Green tea catechins partially protect DNA from ·OH radical-induced strand breaks and base damage through fast chemical repair of DNA radicals
Robert F. Anderson1,3,,
Louisa J. Fisher,
Yukihiko Hara2,,
Tracy Harris,
Wai B. Mak,
Laurence D. Melton and
John E. Packer
Department of Chemistry and
1 Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland 1, New Zealand and
2 Mitsui Norin, 223 Miyabara, Fujieda City, Shizuoka Pref., 426-01, Japan
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Abstract
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The catechins, ()-epicatechin (EC), ()-epigallocatechin (EGC), ()-epicatechin gallate (ECG) and ()-epigallocatechin gallate (EGCG) are believed to be active constituents of green tea accounting for the reported chemoprevention of certain cancers. The molecular mechanisms by which the measured low concentrations (ca. micromolar) of catechins in humans can reduce the incidence of carcinogenesis is not clear. Using an in vitro plasmid DNA system and radiolytically generating reactive oxygen species (ROS) under constant scavenging conditions, we have shown that all four catechins, when present at low concentrations, ameliorate free radical damage sustained by DNA. A reduction in both prompt DNA single-strand breaks and residual damage to the DNA bases, detected by subsequent incubation with the DNA glycosylases formamidopyrimidine (FPG), endonuclease III (EndoIII) and 5' AP endonuclease exonuclease III (ExoIII), was observed. EGCG was found to be the most active of the catechins, with effects seen at micromolar concentrations. Combined fast-reaction chemistry studies support a mechanism of electron transfer (or H-atom transfer) from catechins to ROS-induced radical sites on the DNA. These results support an antioxidant role for catechins in their direct interaction with DNA radicals.
Abbreviations: D0, the radiation dose required on average to produce one DNA single-strand break; E(1), one-electron reduction potiential at pH 7 versus normal hydrogen electrode; EC, (-)-epicatechin; ECG, (-)-epicatechin gallate; EDTA, ethylenediaminetetraacetic acid; EGC, (-)-epigallocatechin; EGCG, (-)-epigallocatechin gallate; EndoIII, endonuclease III; Exoll, exonuclease III; FPG, formidopyrimidine-DNA glycosylase; G(ess), yield of endonuclease-indiced DNA single-stand breaks per absorbed dose (Gy); G(ssb), radiation chemical yield of single-strand breaks per absorbed dose (Gy); G(ssb'), reduction in G(ess) due to the presence of catechins; Gy, absorbed radiation dose (J/kg); ROS, reactive oxygen specie; TAE, Tris/acetic acid/EDTA buffer; Tris, tris(hydroxy-methyl)aminomethane
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Introduction
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Cancer chemoprevention is an important area of research providing a practical approach for the identification of potentially useful classes of compounds which are inhibitors of cancer development. One such class of compounds which has emerged from epidemiological studies is the polyphenols of green tea (1,2), commonly referred to as green tea catechins (Figure 1
). These naturally occurring compounds have been shown to be highly active as anticarcinogens in vivo using several rodent test systems (3) and epidemiological studies on the consumption of green tea point to a protection against stomach cancer (46) and colon (7,8) cancers. Several mechanisms have been identified by which catechins can operate as anticarcinogens, including inhibition of the monooxygenase activity associated with cytochrome P-450 activation of carcinogen-precursors (9), removal of carcinogens through adduct formation (10), the up-regulation of antioxidant enzymes (11) and the scavenging of reactive oxygen species (ROS) (12). Whereas most of these mechanisms assume, as yet, unidentified carcinogens or their precursors in the diet, the formation of high levels of ROS through metabolic processes is well established (13). Biologically important ROS which can damage DNA, thereby altering gene expression, cell growth and differentiation (14,15), include the hydroxyl radical (·OH), superoxide (O2·), peroxyl radical, singlet oxygen, peroxynitrite and hydrogen peroxide. As oxidative DNA damage is considered to be a pathogenic event in the induction of many cancers (16,17), a reduction in the rate of such damage by catechins acting as antioxidants may lead to a reduced risk of cancer. It is known that catechins are efficient scavengers of ROS per se (18,19) and inhibit the formation of 8-oxodeoxyguanosine, a marker for oxidative damage sustained by DNA (20). A reduction in the rate of oxidative DNA damage in humans, in terms of this marker, has also been observed following the ingestion of foods which contain high amounts of glucosinolates (21), but similar studies have not been done with foods containing flavonoids. However, concentrations of only a few micromolar of the tea catechins have been measured in human plasma after the ingestion of green tea (22), similar to the basal plasma level of other glycosidic flavonoids arising from the diet in non-supplemented humans (23). It is unlikely that the reported health benefits from such low concentrations of catechins can arise through their interception of ROS, thus preventing oxidative DNA damage.
