Identification of Potential Aryl Hydrocarbon Receptor Antagonists in

Yao-Ching Hung, Guewha Steven Huang, Vasyl M. Sava, Svetlana Y. Makan, Meng-Yen Hong. Camellia sinensis tea melanin suppresses transformation of the ...
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Chem. Res. Toxicol. 2003, 16, 865-872

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Identification of Potential Aryl Hydrocarbon Receptor Antagonists in Green Tea C. M. Palermo,† J. I. Martin Hernando,‡ S. D. Dertinger,† A. S. Kende,‡ and T. A. Gasiewicz*,† Department of Environmental Medicine, University of Rochester, Rochester, New York 14642, and Department of Chemistry, University of Rochester, Rochester, New York 14627 Received December 5, 2002

Previous investigations have implicated green tea to exert chemopreventive effects in animal models of chemical carcinogenesis, including polycyclic aryl hydrocarbon-induced cancers. In an effort to understand the compound(s) responsible for this protection, the effects of green tea extracts (GTE) and individual green tea catechins on aryl hydrocarbon receptor (AhR) gene induction were determined. Green tea (GT) was organically extracted and subsequently fractionated by column chromatography. The chemical composition of each fraction was determined by NMR. Several fractions inhibited tetrachlorodibenzo-p-dioxin-induced transcription of a dioxin responsive element-dependent luciferase reporter in stably transfected mouse hepatoma cells in a concentration-dependent manner. To determine the GT component(s) responsible for the observed effects, individual catechins were tested in the luciferase reporter system at concentrations found within the active fractions. Of the catechins tested, epigallocatechingallate (EGCG) and epigallocatechin (EGC) were the most potent antagonists, with IC50 values of 60 and 100 µM, respectively. Re-creation of the active fractions using commercially available catechins further confirmed the identification of EGCG and EGC as the active AhR antagonists in green tea. These data suggest that EGCG and EGC are capable of altering AhR transcription and are responsible for most, if not all, of the AhR antagonist activity of GTE.

Introduction The possibility that green tea (GT) may afford protection against cancer and other diseases has recently become an increasing health interest. Although epidemiological studies have so far yielded no clear association between GT consumption and human cancer risk, a number of studies in mammalian cell systems and laboratory animals have provided evidence indicating that the compounds present in GT are capable of affording protection against cancer initiation and its subsequent development (reviewed in ref 1). Unfortunately, the active constituents and the mechanisms responsible for these effects are not fully understood. This information is necessary to better understand the possible function of GT as a chemopreventive agent. GT is produced through a process of steaming, rolling, and drying of fresh leaves of the plant Camellia sinensis. Because of the inactivation of oxidizing enzymes during the steaming process, the chemical composition of GT resembles that of its leaf of origin (2). However, the composition of the leaf depends on a variety of factors including climate, season, horticulture practices, and the age of the plant (2). GT contains polyphenolic compounds, which include flavonols, flavondiols, flavonoids, and phenolic acids. Most of the polyphenols in GT are flavonols, better know as catechins. Catechins are the most predominant group of substances in GT accounting * Author to whom correspondence should be addressed. E-mail: [email protected]. † Department of Environmental Medicine, University of Rochester. ‡ Department of Chemistry, University of Rochester.

for 16-30% of the dry weight (2, 3). The major catechins in GT are (-)-epigallocatechin-3-gallate (EGCG), (-)epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epicatechin (EC) (Figure 1). EGCG is the most predominant catechin accounting for 50-80% of the total catechin in tea (1). On the basis of recent studies, it is now believed that EGCG is responsible for much of the biological activity mediated by GT (1, 4). Previous investigations have implicated GT as providing protection against polycyclic aromatic hydrocarbon (PAH)-induced cancers in animal models (5, 6). Further studies have demonstrated that GT can reduce the mutagenicity of the PAHs benzo[a]pyrene (B[a]P) (7, 8) and 7,12-dimethylbenz[a]anthracene (8). Mechanistic studies have shown that this protective effect of GT may be a result of scavenging of reactive molecular species of carcinogenic metabolites to prevent their reaching the critical target sites (8, 9), moderate enhancement of phase II detoxifying enzymes (10), and/or direct inhibition of phase I enzyme activity (8, 11, 12), which is required for the conversion of many procarcinogens to their highly reactive forms. Recently, a fourth mechanism of action has been suggested in which GT may cause a decrease in the protein levels of these phase I activating enzymes, in particular P450 (CYP1A1), through a direct interaction with the aryl hydrocarbon receptor (AhR) pathway (13). The AhR is a ligand-activated transcription factor responsible for mediating the effects of PAHs and halogenated aromatic hydrocarbons (HAHs) such as 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). In this pathway, TCDD binding to the cytosolic AhR initiates its translocation into the nucleus. Once in the nucleus, the ligand-

10.1021/tx025672c CCC: $25.00 © 2003 American Chemical Society Published on Web 06/12/2003

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Figure 1. Structures of the compounds found in Lipton GTE.

bound AhR dimerizes with the AhR nuclear translocator protein (ARNT) forming a heterodimer capable of recognizing consensus sequences termed dioxin-response elements (DREs), located in the promoter region of CYP1A1 and other responsive genes, thereby activating transcription (14, 15). A previous study demonstrated that green tea extract (GTE) and EGCG in particular act as AhR antagonists, decreasing CYP1A1 transcription in a concentration-dependent manner (13). Although the data strongly suggest that EGCG is the only effective AhR antagonist present in GTE, these studies failed to thoroughly examine the composition of GTE for other potential antagonists, nor did they examine a concentration range of individual catechins relevant to the concentrations found in GTE. To understand the possible role of tea consumption on cancer risk in humans, the active constituents and the molecular mechanisms responsible for these observations need to be clarified. The current paper specifically examines the interaction of GT with the AhR signal transduction pathway as a potential chemopreventive mechanism. Fractionation techniques and NMR analysis combined with a high-throughput reporter gene assay have allowed for the identification of specific compounds within GTE capable of inhibiting AhR-induced gene transcription. From these studies, the GT catechins EGCG and EGC have been identified as potential AhR antagonists.

