* Molecular and Cell Nutrition Laboratory, College of Agriculture, and
Department of Surgery, University of Kentucky, Lexington, Kentucky 40546-0215; and
Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, Iowa 52242
Received May 28, 2003; accepted August 4, 2003
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ABSTRACT |
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Key Words: flavonoids; PCB; oxidative stress; AhR; CYP1A1; vascular endothelial cells.
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INTRODUCTION |
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Flavonoids are thought to promote optimal health, partly via their antioxidant effects in protecting cellular components against free radicals. Diets rich in antioxidants such as phenolic flavonoids reduce the risk of chronic degenerative diseases associated with free radicals. Consumption of polyphenolic flavonoids in the diet was shown to be inversely associated with morbidity and mortality from coronary heart disease (Hertog et al., 1995). Flavonoids may prevent coronary artery disease by inhibiting LDL oxidation, macrophage foam cell formation, and atherosclerosis (Catapano, 1997
; Fuhrman and Aviram, 2001
; Maeda et al., 2003
). Catechin and quercetin are the most abundant flavonoids in edible plants and foodstuffs derived from plants. The catechins have potent inhibitory effects on LDL oxidation in vitro (Miura et al., 1994
) and ex vivo (Miura et al., 2000
). Quercetin can scavenge superoxide radicals and hydroxyl radicals, reduce lipid peroxy radicals, and inhibit lipid peroxidation (Salah et al., 1995
). The catechin intake mainly depends on beverages (e.g., red wine, tea) consumption, while quercetin is found in fruits and vegetables. Among the four forms of green tea, polyphenols-epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG), EGCG is the predominant catechin (40% of total) and has the greatest antioxidant activity (Higdon and Frei, 2003
), and it is also the most widely studied polyphenol for disease prevention (Bagchi, 1999
). For example, Johnson and Loo (2000)
demonstrated that low concentrations of EGCG and quercetin could scavenge free radicals, thereby inhibiting oxidative damage to cellular DNA.
In the present investigation, we hypothesized that the endothelial cytotoxicity of PCB 77 could be modulated by dietary flavonoids, such as EGCG and quercetin, at appropriate concentrations. Our data suggest that the protective properties of these flavonoids, EGCG or quercetin, are at the functional level of AhR against PCB-induced endothelial cytotoxicity.
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MATERIALS AND METHODS |
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Cell culture and experimental media.
Endothelial cells were isolated from porcine pulmonary arteries and cultured as previously described (Hennig et al., 1984; Toborek et al., 2002
). The basic culture medium consisted of M199 (GIBCO Laboratories, Grand Island, NY) containing 10% bovine calf serum (BCS; Hyclone Laboratories, Inc., Logan, UT). The experimental medium was composed of M199 enriched with 5% FBS and the coplanar PCB 77 (3,3', 4,4'-tetrachlorobiphenyl). Epigallocatechin-3-gallate and quercetin (Sigma, St. Louis, MO) were used as polyphenolic flavonoids. PCB 77 and flavonoids were solubilized in DMSO and used at appropriate concentrations. PCB was used at a concentration of 3.4 µM, as it reflects serum concentrations after acute exposure to PCB (Jensen, 1989
). EGCG was used at concentrations ranging from 5 to 50 µM and quercetin was used at the range of 10 to 100 µM. Hydrogen peroxide (Sigma) was used at 200 µM concentration. Alpha naphthoflavone (
-NF, Sigma), a cytochrome P450 inhibitor and antagonist of Ah receptor (Shimada et al., 1998
), was used at a concentration of 10 µM, either alone or with PCB 77. ß-Napthalone (ß-NF, from Sigma), an AhR agonist (Anna et al., 2000
) was used at a concentration of 1 µM, either alone or with EGCG.
Measurement of oxidative stress.
The effects of exposure to PCB 77, EGCG, quercetin, and H2O2 on cellular oxidation were determined by dichlorofluorescein (DCF) assay (Mattson et al., 1995) and modified for use by a fluorescent microplate reader. After treatment of endothelial cells with PCB 77 and/or flavonoids for 6 h, cells were incubated in the presence of 10-µM dichlorodihydrofluorescein diacetate for 30 min at room temperature. Cells were treated with H2O2 for 15 min at a concentration of 200 µM. Before analysis, cells were washed twice with Hanks and once with 10 mM HEPES buffer (pH 7.4), and the fluorescence within the dye-loaded cells was measured using a Spectramax GeminiXS microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA) with excitation and emission wavelengths of 490 and 526 nm, respectively.
