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Department of Food Science Research for Health, National Institute of Health and Nutrition, Tokyo, 162-8636, Japan
1To whom correspondence should be addressed. E-mail: umegaki{at}nih.go.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • EGCg • reactive oxygen species • WIL2-NS cells • chromosomal damage • oxidative stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Intake of antioxidants has been shown to reduce ROS-related disease in humans (2
). Accordingly, antioxidants, particularly in food, have received great attention as primary preventive ingredients against various diseases.
Green tea is a popular beverage, particularly among Japanese and other Asians, and contains large amounts of flavonoids, mainly catechins (flavan-3-ols). The tea catechin (-)-epigallocatechin-3-O-gallate (EGCg), which constitutes 
60% of the catechins in tea (3
), is a major mediator of the antioxidative effect of green tea. Many researchers have reported beneficial effects of EGCg, such as prevention of LDL oxidation (4
) and oxidative damage to DNA (5
). However, there have been few studies on the effects of EGCg at its physiological concentrations. The concentration of EGCg in human plasma has been reported to reach no higher than 1 µmol/L even when an excess of EGCg was consumed (6
,7
). For the practical use of EGCg as a preventive agent against various ROS-related diseases, it is important to clarify the safety and effectiveness of EGCg at its physiological concentration.
Damage to DNA or chromosomes is important because the damage greatly influences cell functions, resulting in various diseases and aging. To evaluate chromosomal damage in humans, lymphocytes are commonly used as experimental targets because they are susceptible to ROS-induced chromosomal damage (8
). However, differences in the genetic background and intake of antioxidants by subjects may modify the responses of lymphocytes to ROS, resulting in conflicting results. Accordingly, it would be useful to use a lymphoblastoid cell line for the detailed elucidation of EGCg effects on chromosomal damage. The cytokinesis block micronucleus (CBMN) assay using WIL2-NS cells, a human B lymphoblastoid cell line, has been shown to be a sensitive assay system to examine ROS-induced chromosomal damage (9
). In this study, we used the CBMN assay system in WIL2-NS cells to examine whether EGCg itself induces chromosomal damage, and whether physiological concentrations of EGCg protect against chromosomal damage induced by various ROS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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EGCg was obtained from Funakoshi (Tokyo, Japan). Fetal bovine serum, RPMI 1640 medium, antibiotic solution (5 x 106 U/L penicillin and 5 g/L streptomycin), L-glutamine, and Hank’s balanced salt solution (HBSS) were purchased from Gibco (Grand Island, NY). Hypoxanthine (HX), xanthine oxidase (XO), and cytochalasin B were obtained from Sigma (St. Louis, MO). H2O2 was obtained from Mitsubishi Gas Chemical (Tokyo, Japan) and tert-butyl hydroperoxide (tert-BuOOH) from Katayama Chemical (Osaka, Japan). Giemsa’s Solution was obtained from Merck (Darmstadt, Germany).
Cell culture and treatment with EGCg and oxidants.
WIL2-NS cells (ATCC No. CRL-8155) obtained from the American Type Culture Collection (Manassas, VA) were cultured and maintained as described previously (9
). The WIL2-NS cells were washed once with HBSS and resuspended in HBSS at a density of 0.5 x 109 cells/L. Cell suspensions (950 µL) were incubated for 60 min with various concentrations of EGCg (in 50 µL) dissolved in HBSS. To evaluate chromosomal damage induced by EGCg itself, the cells were washed with HBSS, then subjected to the CBMN assay as described below. To evaluate EGCg effects on ROS-induced chromosomal damage, the incubated cell suspensions were treated in one of two ways. One was to expose the cells to ROS without elimination of EGCg such that interactions of EGCg and ROS occurred both inside and outside of the cells. The other was to wash the cells twice with HBSS to eliminate the EGCg outside of the cells, and then to expose the cells to ROS. H2O2, tert-BuOOH, and superoxide were used as the ROS. Superoxide was generated by XO in the presence of 25 µL of HX. In the superoxide generation system, 20 U/L of XO with HX (25 µmol/L) generated 13.8 µmol/L per min of superoxide. The ROS treatments were performed at 37°C for 30 min, and then the cells were washed with HBSS for the CBMN assay.