We are studying mechanisms on how such low concentrations of catechins could lead to health benefits in humans other than direct scavenging of ROS. Our initial studies have shown that catechins can undergo electron transfer (or H-atom transfer) to ROS-damaged sites on DNA (24). This antioxidant reaction results in the fast chemical repair of the DNA in so much as eliminating DNA radicals, thereby reducing the amount of both base damage and strand breaks. This mechanism for the abatement of DNA dysfunction arising through ROS attack could be significant as certain DNA radicals, such as the alkylperoxyl radical on the deoxyribose backbone, are known to possess life-times in the order of seconds (25,26) permitting reactions with low concentrations of antioxidants to occur in competition to damage fixation.
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Materials and methods
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All chemicals were of the highest analytical grade available and used as received. Epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG) were produced from green tea and purified by Mitsui Norin. Epicatechin and catechin were obtained from ICN Biomedicals, Ohio.
Plasmid DNA, pBR322, and stock solutions of calf thymus DNA (Sigma-Aldrich, Germany) were prepared and purified as previously described (24). Plasmid DNA Forms I and II were separated on 1% agarose gels by electrophoresis in TAE buffer. Quantification of the bands following ethidium bromide staining (applying a calibrated enhancement of 1.3 to the closed circular image) was done using the NIH Image software package.
Test solutions of plasmid (8 µg/ml = 2.82 nM in base pairs) and added catechins were irradiated in the presence of air using a 1.5x104 GBq 60Co
-ray source providing a dose rate of 30 Gy/min (Fricke dosimetry) at room temperature (22°C). Irradiated samples were withdrawn serially and kept on ice until subsequent analysis. Under these conditions the radiation breaks down the water into quantified amounts (in µmol/J = µM/Gy) of free radicals of which the ·OH radical is the main oxidizing species with the reducing H· atoms and eaq being converted to superoxide which does not damage DNA to any significant extent (27).
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The effects of catechins on DNA radicals, in terms of altering the yield of residual DNA damage, was studied by maintaining a constant ·OH radical scavenging capacity, k. The proportionate protection of the plasmid from single-strand break (ssb) formation and other damage through direct radical scavenging [i.e. intercepting ·OH radicals, by Tris buffer (reaction 3) and catechins, F (reaction 4), before they react with DNA] was maintained by reducing the concentration of Tris buffer (
10 mM, pH 7.4) as increasing concentrations of catechins were added to maintain a k = 1.5x107 s1 (24). By maintaining a low k value, strand breakage and base damage almost exclusively arise directly or indirectly from reactions initiated by a small proportion of the ·OH radical yield, produced on the irradiation of water, which is formed in close proximity to the DNA (28), reactions (5).
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DNA single-strand breaks (conversion of Form I to II) following irradiation in the presence and absence of added catechins were determined in Tris buffer containing 1 mM phosphate and 0.1 mM EDTA. The radiation dose required on average to produce one strand break in Form I, Do (in Gy); values for the loss of Form I due to ssb formation were obtained from the slopes of the regression lines of the percentage of Form I as a function of dose using Do = [(log1037) 2]/slope. Do values were used to calculate the G value (radiation chemical yield) from triplicate experiments for ssb formation (in µM/Gy) where G(ssb) = [DNA] (µM in base pairs)/Do (in Gy)x
(kg/l, assumed to be unity).