Materials and Methods Chemicals. TCDD was purchased from Cambridge Isotope Laboratories (Cambridge, MA). The GTE Polyphenon 100, Trolox, B[a]P, and the individual GT catechins EGCG, EGC, ECG, EC, and GC were purchased from Sigma Chemical Company (St. Louis, MO). GT Extraction and Composition Analysis. Lipton GT (200 g) was added to 800 mL of water at 100 °C and brewed for 20-30 min with the heat turned off. After it was cooled on ice, GT was extracted three times with ethyl acetate. The combined organic layers were washed with brine and dried with Na2SO4. The solvents were removed under reduced pressure to provide a solid residue (14.8 g, GTE). Under these conditions, the weight of the extract was between 8 and 10% of the weight of the tea leaves. MS (APCI+) of this residue showed the presence of catechins and caffeine as major components (Figure 1). The residue (2 g) was chromatographed on silica gel using CHCl3/ MeOH/H2O (70:30:10, lower phase) as the eluent. TLC was

Figure 2. Extraction and fractionation of GT. Lipton GT was brewed for 30 min and organically extracted to obtain Lipton GTE. Lipton GTE was further fractionated using silica gel chromatography to obtain six GT fractions (see Table 1 for composition). carried out as a control using CHCl3/MeOH/H2O (65:35:10, lower phase) as the eluent and visualized with 1% Ce(SO4)2 in 10% H2SO4. Seven fractions, A-G, were originally isolated (Figure 2). Fractions E and F were combined and designated E in the biological tests (E and F were similar by TLC). The composition of each fraction was determined by MS and NMR (Table 1) (16, 17). Reconstitution of Fractions from Individual Chemicals. GT fractions were reconstituted from commercially available catechins. EC, GC, EGC, ECG, and EGCG were dissolved in acetone to obtain solutions of concentrations of 0.20 (EC, EGC, ECG, and EGCG) and 0.25 mg/mL (GC). The re-created fractions (RF) (RF1-RF3) were prepared by combining the necessary volumes of these solutions. RF1 was similar to fraction D with the exception of one compound, epigallocatechin-3methoxy-gallate (EGC(3MeO)G), which is not commercially available. RF2 is similar to E, and RF3 is similar to G. The composition of these fractions is summarized in Table 2. The subsequent 1H NMR analysis of these synthetic mixtures was similar to those of the corresponding fractions (compare Tables 1 and 2). RF1-RF3 were dried and solubolized in DMSO before treatment. Luciferase Reporter Gene Assay. Mouse hepatoma cells, Hepa-2DLuc (18), are a derivative of Hepa1c1c7 cells that have been stably transfected with the reporter plasmid p2Dluc (19),

Aryl Hydrocarbon Receptor Antagonists in Green Tea Table 1. Composition of Lipton GT Fractions Using NMR and MSa GT fraction caffeine A B

C

EG

GC

EGC EGC ECG EGCG (3 MeO)G

1.0 (100) 500

C

1.0 7.5 (11.8) (88.2) 40 300 1.5 1.0 9.0 7.0 (5.8) (4.1) (36.5) (41.1) 20 20 120 90 1.0 3.1 1.2 3.3 (9.1) (28.8) (16.1) (45.9) 30 94 36 100 1.0 2.0 1.0 12.5 (4.3) (8.6) (6.2) (80.8) 14 28 14 176

D E G

2.0 (12.5) 26

a Molar ratios (top numbers in each row) were determined from NMR spectra, and mg of each compound per 100 mg of dried extract (in parenthesis) were calculated. The maximal concentration (µM) of each compound in 100 µg/mL extract is listed as the bottom number in each row. Note that the NMR ratios are approximations of the composition of the fractions; the values in this table cannot be interpreted as exact values.

Table 2. Composition of RFsa RF1 (% in mixture) RF2 (% in mixture) RF3 (% in mixture)

EC

EGC

ECG

0.22 (6.4)

1.44 (41.6) 0.78 (28.8) 0.50 (8.6)

1.60 (46.2) 0.44 (16.2) 0.36 (6.2)

EGCG

GC

1.24 (45.8) 4.70 (80.9)

0.20 (5.8) 0.25 (9.2) 0.25 (4.3)

a EC, GC, EGC, ECG, and EGCG were dissolved in acetone to obtain solutions of concentrations of 0.20 (EC, EGC, ECG, and EGCG) and 0.25 mg/mL (GC). From these solutions, RF1-RF3 were produced. RF1-RF3 were dried and solubilized in DMSO before treatment.