For the analysis of the EGCg content in the cells, the cells were treated with EGCg at 37°C for 60 min. After the cells were washed with HBSS 2–5 times, they were centrifuged at 8000 x g for 30 s. The resulting cell pellets were analyzed.
To examine the direct interaction between EGCg and various ROS, EGCg solutions without cells were mixed with ROS for 30 min, and changes in the concentration of EGCg in the solution were analyzed.
Analytical methods.
Chromosomal damage in WIL2-NS cells was assessed by the CBMN-assay as described elsewhere (9
). Briefly, WIL2-NS cells were washed with HBSS and with RPMI 1640 to remove EGCg and/or oxidants, then resuspended in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 1% (v/v) antibiotic solution, 2 mmol/L glutamine, 4.44 mg/L cytochalasin B at a cell density of 0.5 x 109 cells/L. After 42 h of culture, the cells were harvested. Slides were prepared using a cytocentrifuge (Shandon Southern Products, Cheshire, UK), air dried and fixed with absolute methanol, and then stained with 4% (v/v) Giemsa’s solution in water for 30 min. Chromosomal damage rates were expressed as the number of micronucleated binucleate cells (MNed BN cells) per 1000 binucleated cells (BN cells), and the nuclear division index (NDI) was calculated as reported previously (9
).
EGCg was analyzed by HPLC with an electrochemical detector (ECD) as described elsewhere (6
). Briefly, samples were subjected to solid phase extraction, and the resulting samples were applied to the HPLC (Shimadzu LC-10AD; Shimadzu, Kyoto, Japan) with an ECD (Coulochem II; ESA Bedford, MA) equipped with analytical cells (detector 1, -150 mV; detector 2, +150 mV). The separating conditions were as follows: column, IRICA RP-18T (4.6 x 250 mm, 5-µm particles; IRICA Instruments, Kyoto, Japan); column temperature, 35°C; mobile phase, 50 mmol/L phosphoric acid, 0.05 mmol/L EDTA and 14% (v/v) acetonitrile (pH 2.5); flow rate, 1 mL/min.
Changes in the concentration of H2O2 in the solution were also measured by the phenol red method (10
).
Statistical analysis.
The data are presented as means ± SE for triplicate experiments. Statistical analyses of the data were carried out using ANOVA followed by the post hoc test of Fisher’s protected least significant difference test. To evaluate EGCg incorporation into WIL2-NS cells and direct reaction between oxidants and EGCg, unpaired t test were applied. Differences with a P value <0.05 were considered significant. The statistical analyses were performed using a computer program (StatView Version 5.0; SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. However, 100 µmol/L EGCg induced chromosomal damage. The nuclear division index, a measure of cell division, decreased at EGCg concentrations of at least 10 µmol/L. When production of H2O2 was also examined by incubating EGCg in a solution without cells, EGCg produced H2O2 at the concentrations of at least 30 µmol/L, but not at concentrations of < 10 µmol/L or less (Table 1)
. Interestingly, the concentrations that produced H2O2 and those that increased in the MNed BN cells were consistent.
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When WIL2-NS cells were exposed to ROS (H2O2, tert-BuOOH or superoxide) for 30 min, chromosomal damage dose-dependently increased up to the value of 150 MNed BN cells/1000 BN cells (data not shown). WIL2-NS cells were pretreated with various concentrations (0, 0.3, 1 and 10 µmol/L) of EGCg for 60 min, and then exposed to H2O2 (20 µmol/L), tert-BuOOH (1 mmol/L), or superoxide (20 mU of XO) to induce chromosomal damage. Regardless of the type of ROS, EGCg dose-dependently prevented the ROS-induced chromosomal damage (Fig. 1
, A1–C1). With tert-BuOOH, the increase in the chromosomal damage was significantly attenuated even at 0.3 µmol/L of EGCg (Fig. 1
B1).
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, A2 and C2). In contrast, no preventive effect by EGCg was detected when cells were exposed to 1 mmol/L tert-BuOOH (Fig. 1
B2).