Reverse transcriptase-polymerase chain reaction (RT-PCR).
Total RNA was extracted by use of RNA isolation reagent (RNA Stat-60, Tel-Test, Inc., TX) and reverse transcribed at 42°C for 60 min in 20 µl of 5 mM MgCl2, 10 mM TrisHCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1 mM dNTP, 1 unit/µl of recombinant RNasin ribonuclease inhibitor, 10 U/100 µg of AMV reverse transcriptase, and 0.5 µg of oligo(dT) primer. Specific primer sequences were synthesized by IDT Technologies, Inc. (Coralville, IA). The primers used for CYP1A1 were: sense 5'-TGGAG AGGCA AGAGT AGTTG G-3' and antisense 5'-GGCAC AACGG AGTAG CTCAT A-3'. Oligonucleotide primers to amplify the porcine housekeeping gene ß-actin were used according to Barchowsky et al. (1998). The PCR mixture consisted of a Taq PCR Master Mix (Qiagen, Valencia, CA), 2 µl of the reverse-transcriptase product, and 20 pmol of primer pairs in a total volume of 50 µl. Thermocycling was performed with the following temperature cycles: initial denaturation at 94°C for 4 min, 28 cycles of 1-min denaturation at 94°C, 1-min annealing at 57°C, and 1-min synthesis at 72°C, followed by a final extension step of 7 min at 72°C. PCR products were separated by 2% agarose gel electrophoresis, stained with SYBR Green I (Molecular Probes, Eugene, OR), and visualized using phosphoimaging technology (FLA-2000, Fuji, Stamford, CT).
CYP1A activity.
Ethoxyresorufin-O-deethylase (EROD), a specific measure of CYP1A activity, was determined in intact porcine pulmonary artery endothelial cells grown in 48-well plates, as described by Kennedy and Jones (1994), with 5 µM ethoxyresorufin in growth medium as a substrate in the presence of 1.5 mM salicylamide, to inhibit conjugating enzymes (Ciolino et al., 1999
). The assay was performed at 37°C. The fluorescence of resorufin generated by the conversion of ethoxyresorufin by CYP1Awas measured first, immediately after addition of reagents and then after 60 min in a Spectramax fluorescence plate reader with excitation of 530 nm and emission at 595 nm.
Electrophoretic mobility shift assay.
Nuclear extracts from endothelial cells were prepared according to the method of Dignam et al. (1983). Binding reactions were performed in a 20 µl volume containing 5 µg of nuclear protein extracts, 10 mM TrisCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.1 mM dithiothreitol, 10% glycerol, 0.5 µg of poly(dI-dC) (nonspecific competitor), and 40,000 cpm of 32P-labeled specific oligonucleotide probe. A complementary pair of synthetic oligonucleotides containing the sequences 5'-GATCC GGCTC TTGTC ACGCA ACTCC GAGCT CA-3' and 5'-GATCT GAGCT CGGAG TTGCG TGAGA AGAGC CG-3' were synthesized, annealed, and labeled at their 5' ends by using T4 polynucleotide kinase and [g32P]ATP (Chen and Tukey, 1996
). The resultant proteinDNA complexes were resolved on a 5% polyacrylamide gel using 0.25x TBE buffer (50 mM TrisHCl, 45 mM boric acid, 0.5 mM EDTA, pH 8.4). Competition studies were performed by the addition of a molar excess of unlabeled oligonucleotide to the binding reaction. Densitometric quantitation of these and other gels were performed using UN-Scan-It software (Silk Scientific, Inc., Orem, UT).
Statistical analysis.
The data were analyzed using SYSTAT 7.0 (SPSS, Inc., Chicago, IL). Comparisons between treatments were made by one-way ANOVA with post-hoc comparisons of the means made by the Bonferroni least-significant-difference method. Statistical probability of p 0.05 was considered significant.