containing two copies of the DRED consensus sequence (20) and a minimal promoter upstream of the firefly luciferase gene. Cells were maintained in modified Eagle’s medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum, sodium pyruvate, L-glutamine, sodium bicarbonate, and Gentamicin (MEM+) at 37 °C in a humid atmosphere with 5% CO2. Cytodex microcarrier beads (Sigma) were hydrated and autoclaved in PBS (Gibco, Grand Island, NY) at a dry weight of 3 mg/mL. For each experiment, beads were mixed to achieve a homogeneous suspension, and 10 mL was transferred to a 50 mL polypropylene tube. The beads were washed and resuspended in MEM+. Hepa-2DLuc cells were added to the bead suspension to achieve 7.5 × 105 cells per 30 mg beads per 10 mL MEM+. The cell/bead suspension was transferred to a 100 mm nontissue culture dish and incubated for 48 h before treatment. Nontissue culture-treated dishes were employed to promote attachment of the cells to the microcarrier beads. After 48 h of attachment and growth, the cell/bead suspension was transferred to a 50 mL polypropylene tube and the volume was doubled with MEM+. The cell/bead suspension was allowed to settle, and the supernatant was removed to a final volume of 5 mL (a bead density of 6 mg/mL). A series of polypropylene microcentrifuge tubes were prepared that contained 500 µL of MEM+ and a range of GTE or individual catechin concentrations (prepared in DMSO; final concentration, 0.2% v/v). DMSO was also added to 500 µL of MEM+ as a vehicle control. To these tubes, 500 µL aliquots of cell/bead suspension were added, halving the concentration of each test compound, and resulting in a final bead concentration of 3 mg/mL. Aliquots (100 µL) of these mixtures were transferred to wells of a 96 well CulturPlate (Packard, Meriden, CT), four replicate wells per treatment. The plate was incubated for 4 h at 37 °C after which 100 µL of Steady-Glo Luciferase reagent (Promega, Maidson, WI) was

Chem. Res. Toxicol., Vol. 16, No. 7, 2003 867 added to each well. The luminescence was read on the Packard Lumicount. AhR antagonist treatment was performed according to the above protocol with the following exceptions. A series of polypropylene microcentrifuge tubes were prepared containing 500 µL of either 300 pM TCDD or 50 nM B[a]P solution in MEM+ (final treatment concentrations of 150 pM and 25 nM, respectively). To these, antagonist was added in 1 µL aliquots from 1000× stocks to yield twice the final desired treatment concentrations. DMSO was added to MEM+ as a vehicle control. The cell/bead suspension (500 µL) was then added to the microcentrifuge tube halving the chemical concentrations and achieving a final bead density of 3 mg/mL. This method was found to give reproducible data in replicate experiments performed on separate days (data not shown). SDS-PAGE and Western Blot Analysis. Hepa-2DLuc cells were plated onto six well plates at a density of 5 × 105 cells/ well and incubated overnight at 37 °C in a humid atmosphere with 5% CO2. Cells were treated with either vehicle (DMSO), TCDD (150 pM), test compound alone, or test compound in the presence of TCDD (150 pM) for 4 h. Cells were lysed (0.2% Trition, 5 mM EDTA in PBS), and the total protein was quantified using the Coomassie Plus Protein Assay Reagent (Pierce, Rockford, IL). Protein (30 µg) was separated by SDSPAGE (7.5% acrylamide resolving gel) and transferred to PVDF membrane (Millipore, Bedford, MA). Membranes were probed with antibodies recognizing Luciferase (Sigma) and actin (Sigma). The secondary antibody was anti-rabbit IgG coupled to horseradish peroxidase (Jackson Immuno Research, West Grove, PA). Both primary and secondary antibodies were used at a dilution of 1:5000 in TBST (50 mM Tris, 300 mM NaCl, 0.5% Tween 20, pH 7.5) containing 5% milk. Proteins were visualized by chemiluminescence (KPL, Gaithersburg, MD). The luciferase protein used as a positive control in blotting was synthesized by the in vitro TNT Coupled Reticulocyte Lysate System (Promega) following the manufacturer’s recommended procedure. As determined by lactate dehydrogenase exculsion and Calcein-AM inclusion assays, EGCG and EGC were not cytotoxic to these cells after 24 h of treatment at the concentrations used in these studies (data not shown).

Results Effect of GT Fractions and Individual Tea Catechins on a DRE-Dependent Reporter Gene in Mouse Hepatoma Cells. To assess the ability of GTE to influence AhR-mediated transcription, the efficacy of GTE to inhibit TCDD-induced luciferase activity in stably transfected mouse hepatoma cells was determined. This reporter construct contains two copies of the DRED consensus sequence (20) and a minimal promoter upstream of the luciferase gene. In this system, GTE significantly inhibited TCDD-induced luciferase activity (Figure 3A), with an IC50 value of approximately 50 µg/ mL. Notably, this IC50 value was similar to that observed with Polyphenon 100, a commercially available mixture of polyphenolic compounds (Figure 3A). GTE was also capable of inhibiting luciferase induction by the less potent AhR agonist B[a]P (Figure 3B). These data support the hypothesis that GT contains AhR antagonists. To identify the GT component(s) responsible for the antagonist activity, GTE was further fractionated using column chromatography (Figure 2). Six fractions were isolated. Fraction A was eliminated based on the presence of only traces of an unidentifiable component. Fraction B was insoluble in DMSO and determined to contain caffeine (Table 1). Fractions E and F were combined due to similarities in composition and designated E in the biological tests. Fractions C-E and G were tested for the ability to induce AhR-mediated luciferase transcription

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Figure 3. Lipton GTE and polyphenol 100 inhibit AhR-induced luciferase activity in a dose-dependent manner. Hepa-2Dluc cells were treated with 150 pM TCDD (A) or 25 nM B[a]P (B) in the presence of increasing concentrations of GTE for 4 h (n ) 4). Values are presented as percent of observed luciferase induction in the presence of 150 pM TCDD alone ( SD. Representative data from one of three separate experiments are shown.