DNA base damage was evaluated as increased amount of strand breakage G(ssb') formed upon incubating irradiated plasmid (in the presence and absence of catechins) with the DNA glycosylases formamidopyrimidine-DNA glycosylase (FPG), endonuclease III (EndoIII) and 5' AP endonuclease exonuclease III (ExoIII). While all three proteins recognize sites of base loss with unmodified deoxyribose moiety and deoxyribose oxidized in the 4' position, ExoIII additionally recognizes deoxyribose oxidized in the 1' position, FPG recognizes 7,8-dihydro-8-oxoguanine and formamidopyrimidines and EndoIII recognizes 5,6-dihydropyrimidines (29,30). EndoIII protein was kindly supplied by Dr R.P.Cunningham (State University New York), FPG protein was purified from an over-expressing strain of Escherichia coli, a kind gift from Dr Y.W.Kow (Emory University School of Medicine, Georgia) and ExoIII was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Irradiated samples were incubated with each protein prior to gel electrophoresis as previously described (24), in a total volume of 20 µl for 30 min at 37°C, as well as incubation buffers alone. The difference in G values between the control and protein-treated cases gives the G value representing the increase in strand breakage arising from the action of each protein, G(ess). The difference in these G values when each of the catechins are present at the time of the irradiations gives the catechins-induced reduction in strand breaks and base damage which is recognized by the proteins. Subtracting this second calculated value from G(ess) yields the G(ssb') value for base damage (recognized by the proteins) which is influenced by the catechins.
Pulse radiolysis studies and the associated dosimetry was carried out using a Dynaray 4 linear accelerator as previously described (31). Pulses of electrons (typically 24 Gy in 200 ns) were used to initiate radical reactions and time-resolved measurements were made of electron transfer from catechins to radical sites on DNA formed by ·OH radical attack. Solutions of calf thymus DNA (2 mM in base pairs) and various concentrations of catechins (20200 µM) were irradiated at pH 7.4 (5 mM phosphate) in 4:1 (v/v) N2O/O2 mixture-saturated solutions. Under these conditions the yield of ·OH radicals is increased (to 0.56 µM/Gy) and react with the DNA leading to a range of DNA radicals, some of which react with oxygen to presumably form peroxyl radicals, reaction (7). Such radicals have been shown previously to be reduced by flavonoids (24) through electron transfer (or H-atom transfer), as both a fast phase independent of oxygen being present, and a slow kinetic phase only present in oxygen-containing solutions, reaction 8.
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Results
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In order to prepare DNA test solutions at a constant radical scavenging capacity, k, when adding catechins, it was necessary to determine the second-order rate constants for the reaction of ·OH radical with catechins. This was done as previously described using pulse radiolysis where competition kinetics plots are constructed for the yield of each of the catechin radicals in mixtures containing dimethylsulphoxide in N2O-saturated solution (24). The rate constants for reaction (4), calculated relative to the rate constant for the scavenging of ·OH radicals by dimethylsulphoxide of 6.6x109 M1 s1 (32), are presented in Table I
together with the literature values of the one-electron reduction potentials, E(1), of the catechins. The E(1) of ECG was determined utilizing the N·ISOdia3 radical and using methyl gallate, E(1) = 0.56 V, as the reference compound, in a redox equilibrium as previously described (33).