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RESULTS |
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Quercetin and EGCG Inhibit PCB 77-Induced Oxidative Stress in Endothelial Cells
The effects of PCB 77 alone, and along with catechin (EGCG) and quercetin, on oxidative stress were determined by DCF fluorescence. Figure 1A shows that PCB 77, at 3.4 µM concentration, markedly induced oxidative stress. EGCG was used at different concentrations, i.e., 5, 10, 25, and 50 µM along with 3.4 µM PCB 77. PCB 77-induced oxidative stress was significantly reduced at all EGCG concentrations tested. The EGCG-mediated reduction in oxidative stress was concentration-dependent, with maximal inhibition at 50 µM. Similarly, quercetin was used at different concentrations, varying from 10 to 100 µM, along with 3.4 µM of PCB 77. Quercetin also downregulated PCB 77-mediated oxidative stress (Fig. 1B
), with maximal inhibition at the 100 µM concentration. The downregulation of oxidative stress by either addition of catechin or quercetin to PCB-containing cell cultures was similar to the control levels of these flavonoids alone (Fig. 1C
). Treatment of cells with EGCG up to 50 µM and quercetin up to 100 µM did not affect the cell viability as ascertained by MTT assay (data not shown).
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To validate that the flavonoids used in this study can scavenge free radicals in our culture model, cells were treated with H2O2. Exposure to H2O2 resulted in a marked increase in cellular oxidative stress. However, when cells were pretreated with EGCG (at 50 µM) or quercetin (at 100 µM) for 6 h and subsequently challenged with H2O2 for 15 min, induction of oxidative stress was suppressed (data not shown). Similarly, cells exposed to the AhR antagonist -NF at 10 µM concentration, either alone or together with PCB 77, showed reduced oxidative stress when compared either to control cells or cells treated with PCB 77 alone (data not shown).
Quercetin and EGCG Inhibit PCB 77-Induced CYP1A Enzymatic Activity and CYP1A1 mRNA Expression
The enzymatic activity of CYP1A in intact endothelial cells treated with EGCG or quercetin together with PCB 77 were assayed by measuring EROD activity. Compared to PCB treatment, incubation of cells with PCB 77 and flavonoids caused a concentration-dependent decrease in EROD activity (Figs. 2A and 2B
). EGCG at 50 µM concentration and quercetin at 100 µM concentration were able to markedly inhibit CYP1A activity. Cells treated with
-NF, either alone or with PCB 77, showed a marked reduction in EROD activity (Fig. 3A
). When cells were treated with ß-NF, an AhR agonist, CYP1A activity was increased. In contrast, cotreatment with ß-NF and EGCG showed a marked reduction in CYP1A activity (Fig. 3A
). When endothelial cells were treated with either EGCG (50 µM) or quercetin (100 µM) alone, inhibition of CYP1A1 activity was observed (see Fig. 3B
).
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DISCUSSION |
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These findings have been confirmed in the present study. Furthermore, our data clearly demonstrate that dietary flavonoids like EGCG and quercetin can downregulate the action of PCB 77, both by reducing the induction of oxidative stress as well as the activity and expression of CYP1A1. The marked reduction in oxidative stress caused by EGCG and quercetin, along with H2O2, also confirmed the potent antioxidant properties of these flavonoids.
Flavonoids show antioxidant properties and are potent scavengers of free radicals (Higdon and Frei, 2003; Sugihara et al., 2001
). For example, EGCG was shown to reduce oxidation by hydrogen peroxide, both in a cell-free system (Wei et al., 1999
), and in terms of DNA single-strand-break damage (Johnson and Loo, 2000
). Kashima (1999)
demonstrated the scavenging effects of catechins on superoxide and hydroxyl radicals. Nakagawa and Yokozawa (2002)
found that green tea catechins directly scavenged NO and O2-, suggesting that ONOO- formation was inhibited as a result of the removal of NO and O2-. Quercetin is a scavenger of O2-, NO., HO., and peroxyradicals, as well as an iron chelator (Chen et al., 1990
, Hussein et al., 1987
). Quercetin was able to also prevent cyclosporine-induced production of reactive oxygen species (Satyanarayana et al., 2001
). Furthermore, both resveratrol and quercetin were effective in inhibiting a cellular increase in endothelin-1 as a response to hydrogen peroxide treatment (Ruef et al., 2001
).
The mechanism by which EGCG and quercetin can inhibit oxidative damage could be related to their chemical structures. EGCG and quercetin may work by providing hydrogen atoms from their phenolic hydroxyl groups to scavenge hydroxyl radicals (Shahidi and Wanasundara, 1992). Another possible way that EGCG and quercetin might inhibit oxidative damage could be by chelating metal ions, which can facilitate formation of hydroxyl radicals (Halliwell and Gutteridge, 1990
).