and to antagonize TCDD-induced luciferase activity. Fraction C demonstrated minimal agonist activity (Figure 4A), with no antagonist activity (Figure 4B). Fractions D, E, and G had significant antagonist activity (Figure 4B) with similar IC50 values (∼50 µg/mL). However, only fractions E and G were capable of inhibiting reporter gene transcription to nearly background levels, suggesting the presence of more potent antagonist(s) within these two fractions. Interestingly, these fractions appear to be slightly more potent inhibitors of TCDDinduced transcription than GT alone. This is believed to be due to the separation of the minor agonists in fraction C from the antagonists within these fractions. Fractions D, E, and G were not capable of inducing reporter gene activity alone in the reporter cells up to 100 µg/mL (Figure 4A). To identify the compound(s) responsible for the observed AhR antagonist activity, the composition of each of the GT fractions (A-G) was determined by NMR and MS (Table 1). From the NMR molar ratios, it was possible to calculate approximate concentrations of individual components within each 100 µg/mL GT fraction. These components could then be assessed individually for their ability to inhibit TCDD-induced luciferase induction at concentrations relevant to those found within the GT fraction. These data demonstrated that EGCG, EGC, ECG, and GC were able to block TCDD-induced luciferase activity (Figure 5), with a rank order potency of EGCG > GC > ECG > EGC. However, after considering the maximum concentration (see Table 1) of each catechin within any given 100 µg/mL GT fraction tested, it was concluded that GC and ECG were present at concentrations too low to contribute substantially to the antagonist activity of the individual fractions. Conversely, EGCG

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Figure 4. GT fractions and individual tea catechins inhibit TCDD-induced luciferase activity in a dose-dependent manner. (A) Hepa-2Dluc cells were treated with increasing concentrations of the indicated GT fraction for 4 h (n ) 4). Values are presented as percent of observed luciferase induction in the presence of 1 nM TCDD alone ( SD. Note: some error bars are too small to see. (B) Hepa-2Dluc cells were treated with increasing concentrations of the indicated GT fractions in the presence of 150 pM TCDD for 4 h (n ) 4). Values are presented as percent of observed luciferase induction in the presence of 150 pM TCDD alone ( SD. Lipton GT fractions D, E, and G are shown with IC50 values of 50, 65, and 65 µg/mL, respectively. Fraction C has no antagonist activity at 50 µg/mL. Representative data from one of three separate experiments are shown.

Figure 5. Individual tea catechins inhibit TCDD-induced luciferase activity in a dose-dependent manner. Hepa-2Dluc cells were treated with 150 pM TCDD and increasing concentrations of individual catechin for 4 h (n ) 4). Values are presented as percent of observed luciferase induction in the presence of 150 pM TCDD alone ( SD. Representative data from one of three separate experiments are shown.

and EGC were present in the GT fractions at concentrations capable of eliciting antagonist activity. Therefore, EGCG and EGC were identified as the most significant potential AhR antagonists present in this particular preparation of GTE. Of these two, EGCG (at 200 µM) was capable of inhibiting TCDD-induced reporter gene transcription to background levels, suggesting this was the main catechin responsible for the AhR antagonist activity observed in the GT fractions. Neither EGCG nor EGC had any agonist activity in this assay up to 200 µM (data not shown). It is possible that a minor component within these GT fractions was responsible for the AhR antagonist activity.

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Figure 7. Antioxidant Trolox does not inhibit TCDD-induced luciferase activity. Hepa-2Dluc cells were treated with 150 pM TCDD and increasing concentrations of Trolox for 4 h (n ) 4). Values are presented as percent of observed luciferase induction in the presence of 150 pM TCDD alone ( SD. Representative data from one of two separate experiments are shown.

Figure 6. Antagonist activity of RFs in Hepa-2Dluc cells is comparable to the original counterparts. (A) Hepa-2Dluc cells were treated with increasing concentrations of the indicated RF for 4 h (n ) 4). Values are presented as percent of observed luciferase induction in the presence of 1 nM TCDD alone ( SD. Note: some error bars are too small to see. (B) Hepa-2Dluc cells were treated with increasing concentrations of the indicated RF in the presence of 150 pM TCDD for 4 h (n ) 4). Values are presented as percent of observed luciferase induction in the presence of 150 pM TCDD alone ( SD. Compare RF1 to fraction D, RF2 to fraction E, and RF3 to fraction G. Representative data from one of three separate experiments are shown.

The presence of a small amount of diasterioisomers of these catechins (e.g., GCG for EGCG) or other minor components in these fractions cannot be ruled out due to limitations in the detection methods. Furthermore, the compound EGC(3MeO)G was not commercially available and not tested in the above assays. If a minor component is capable of inhibiting TCDD-induced luciferase activity at these low concentrations, it would be important to identify. To address this issue, fractions were re-created from commercially available catechins to mimic the composition of the GT fractions with antagonist activity (fractions D, E, and G). The composition of these fractions is summarized in Table 2. If the active components have been appropriately identified, these RFs (RF1-RF3) should lack agonist activity and have comparable antagonist activity as those in Figure 4B. As expected, the RFs were not capable of inducing luciferase reporter gene induction when given alone (Figure 6B). Furthermore, the ability of the RFs to inhibit TCDD-induced luciferase activity was comparable to the antagonist activity of the initial fractions (compare Figure 6B to Figure 4B). RF2 and RF3 (corresponding to fractions E and G, respectively) were able to inhibit luciferase activity almost to background levels. RF1 was capable of inhibiting luciferase activity to a similar degree as fraction D further suggesting that EGC(3MeO)G is not a potent AhR antagonist. These experiments support the above data indicating that EGCG and EGC have most, if not all, of the AhR antagonist activity. Antioxidant Effects on Luciferase Reporter Gene Activity. GT has been identified to have very strong antioxidant activity in both in vitro and in vivo systems (21) being more potent than both vitamin C and vitamin