To examine the direct interactions between EGCg and ROS, each ROS was added to a solution of 3 µmol/L EGCg incubated for 30 min, and then the changes in the concentration of EGCg were analyzed. The concentration of EGCg was not decreased by incubation with H2O2, but was significantly decreased by incubation with tert-BuOOH and superoxide (Fig. 2
). To further confirm the lack of interaction between EGCg and H2O2, changes in the concentration of H2O2 were analyzed instead of changes in EGCg. No decrease in the concentration of H2O2 was detected in mixtures of various concentrations of H2O2 and EGCg (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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0.3–1µmol/L) at 2 h (6
,7
). In this study, EGCg induced chromosomal damage at 100 µmol/L but did not induce such damage at physiological concentrations. Furthermore, physiological concentrations of EGCg protected against chromosomal damage induced by H2O2, tert-BuOOH and superoxide. These findings strongly suggest that EGCg has no genotoxicity even when an excess amount is consumed and has the beneficial effect of minimizing ROS-induced chromosomal damage, which is believed to be involved in various diseases and aging.
The induction of chromosomal damage by EGCg at high concentrations was due to its production of H2O2. It has already been reported that high concentrations of EGCg have oxidizing effects: induction of oxidative DNA damage at 100 µmol/L (5
), reduction of cell-cycle progression at 200 µmol/L (11
), and production of H2O2 at 100 µmol/L (12
). Those reports are consistent with these findings. It is noteworthy that high concentrations of EGCg are unattainable in vivo, particularly in plasma, even if people consume an excess of EGCg. In addition, antioxidants other than EGCg and catalase, which eliminate H2O2, also are present in the blood. Therefore, it is unlikely that EGCg itself induces chromosomal damage under physiological conditions.
In this study, we showed that EGCg at physiological concentrations dose-dependently prevented ROS-induced chromosomal damage in WIL2-NS cells. There are two sites at which EGCg may protect against ROS-induced chromosomal damage. One is outside and the other is inside of the cells. To clarify the effect of EGCg, it was necessary to show the presence of EGCg in the cells. It has been shown that 3H-labeled EGCg is incorporated into cells (13
). In this study, when WIL2-NS cells were incubated with 0.3 µmol/L EGCg, 3.1 pmol of EGCg was detected in 106 cells. When the concentration of EGCg in the cell was calculated using the approximate cell volume, the estimated concentration was 6 µmol/L. EGCg (0.3 µmol/L) is the concentration obtained by a normal daily intake of green tea. These findings suggest that a large amount of EGCg, which protects against ROS-induced chromosomal damage, can be present within WIL2-NS cells, circulating lymphocytes, and other blood cells.
When EGCg and either superoxides or tert-BuOOH was mixed, EGCg in the mixture decreased, suggesting that EGCg directly scavenged superoxide and tert-BuOOH, resulting in a decrease in the concentration of ROS that could induce chromosomal damage in WIL2-NS cells. Direct interaction between EGCg and superoxide (14
) or peroxyradicals (15
) has also been shown by other researchers. When the EGCg existing outside of the cells was eliminated, the preventive effect of EGCg against chromosomal damage was diminished only in the case of tert-BuOOH exposure, but not in the case of superoxide exposure. These findings suggest that the preventive effect of EGCg mainly operates outside of the cells in the case of tert-BuOOH exposure, and both inside and outside of the cells in the case of superoxide exposure.
We did not detect a direct interaction between EGCg and H2O2 when we evaluated not only the decrease in EGCg, but also the decrease in H2O2 after mixing these two reagents. This may be related to the low reactivity of H2O2 (16
). Nevertheless, the protective effect of EGCg against H2O2-induced chromosomal damage was observed even when the EGCg present outside of the cells was eliminated by washing the cells. This suggests that the protective effect of EGCg against H2O2 occurs mainly inside of the cells. For H2O2–induced DNA damage, a requirement for iron and prevention by iron-chelation have been reported (17
). It has been reported that the cytoprotective effect of EGCg is correlated with iron chelation (18
), and the formation of the hydroxyl radical via the Fenton reaction is prevented by EGCg through its iron-cheating activity (14
). Furthermore, green tea extract has been shown to prevent DNA damage by ferrous ion (19
). These findings suggest that the preventive effect of EGCg against H2O2-induced chromosomal damage is related to the prevention of hydroxyl radical formation within the cells by way of the iron-chelating activity of EGCg. Further detailed studies will be needed to clarify the precise mechanism by which EGCg prevents the ROS-induced chromosomal damage that occurs inside the cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received 27 February 2002. Initial review completed 25 March 2002. Revision accepted 10 April 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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