signals but not with the C8a carbon, showing that the signal at 6.12 ppm was the H6 proton. We then identified 1a and 2a to be the 8-chlorinated ECg and 6-chlorinated ECg, respectively. The NMR assignment data for 1a-3a are summarized in Supporting Information Tables 1 and 2. Similar 1H NMR profiles indicating the chlorination of 6and/or 8-positions of EGCg were also observed for 1b-3b (Supporting Information, Table 3). The 2H signal for H6 and H8 of EGCg changed into the 1H signal in 1b and 2b and disappeared in 3b. Although we could not obtain the informative HMBC spectrum for identifying the chlorination positions of 1b-3b, HPLC (UV and MS) studies showed the similar reaction profiles between 1a-3a (for ECg) and 1b-3b (for EGCg). On the basis of the information, we presumed 1b and 2b to be the 8- and 6-chlorinated EGCg, respectively, and 3b to be 6,8dichlorinated EGCg. ECg Inhibits HOCl-Derived Cellular DNA Damage. To examine whether the galloylated catechins can inhibit the chlorination of DNA in living cells, the inhibitory effect of ECg on the chlorination of DNA was evaluated in HL-60 cells. First, we analyzed the cellular accumulation of ECg in HL-60 cells

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Figure 8. Formation of chlorinated ECg derivatives in the HL-60 cells treated with ECg and HOCl. (A) MS/MS fragmentation patterns of mono- (left) and di- (right) chlorinated ECg derivatives. (B) HPLCMS/MS analysis of mono- and dichlorinated ECg derivatives in HL60 cells. Cells were pretreated with 1 mM ECg for 2 h, washed, and then treated with 1 mM HOCl for 15 min. The ECg derivatives were extracted and then analyzed by HPLC-MS/MS. Figure 7. ECg inhibits HOCl-induced DNA chlorination in HL-60 cells. (A) Cellular accumulation of ECg in HL-60 cells (8 × 106 cells) treated with 0.2 mM ECg at 37 °C for 0-24 h analyzed by HPLC-MS/MS. Left, chromatographic profiles of authentic ECg (upper) and a cell sample (incubation for 2 h) at m/z 440.9 f 168.7. Right, a time-course experiment for the accumulation of ECg in HL-60 cells. Each point represents the mean of duplicate determinations. (B) Immunocytometric detection of chlorinated dC residues in HL-60 cells exposed to HOCl. Cells were treated with different concentrations of HOCl for 15 min, fixed, and then treated with mAb2D3 followed by treatment with FITClabeled secondary antibody. The fluorescence intensity of each cell was analyzed by a flow cytometer. (C) Inhibition of cellular DNA chlorination by ECg. Cells were pretreated with ECg for 2 h, washed, and then treated with HOCl (0.5 mM) for 15 min. The chlorinated DNA was analyzed by immunocytometry. The relative fluorescence intensities in B and C are shown as means (n ) 3) in panels D and E, respectively.

by HPLC-MS/MS (Figure 7A, left). An authentic ECg showing the typical MS/MS spectrum at m/z 440.9f168.7 was eluted at 16.2 min. Under this analytical condition, we detected ECg in the extracts of HL-60 cells treated with 200 µM ECg. A time-course experiment showed that the accumulation of ECg in the HL-60 cells reached a maximum at 2 h (Figure 7A, right). The evaluation of the DNA chlorination was performed by an immunocytometric analysis using the anti-Cl2dC mAb2D3 (11). The treatment of HL-60 cells with HOCl dose dependently increased the immunofluorescence with mAb2D3 and reached a plateau at 0.5 mM (Figure 7B). Pretreatment of different concentrations of ECg for 2 h dose dependently inhibited the DNA chlorination in HL-60 cells treated with 0.5 mM HOCl (Figure 7C). The relative fluorescence intensity values are shown in Figure 7D,E. Under this experimental condition, cytotoxicity of ECg was not observed at least within 250 µM (Supporting Information, Figure S6).

To examine whether ECg can inhibit DNA chlorination by scavenging HOCl in the cells, the chlorinated ECg derivatives in the ECg/HOCl-treated HL-60 cells were analyzed by HPLCMS/MS. The MS/MS fragmentation patterns for the mono- and dichlorinated ECg derivatives are shown in Figure 8A. The major fragmentations were observed at the 3-galloyl moiety: 8-chloro-ECg, m/z 474.9 f 124.9, 168.7, and 322.9; 6,8dichloro-ECg, m/z 508.8 f 168.8 and 357.0 (m/z 284.9 could not be identified). Authentic standards could be separated and detected by monitoring the specific MS/MS fragmented ions at m/z 474.89 f 168.7 (monochloro) and m/z 508.8 f 357.0 (dichloro) (Figure 8B, upper). We successfully detected the peaks corresponding to the authentic standards in the extracts of HL-60 cells treated with ECg and HOCl (Figure 8B, lower). These results showed that ECg indeed inhibited the DNA chlorination in living cells by scavenging HOCl and/or chlorinating species to form mono- and dichlorinated ECg derivatives.