DNA strand breakage in plasmid pBR322 following irradiation in the presence and absence of each of the four major catechins in green tea was followed by gel electrophoresis. Examples of the radiation doseresponse curves (average of three separate experiments) for the loss of supercoiled Form I in the presence and absence of 100 µM EGCG, at a constant k of 1.5x107 s1, are presented in Figure 2
. D0 values from such plots were used to calculate the yield of strand breaks (Gssb) for each of the four catechins at both low and high concentration (10 and 100 µM) and are presented in Figure 3
. All catechins were found to be active in reducing the amount of DNA strand breaks, with EGCG being slightly more active than the other compounds. Adding catechins (100 µM) to irradiated controls immediately after irradiation had no modifying effect. Four concentrations of EGCG were tested, at the 1, 2, 10 and 100 µM level for DNA strand breaks and base damage by incubating irradiated plasmid with the endonucleases FPG, ExoIII and EndoIII. While the low concentrations are at physiological levels, the highest concentration results in the maximum, or near to the maximum, in the reduction of single-strand breaks when present during irradiation. Averaged doseresponse curves for EGCG following post-irradiation incubation with the proteins are also presented in Figure 2
and the calculated G (ssb') [together with G(ssb)] values presented in Figure 4
. The most active catechin, EGCG, reduces the amount of prompt DNA strand breaks, at the physiologically significant concentrations of 12 µM, as well as damage to both purine and pyrimidine bases, but higher concentrations are necessary to affect AP sites (Figure 4
).

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Fig. 3. Yields of DNA strand breaks due to the direct action of ·OH radicals and their reduction in the presence of catechins EC, ECG, EGC and EGCG (100 µM). Error bars represent the standard deviation derived from the average of the Do values.
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Fig. 4. Yields of DNA strand breaks due to the direct action of ·OH radicals and their reduction in the presence of EGCG (1, 2, 10 and 100 µM) and the formation of additional DNA strand breaks upon incubation with FPG, EndoIII and ExoIII endonucleases which recognize a variety of residual base damages. Error bars represent the standard deviation derived from the average of the Do values.
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The kinetics of electron transfer from catechins to the DNA radicals formed in N2O/O2-saturated solutions were determined by observing the formation of the one-electron oxidized catechin species which have absorption peaks at 310 nm (33). For comparison purposes between the catechins, the pseudo first-order rate constants at DNA and catechin concentrations of 2 mM and 40 µM, respectively, are presented in Table I
. The percentage of electron transfer, relative to the maximum observable level of oxidation of each catechin induced upon reaction with the SeO3· radical, were determined for each catechin. Plateaus in the percentage electron transfer were reached at ca. 100200 µM concentrations, as previously seen for other flavonoids (24). These percentage electron transfer values for the fast kinetic phase and for the total observed transfer (the sum of both kinetic phases) are also presented in Table I
. Qualitatively, the rates of electron transfer, as well as the percentage of electron transfer, for both kinetic phases, are higher for catechins whose radicals are of lower one-electron reduction potential. As previously reported (24), only a proportion of the DNA damage arising through ·OH radical attack can be reduced by fast chemical reaction with the flavonoids. The present data indicates that ~40% of the damage sustained to DNA through ·OH radical attack can be reduced in this way.
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Discussion
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This in vitro study shows that catechins of green tea are highly active in reducing the amount of oxidative damage sustained by DNA through ·OH radical attack. Catechins, when compared with other classes of flavonoids (24), are found to be very active in reducing the amount of strand breakage and residual base damage by a mechanism other than direct scavenging of ·OH radicals before they react with DNA. Pulse radiolysis data, both in this and a previous study (24), support the mechanism of electron transfer (or H-atom transfer) from catechins to radical sites on DNA. Both a high percentage and increased rate of electron transfer qualitatively correlate with increased efficiency in reducing DNA damage. Restitution of the DNA in this way results in the strand remaining intact and the range of free radical-induced base damage being reduced to forms which are no longer recognized by a range of endonucleases as damaged sites. While it is likely that the fast chemical reduction of DNA damage through the proposed mechanism results in a high degree of fidelity in repair, our studies so far do not address this. A system in which the functioning of a protein transcribed from a DNA test system, following free radical damage both in the presence and absence of catechins, could yield more specific information. However, clearly, catechins and other classes of flavonoids exert an antioxidant effect in the protection of DNA other than by direct scavenging of ·OH radicals per se. Any reduction in the rate of damage to DNA may result in a decreased risk of cancer (16,17).