E at scavenging free radicals (2, 4). Of the catechins in tea, EGCG is the most potent tea antioxidant exhibiting effects in cell culture models at low micromolar concentrations. The observation that EGCG is the most active compound in our system at relatively high micromolar concentrations raises the possibility that the inhibition of TCDD-induced luciferase activity could result from an antioxidant effect on cellular function or signal transduction pathways unrelated (or related) to the AhR. It was therefore important to address if other potent antioxidants could alter the experimental outcome. To address this question, Hepa-2Dluc cells were treated for 4 h with the antioxidant Trolox in the absence and presence of 150 pM TCDD (nonsaturating dose). Trolox, a water soluble form of vitamin E, is a very efficient radical scavenger and potent inhibitor of lipid peroxidation. Treatment with Trolox did not alter the ability of TCDD to induce reporter gene activity (Figure 7). Furthermore, Trolox alone had no effect on background luciferase expression (data not shown). From these studies, it was concluded that the antioxidant properties of these compounds are unlikely to be playing a role in the inhibition of TCDD-induced luciferase reporter gene activity. Inhibition of Luciferase Transcription. Although the above data strongly suggest that EGCG and EGC are AhR antagonists, it is possible that the observed decrease in luciferase response is not due to direct inhibition at the level of transcription but due to direct inhibition of enzyme activity itself or other indirect effects. Therefore, to further confirm that EGCG and EGC are functioning to inhibit TCDD-induced gene expression, changes in luciferase protein were examined by western blotting. After 4 h of treatment, both EGCG and EGC are capable of inhibiting TCDD-induced luciferase protein in a concentration-dependent manner (Figure 8). Furthermore, EGCG and EGC failed to directly inhibit in vitro translated luciferase enzyme activity further suggesting that these compounds are functioning as AhR antagonists (data not shown).

Discussion The chemopreventive properties of GT have been demonstrated in numerous animal models of chemical carcinogenesis. Multiple mechanisms have been proposed for this protection, and it is likely that GT is functioning through more than one pathway. To understand the most relevant mechanisms, the effective concentrations and

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Figure 8. EGCG and EGC inhibit TCDD-induced luciferase protein expression. Hepa-2DLuc cells were treated with DMSO (D), 150 pM TCDD (T), or 150 pM in the presence of the indicated catechin (µM) for 4 h. Proteins were separated on 7.5% SDS-PAGE and blotted for luciferase (top) and actin (bottom) as a loading control. Representative data from one of four separate experiments are shown.

active constituents must be carefully and systematically identified. Using a DRE-dependent reporter gene assay, we identified GTE to have AhR antagonist activity with no significant agonist activity in mouse hepatoma cells. Yet, because of the complexity of the GT mixture, it was not clear if this activity resulted from the action of one compound or multiple compounds, if this was a direct effect or an indirect effect, or if the antagonist response could be intensified by eliminating agonist effects of other compounds present in the mixture. This paper addresses these questions by identifying any and all potential AhR ligands present in GT. To our knowledge, this is one of the few papers to systematically fractionate GTE in an effort to thoroughly identify the constituents responsible for an observed biological effect. Column chromatography coupled with MS and NMR were utilized to fractionate and identify compounds in GT that affected AhR activity. This approach allowed for an assessment of all GT components as possible AhR ligands eliminating the possibility of overlooking a minor component that may be very important in eliciting the biological response of GTE. Through fractionation, ligands with different biological activities were separated. In doing so, minor agonist activity (∼20% of saturating TCDD in fraction C) was identified that was not observed in whole GTE. In addition, concentrations of individual components within each fraction could be approximated using 1H NMR molar ratios. This information was necessary to properly identify the individual compounds responsible for the activity of the mixture. Although our results suggest that EGCG and EGC are the main AhR antagonists in Lipton GTE, some AhR activity, albeit relatively weak, remains to be accounted for. For example, fraction C exhibited minor agonist activity (∼20% of saturating TCDD), which was not observed with whole GTE. Compositional analysis identified EC as the main constituent of this fraction. However, treatment of the reporter cells with pure EC alone (up to 100 µM) resulted in no induction of AhR activity (data not shown). These results suggest that a compound below our limit of detection is responsible for this minimal induction. It is important to note that a previous paper (13) examined the AhR binding activity of GTE in HepG2 cells with similar yet slightly contradictory findings to those reported here. In agreement with our findings, those studies demonstrated GTE to exhibit AhR antagonist activity, identifying EGCG as an active component. Contradictory to our findings, those studies reported weak agonist activity of GTE, no effect of EGC on AhR function, and more potent antagonist activity by GTE