Discussion In this study, we showed the inhibitory effects and the reaction mechanisms of tea catechins on the HOCl-induced chlorination of dC residues in DNA in vitro and in living cells. Among the four major tea catechins, the galloylated catechins, ECg and EGCg, exhibited much stronger inhibitory effects. The inhibitory mechanism was then suggested to be the direct scavenging reaction between the catechins and the HOCl (or chloramines) to generate the chlorinated catechin derivatives. We identified the precise chemical structures of the derivatives chlorinated at the 6- and/or 8-positions by NMR studies, suggesting that one molecule of the galloylated catechins efficiently scavenges one

Galloylated Catechins Inhibit HOCl-Induced DNA Damage

or two chlorine atom(s) of the chlorinating species. Although similar chlorination reactions at the 6- and/or 8-positions of flavonoids, isoflavones (daidzein and genistein), and quercetin have been reported (15, 16, 22), the inhibitory effect of the galloylated catechins on the DNA chlorination was much higher than quercetin (Figure 1). On the other hand, we could not detect the formation of similar chlorinated derivatives of the nongalloylated catechins, such as EC and EGC, under our experimental conditions, indicating that the susceptibility of the catechin compounds to the chlorination reaction closely reflects their inhibitory effects on the DNA chlorination. The current results showed that the 3-galloylation of catechins may enhance the reactivity of the 6- and/or 8-positions of the catechins with chlorinating species. It has been reported that the 3-galloylation of catechins enhances the radical scavenging activity (23). In addition, atomic orbital energy analyses suggest that the galloylated catechins are more susceptible to a nucleophilic attack than other catechins (24). Although a precise mechanism for the enhancement of the inhibitory effects by the 3-galloylation remains unclear, catechins with the 3-galloyl moiety could be the potential inhibitors for inflammation-derived DNA damage in vivo. We have demonstrated that tea catechins can not only directly scavenge HOCl but also reduce chloramines to the native form (Figure 3). The chloramines formed in proteins, nucleic acids, and lipids (aminophospholipids) could secondarily chlorinate/ oxidize neighbor molecules. Unstable chloramines are suggested to initiate an oxidation reaction via nitrogen-centered radicals and to transfer their chlorine atoms to other molecules (9, 10, 25). In living cells, it is generally thought that the modified/damaged nucleobases and proteins can rapidly be enzymatically repaired and/or removed from the cells. Our current results suggest an alternative nonenzymatic repair mechanism such that tea catechins could chemically repair the chlorinated nucleobases. The thiol and thioether compounds, such as reduced glutathione and methionine, were recognized as efficient endogenous scavengers of HOCl and chloramines (26, 27). At the site of inflammation, neutrophil infiltration and the following generation of reactive oxygen species including HOCl and chloramines reduce the glutathione levels in the target tissues (11). Thus, the catechins, especially the galloylated derivatives, could be the supplementary antioxidants derived from the daily human diet. In this study, we focused on four major catechin derivatives found in tea (28), which might be a potential source of galloylated catechins. Catechins can be absorbed and then found in plasma and tissues in humans; the average peak plasma catechin concentrations in healthy volunteers given a single dose of 1.5 mmol of ECg or EGCg were 3.1 and 1.3 µM, respectively (29). We performed several model reactions in vitro using relatively higher concentrations of catechins to clarify the precise reaction pathways. It has been reported that the galloylated catechins also act as a prooxidant that induces cellular DNA damage in the presence of transition metals (30). Although we have not determined the levels of DNA damage, such as 8-oxo2′-deoxyguanosine, in HL-60 cells exposed to ECg, the HOClinduced DNA chlorination in the cells was indeed inhibited by ECg at the concentrations without cytotoxicity (Figure 7 and Supporting Information, Figure S5). Although it remains unknown whether plasma levels of catechins could inhibit HOCl-induced DNA damage in vivo, recent studies have shown that polyphenols might accumulate in the inflammatory tissues (31, 32). In addition, a specific receptor for EGCg that triggers the signaling pathways has been identified (33, 34). These observations raise the possibility that dietary polyphenols

Chem. Res. Toxicol., Vol. 21, No. 7, 2008 1413

including catechins could effectively act as antioxidative and anti-inflammatory agents in tissues. Further studies for tissue/ cellular localization of tea catechins might clarify in the future whether lower concentrations of tea catechins indeed can act as antioxidative/inflammatory agents in vivo. Although numerous studies of tea catechins on the beneficial effects (including the prevention of cardiovascular diseases and cancers) in vivo have been reported (35, 36), our current results also provide one mechanism for the beneficial health effect of tea catechins, especially inflammation-derived DNA damage. Acknowledgment. We thank Harue Kumon (Nagoya University) for her technical support. This study was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science Research Fellow (to Y.K.). Supporting Information Available: 1H NMR spectrum for N-CldC (Figure S1), the reactivity of N-CldC and galloyl moiety (Figure S2), LC-MS profiles of chlorinated ECg/EGCg (Figures S3 and S4), 1H NMR spectra for chlorinated ECgs (Figure S5), cytotoxicity of ECg in HL-60 cells (Figure S6), and NMR assignment tables for chlorinated ECg/EGCg (Tables S1-S3). This material is available free of charge via the Internet at http:// pubs.acs.org.

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