We have shown that EGCG at 50 µM and quercetin at 100 µM concentrations neutralize the oxidative stress induced by PCB 77, thus acting as antioxidants. Similar effects were observed when CYP1A1 activity was measured by EROD assay, as well as by CYP1A1 mRNA expression studied by the RT-PCR method. However, our data do not rule out the involvement of other stress response-signaling pathways. For example, AhR agonists are known to increase cyclooxygenase expression (Kietz and Fischer, 2003), and quercetin and EGCG have been reported to inhibit cyclooxygenase activity or expression (Chen et al., 2001
; Raso et al., 2001
). Preliminary studies in our laboratory (data not shown) do not show a significant inhibition of COX-2 mRNA expression by catechin or quercetin in our endothelial cell cultures. Similar results were reported by Banerjee et al. (2002)
, using quercetin and a human lung adenocarcinoma-cell line.
Inhibition of CYP1A1 activity by catechins has already been reported (Moon et al., 1998). Simultaneous treatment of cells with green tea extracts (GTEs) plus TCDD inhibited the induced transcription of the CYP1A1-luciferase gene in a concentration-dependent manner, and this correlated with a decrease in CYP1A1 mRNA and protein levels (Williams et al., 2000
). It was further demonstrated in HepG2 cells that GTEs inhibited TCDD-induced binding of the AhR to DNA and subsequent CYP1A transcription. The authors concluded that the inhibition of TCDD-induced CYP1A1 expression was most likely due to the ability of components of GTEs to interact directly with the receptor and function as antagonists of the AhR (Williams et al., 2000
). In the present study, we have shown that EGCG at 50 µM and quercetin at 100 µM concentration, were able to downregulate PCB-induced CYP1A1 mRNA expression as well as its activity. Furthermore, these flavonoids were able to prevent AhR-DNA binding activity, thus blocking CYP1A1 induction.
To demonstrate that the protective effects of EGCG and quercetin are initiated upstream from CYP1A1, i.e., at the functional level of AhR, EROD activity and CYP1A1 expression were investigated with the use of -NF,
CYP1A1 inhibitor, and AhR antagonist. Treatment of cells with
-NF alone or with
-NF plus PCB 77 inhibited the EROD activity and CYP1A1 expression relative to PCB treatment alone. Similar effects were observed when cells were treated with EGCG plus PCB 77 and quercetin plus PCB-77. ß-NF upregulated the CYP1A1 expression and activity, proving its agonist activity towards AhR; while in combination with EGCG, it showed a decrease in the induction of CYP1A1. These data strongly suggest that flavonoids act as antioxidants through inhibition of AhR function.
In our cell system, -NF appeared to act as a partial agonist by increasing the AhR-DNA binding as well as partial induction of CYP1A1 mRNA expression. Even though AhR-DNA binding was observed in the presence of
-NF, a significant reduction in CYP1A1 mRNA expression was observed when compared to cultures treated with either PCB-77 or ß-NF, thus indicating the inhibitory property of
-NF on CYP1A1 expression. Our AhR-DNA binding data, using our endothelial cell model, agree with the report of Casper et al. (1999)
, using T47D cells that showed AhR-DNA binding with
-NF treatment, but with subsequent CYP1A1 mRNA inhibition.
Results from this study indicate that PCB 77 activates AhR and produces CYP1A1 leading to oxidative stress and subsequent endothelial-cell damage. Our data provide evidence that PCB 77-mediated cellular oxidative stress could be blocked with either EGCG or quercetin in a concentration-dependent manner. Our DNA binding studies of the AhR suggest that a major portion of the overall protective mechanism(s) is initiated at the functional level of AhR. Both EGCG and quercetin were able to block AhR activation by PCB 77, thereby demonstrating AhR antagonist action. Moreover, the presence of EGCG downregulated the -NF plus PCB-induced DNA binding, suggesting that a significant protective property of EGCG is initiated at the level of the AhR. Furthermore, our data provide evidence that nutrition, e.g., flavonoids found in foods, can protect against environmental contaminants such as PCB 77 at both functional levels of AhR and/or CYP1A1.
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ACKNOWLEDGMENTS |
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NOTES |
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