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than EGCG alone. These different observations can be accounted for by many factors including variations in cell lines, promoter constructs, and treatment times. It is well-known that GT composition is highly variable. The GTE (100 µg/mL) used in the previous study was purchased from Sigma. The GTE (100 µg/mL) used here was extracted from Lipton GT. Therefore, it is highly possible that the different AhR agonist/antagonist activity of GTE observed between studies reflects the different compositions among the teas used. Analysis of the GTE utilized in the previous paper would help clarify these differences. In addition to compositional variation, different treatment concentrations must also be considered. In the previous study, the highest concentration of individual catechin used was 10 µM. However, in the present paper, much higher concentrations were used (up to 200 µM) based on the compositional analysis of the extract. It was at these higher concentrations (100 µM) that EGCG demonstrated more potent antagonist activity than GTE. Similarly, the previously published finding that EGC does not alter AhR activity was based on a 10 µM treatment. Again, our finding that EGC has AhR antagonist activity resulted from treatment at much higher concentrations (200 µM). Finally, the previous experiments were performed in human cell lines. Differences in the findings of the present study with mouse cells as compared with the studies performed in human cells may result from variations in ligand binding affinity of the GT compounds for the AhR. To assess this issue requires further investigation requiring direct measurements of the competitive binding behaviors of the GT compounds to the AhR. To date, binding assays in mouse cells have been difficult to perform most likely due to the low affinity of these compounds. However, preliminary data demonstrate that EGC may compete with TCDD for binding to the AhR, whereas EGCG cannot (data not shown). This suggests that these compounds may be functioning through very different and complex mechanisms. In addition to inhibiting phase I enzymes, GT has been implicated in providing protection against cancer through numerous other mechanisms including the modulation of signal transduction pathways that lead to inhibition of proliferation and transformation (22-26), induction of apoptosis of preneoplastic and neoplastic cells (27, 28), inhibition of tumor invasion and angiogenesis (29, 30), and increasing DNA repair mechanisms (31). However, to understand the physiological relevance of the proposed mechanism, the effective concentrations must be considered. Pharmacokinetic studies in humans suggest that the highest concentration of catechin attained in the plasma after consumption of ∼2 cups of tea is within the low micromolar range (0.1-1 µM) (32, 33). Furthermore, it is believed that most of this catechin is not in the free form but exists predominately as glucuronide and sulfate conjugates, resulting in even lower free plasma concentrations (33-35). A few mechanisms have been proposed to occur at such low concentrations including inhibition of protein kinase activities (36, 37) and telomerase inhibition (38). Here, we demonstrate antagonist activity of EGCG and EGC between 50 and 200 µM. On the basis of these observations, antagonizing AhR signal transduction may not be a physiologically relevant mechanism of chemoprotection. In addition, although our data demonstrate that GTE was capable of antagonizing the lower affinity AhR ligand B[a]P, fairly high concentrations were

Aryl Hydrocarbon Receptor Antagonists in Green Tea

still required to see an effect (Figure 1B). This further supports the conclusion that inhibition of AhR signal transduction by GTE requires high concentrations and is therefore unlikely to occur in vivo. However, before eliminating the AhR signal transduction pathway as a potential chemopreventive target of GT, other observations must be considered. It has been reported that many GT catechins including EGCG directly inhibit CYP1A1 enzyme activity (8, 11). Therefore, it is possible that the presence of multiple catechins may result in CYP1A1 inhibition through both direct enzyme inhibition and transcriptional inhibition, resulting in a synergistic effect through actions at multiple cellular targets. Furthermore, the findings reported here that multiple catechins function directly to inhibit AhR-mediated transcription suggest that exposure to multiple catechins could result in additive effects. Both synergistic or additive mechanisms of action suggest that catechins could be more effective at lower concentrations. In addition, it is possible that cellular concentrations of particular catechins may be higher than plasma concentrations and that the human AhR may be more sensitive to these catechins than mouse AhR. Furthermore, the body burden of TCDD is usually within the low picomolar range (15-30 pM), much less than the 150 pM used in these studies (39). The role of the AhR in mediating carcinogenesis is not clearly understood. Some mechanisms reported in the literature include effects on cell proliferation (40-43), NF-κB activity (44), Cox-2 gene expression (45, 46), Bax protein levels (47), and cross-talk with the estrogen receptor (48, 49). Furthermore, it is widely accepted that the AhR mediates the toxicity of numerous HAHs and PAHs, many of which, like TCDD, are known tumor promoters. In addition, the AhR is responsible for inducing transcription of the P450 family of enzymes, responsible for the bioactivation of numerous carcinogens (50). On the basis of these observations, antagonizing AhR signaling may serve as a potential chemopreventive mechanism by inhibiting the toxicity of dioxin and dioxinlike compounds and by reducing the carcinogenicity of certain promutagens. Previous data from this laboratory have identified synthetic flavonoids as potent AhR antagonists (18, 19, 51). These compounds bind to the TCDD ligand binding site on the AhR with high affinity (18). Further evaluation of their mechanism of action has revealed that binding of antagonist to the AhR inhibits the formation of a complex that can initiate receptor transformation and nuclear localization, bind DNA under cell free conditions, and activate transcriptional activity in whole cells. However, the structural basis and exact effects of antagonists on the AhR complex remain to be elucidated. Identification of EGCG and EGC as potential AhR antagonists provides us with more insight into the structure-activity relationships that govern AhR agonist vs antagonist responses. Future studies will focus on the exact mechanism by which these compounds alter AhR structure, function, and complex formation to ultimately inhibit AhR transcription of endogenous genes and help in the identification or even synthesis of more potent chemopreventive agents.

Acknowledgment. This work was supported by NIH Grant ES09702, Training Grant ES07026, Center Grant ES01247, and a grant from the American Institute for Cancer Research.

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References (1) Yang, C. S., Maliakal, P., and Meng, X. (2002) Inhibition of carcinogenesis by tea. Annu. Rev. Pharmacol. Toxicol. 42, 2554. (2) Graham, H. N. (1992) Green tea composition, consumption, and polyphenol chemistry. Prev. Med. 21, 334-350. (3) Yamanishi, T., Hara, Y., Luo, S., and Wickremasinghe, R. L. (1995) Special issue on tea. Food Rev. Int. 11, 371-546. (4) Dufresne, C. J., and Farnworth, E. R. (2001) A review of the latest research findings on the health promotion properties of tea. J. Nutr. Biochem. 12, 404-421. (5) Wang, Z. Y., Kahn, W. A., Bickers, D. R., and Mukhtar, H. (1989) Protection against polycyclic aromatic hydrocarbon-induced skin tumor initiation in mice by green tea polyphenols. Carcinogenesis 10, 411-415. (6) Hirose, M., Hoshiya, T., Akagi, K., Futakuchi, M., and Ito, N. (1994) Inhibition of mammary gland carcinogenisis by green tea catechins and other naturally occurring antioxidants in female Sprgue-Dawley rats pretreated with 7,12-dimethybenz[a]anthracene. Cancer Lett. 83, 149-156. (7) Jiang, T., Glickman, B. W., and de Boer, J. G. (2001) Protective effect of green tea against benzo[a]pyrene-induced mutations in the liver of Big Blue transgenic mice. Mutat. Res. 480-481, 147151. (8) Bu-Abbas, A., Clifford, M. N., Walker, R., and Ioannides, C. (1994) Marked antimutagenic potential of aqueous green tea extracts: Mechanism of action. Mutagenesis 9, 325-331. (9) Gordon, M. H. (1996) Dietary antioxidants in disease prevention. Nat. Prod. Rep. 13, 265-273. (10) Sohn, O. S., Surace, A., Fiala, E. S., Richie, J. P., Jr., Colosimo, S., Zang, E., and Weisburger, J. H. (1994) Effects of green and black tea on hepatic xenobiotic metabolizing systems in the male F344 rat. Xenobiotica 24, 119-127. (11) Wang, Z. Y., Das, M., Bickers, D. R., and Mukhtar, H. (1988) Interaction of epicatechins derived from green tea with rat hepatic cytochrome P-450. Drug Metab. Dispos. 16, 98-103. (12) Muto, S., Fujita, K. i., Yamazaki, Y., and Kamataki, T. (2001) Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450. Mutat. Res. 479, 197-206. (13) Williams, S. N., Shih, H., Guenette, K., Backney, W., Denison, M. S., Pickewell, G. V., and Quattrochi, L. C. (2000) Comparitive studies on the effects of green tea extracts and individual tea catechins on human CYP1A gene expression. Chem-Biol. Interact. 128, 211-229. (14) Schmidt, J. V., and Bradfield, C. A. (1996) AH receptor signaling pathway. Annu. Rev. Cell Dev. Biol. 12, 55-89. (15) Whitlock, J. P., Jr. (1993) Mechanistic aspects of dioxin action. Chem. Res. Toxicol. 6, 754-763. (16) Davis, A. L., Cai, Y., Davies, P. A., and Lewis, J. R. (1996) 1H and 13C NMR assignments of some green tea polyphenols. Magn. Reson. Chem. 34, 887-890. (17) Dalluge, J. J., and Nelson, B. C. (2000) Determination of tea catechins. J. Chromatogr. A 881, 411-424. (18) Henry, E. C., Kende, A. S., Rucci, G., Totleben, M. J., Willey, J. J., Dertinger, S. D., Pollenz, R. S., Jones, J. P., and Gasiewicz, T. A. (1999) Flavone antagonists bind competitively with 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor but inhibit nuclear uptake and transformation. Mol. Pharmacol. 55, 716-725. (19) Gasiewicz, T. A., Kende, A. S., Rucci, G., and Willey, J. J. (1996) Analysis of structural requirements for Ah receptor antagonist activity: Ellipticines, flavones and related compounds. Biochem. Pharmacol. 52, 1787-1803. (20) Lusska, A., Shen, E., and Whitlock, J. P., Jr. (1993) Protein-DNA interactions at a dioxin-responsive enhancer: Analysis of six bona fide DNA-binding sites for the liganded Ah receptor. J. Biol. Chem. 268, 8878-8884. (21) Wiseman, S. A., Balentine, D. A., and Frei, B. (1997) Antioxidants in Tea. Crit. Rev. Food Sci. Nutr. 37, 705-718. (22) Ahmad, N., Cheng, P., and Mukhtar, H. (2000) Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate. Biochem. Biophys. Res. Commun. 275, 328-334. (23) Dong, Z., Ma, W.-Y., Huang, C., and Yang, C. S. (1997) Inhibition of tumor promoter-induced AP-1 activation and cell transformation by tea polyphenols, (-)-epigallocatechin gallate and theaflavins. Cancer Res. 57, 4414-4419. (24) Chung, J. Y., Huang, C., Meng, X., Dong, Z., and Yang, C. S. (1999) Inhibition of activator protein 1 activity and cell growth by purified green tea and black tea polyphenols in H-rastransformed cells: Structure-activity relationship and mechanisms involved. Cancer Res. 559, 4610-4617.

872

Chem. Res. Toxicol., Vol. 16, No. 7, 2003

(25) Balasubramanian, S., Efimova, T., and Eckert, R. (2002) Green tea polyphenol stimulates a Ras, MEKK1, MEK3, and p38 cascade to increase activator protein 1 factor-dependent involucrin gene expression in normal human keratinocytes. J. Biol. Chem. 277, 1828-1836. (26) Ahmad, N., Gupta, S., and Mukhtar, H. (2000) Green tea polyphenol epigallocatechin-3-gallate differentially modulates nuclear factor kB in cancer cells versus normal cells. Arch. Biochem. Biophys. 376, 338-346. (27) Hayakawa, S., Saeki, K., Sazuka, M., Suzuki, Y., Shoji, Y., Ohta, T., Kaji, K., Yuo, A., and Isemura, M. (2001) Apoptosis induction by epigallocatechin gallate involves its binding to Fas. Biochem. Biophys. Res. Commun. 285, 1102-1106. (28) Ahmad, N., Feyes, D. K., Nieminen, A.-L., Agarwal, R., and Mukhtar, H. (1997) Green tea constituent epigallocatechin-3gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J. Natl. Cancer Inst. 89, 1881-1886. (29) Fotsis, T., Pepper, M. S., Aktas, E., Breit, S., and Rasku, S. (1997) Flavanoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis. Cancer Res. 2916-2921. (30) Demeule, M., Brossard, M., Page, M., Gingras, D., and Beliveau, R. (2000) Matrix metalloproteinase inhibition by green tea catechins. Biochim. Biophys. Acta. 1478, 51-60. (31) Anderson, R. F., Fisher, L. J., Hara, Y., Harris, T., Mak, W. B., Melton, L. D., and Packer, J. E. (2001) Green tea catechins partially protect DNA from OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals. Carcinogenesis 22, 1189-1193. (32) Yang, C. S., Chen, L., Lee, M.-J., Balentine, D. A., Kuo, M. C., and Schantz, S. P. (1998) Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol., Biomarkers Prev. 7, 351-354. (33) Chow, S. H.-H., Cai, Y., Alberts, D. S., Hakim, I., Dorr, R., Shahi, F., Crowell, J. A., Yang, C. S., and Hara, Y. (2001) Phase I pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and Polyphenon E. Cancer Epidemiol., Biomarkers Prev. 10, 53-58. (34) Antonio, L., Grillasca, J.-P., Taskinen, J., Elovaara, E., Burchell, B., Piet, M.-H., Ethell, B., Ouzzine, M., Fournel-Gigleux, S., and Magdalou, J. (2002) Chracterization of catechol glucuronidation in rat liver. Drug Metab. Dispos. 30, 199-207. (35) Kohri, T., Matsumoto, N., Yamakawa, M., Suzuki, M., Nanjo, F., Hara, Y., and Oku, N. (2001) Metabolic fate of (-)-[4-3H]epigallocatechin gallate in rats after oral administration. J. Agric. Food Chem. 49, 4102-4112. (36) Liang, Y.-C., Lin-Shiau, S.-Y., Chen, C.-F., and Lin, J.-K. (1999) Inhibition of cyclin-dependent kinases 2 and 4 activities as well as induction of Cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (-)-epigallocatechin-3-gallate. J. Cell Biochem. 75, 1-12. (37) Lamy, S., Gingras, D., and Beliveau, R. (2002) Green tea catechins inhibit vascular endothelial growth factor receptor phosphorylation. Cancer Res. 62, 381-385. (38) Naasani, I., Seimiya, H., and Tsuruo, T. (1998) Telomerase inhibition, telomere shortening, and senescence of cancer cells

Palermo et al.

(39)

(40) (41)

(42)

(43) (44) (45)

(46)

(47)

(48) (49)

(50) (51)

by tea catechins. Biochem. Biophys. Res. Commun. 249, 391396. Poland, A., and Knutson, J. C. (1982) 2,3,7,8-Tetrachlorodibenzop-dioxin and related halogenated aromatic hydrocarbons. Examinations of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22, 517-554. Ma, Q., and Whitlock, J. P., Jr. (1996) The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. Mol. Cell. Biol. 16, 2144-2150. Kolluri, S. K., Weiss, C., Koff, A., and Go¨ttlicher, M. (1999) p27kip1 induction and inhibition of proliferation by the intracellular Ah receptor in developing thymus hepatoma cells. Genes Dev. 13, 1742-1753. Puga, A., Barnes, S. J., Dalton, T. P., Chang, C.-Y., Knudsen, E. S., and Maier, M. A. (2000) Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcriptoin and cell cycle arrest. J. Biol. Chem. 275, 2943-2950. Reiners, J. J., Jr., Clift, R., and Mathieu, P. (1999) Suppression of cell cycle progression by flavonoids: Dependence on the aryl hydrocarbon receptor. Carcinogenesis 20, 1561-1566. Tian, Y., Ke, S., Denision, M. S., Rabson, A. B., and Gallo, M. A. (1999) Ah receptor and NF-kB interactions, a potential mechanism for dioxin toxicity. J. Biol. Chem. 274, 510-515. Wo¨lfle, D., Marotzki, S., Dartsch, D., Marquardt, H., and Marquardt. (2000) Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Carcinogenesis 21, 15-21. Puga, A., Hoffer, A., Zhou, S., Bohm, J. M., Leikauf, G. D., and Shertzer, H. G. (1997) Sustained increase in intracellular free calcium and activation of cyclooxygenase-2 expression in mouse hepatoma cells treated with dioxin. Biochem. Pharmacol. 54, 1287-1296. Matikainen, T., Perez, G. I., Jurisicova, A., Pru, J. K., Schlezinger, J. J., Ryu, H.-Y., Laine, J., Sakai, T., Kormeyer, S. J., Casper, R. F., Tilly, S., and Tilly, J. L. (2002) Aromatic hydrocarbon receptordriven Bax gene expression is required for premature ovarian failure caused by biohazardous environmental chemicals. Nat. Genet. 28, 355-360. Safe, S. (2001) Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol. Lett. 120, 1-7. Klinge, C. M., Kaur, K., and Swanson, H. I. (2000) The aryl hydrocarbon receptor interacts with estrogen receptor alpha and orphan receptors COUP-TF1 and ERRR1. Arch. Biochem. Biophys. 373, 163-174. Gonzalez, F. J., and Gelboin, H. V. (1994) Role of human cytochromes P450 in the metabolic activation of chemical carcinogens and toxins. Drug Metab. Rev. 26, 165-183. Gasiewicz, T. A., and Rucci, G. (1991) R-Naphthoflavone acts as an antagonist of 2,3,7,8-tetrachlorodibenzo-p-dioxin by forming an inactive complex with the Ah receptor. Mol. Pharmacol. 40, 607-612.

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