Reaction of ·OH radicals with DNA gives rise to a wide range of radical intermediates on all of the DNA bases, as well as H-atom abstraction from different sites on the ribose moiety. In the case of thymidine, for example, 65% addition occurs on the C-5 position to yield the corresponding 6-yl radical, 20% 5-yl radical formation and 10% allylic radical through H-atom abstraction from the methyl group (34). Carbon-centred radicals may react at near diffusion-controlled rates with oxygen to produce peroxyl radicals, reaction (7). Peroxyl radicals formed at the 5 or 6 position on pyrimidines have been proposed to be precursors of DNA strand breaks (35,36) and this proposal is directly supported by the demonstration of DNA strand breakage upon the in situ production of a 5-peroxyl radical on thymidine in DNA (37). DNA strand breaks arising from peroxyl radical formation on the ribose have been shown in the action of bleomycin (38). Our proposal is that catechins exert an antioxidant effect on peroxyl radicals, thus preventing DNA strand breaks or radical-induced base damage, through electron (or H-atom) transfer to form the hydroperoxide. Hydroperoxides, formed in free solution and on lipids, are known to induce DNA damage and mutations through a Fenton-type reaction with transition metals to produce peroxyl radicals (39). Such a reaction seems to be also possible with adventitious metal ions bound to DNA, with the peroxyl radicals produced from diffusing hydroperoxides being able to be scavenged by certain antioxidants (40). Our results support a similar antioxidant mechanism for catechins but in addition with them acting directly on peroxyl radicals formed on the DNA. The results in Figure 4
indicate that EGCG does repair a similar proportion of the radical precursors for both strand breaks and base damage when present at high concentration. However, since FPG and EndoIII proteins both possess activity for AP sites, and that EGCG is relatively inactive at low concentrations on the radical precursors sites recognized by ExoIII, it can be deduced that purine and pyrimidine damaged sites are more efficiently repaired by catechins.
The significance of our findings await definitive studies on the uptake of the different classes of flavonoids and their distribution in cells. Flavonoids from green tea are aglycosidic and as such are relatively small uncharged molecules which should pass through cellular membranes, but it is presently unknown if they can be concentrated inside the cell. Modest binding to DNA has been reported for the flavonol class of flavonoids, e.g. quercetin (4143), which possess planar A and C rings; however, it is not known if similar weak binding can occur between catechins and DNA. However, even if there is little increased uptake in cells over the concentrations measured in plasma, our data suggest that catechins can have activity in ameliorating DNA damage at the micromolar level. Concentrations of antioxidants in the micromolar range cannot compete with cellular constituents, which are present in much higher concentrations, for the scavenging of highly reactive ROS species such as the ·OH radical. Hence, any observed health benefits from catechins are highly unlikely to arise from their direct scavenging of radicals, although this still has to be ruled out as an operative antioxidant mechanism in vitro and in vivo for catechins. It is known that low concentration of certain phenolic antioxidants (related to the B ring of catechins) can induce detoxification enzymes (e.g. glutathione S-transferases and superoxide dismutase) (44) which can act against the toxic and neoplastic effects of carcinogens. An induction of superoxide dismutase may decrease the concentration of ROS through scavenging O2. that is initially produced. However, such enzymes do not act on DNA damage and our findings could represent another biochemical mechanism to counter the consequences of ROS-induced DNA damage.
Amelioration of free radical damage on DNA by micromolar concentrations of catechins must also be viewed in the context of the other antioxidants present in the cell. Concentrations of vitamins C and E in human plasma have been reported as 42 ± 12 and 31 ± 3 µM, respectively (45), with vitamin C being concentrated to millimolar levels in certain cells, such as in the brain (46). Glutathione, the primary non-protein thiol in mammalian cells, is also found at several millimolar concentrations (47). All three antioxidants possess E(1) values for their radicals in a similar region to catechins, 0.430.57 V (33,48), viz. vitamin C, 0.28 V (49), Trolox© (a water soluble form of vitamin E), 0.48 V (50) and glutathione, 0.75 V (51). We have found recently that these antioxidants do not ameliorate free radical damage to DNA in our plasmid test system (52), despite it being thermodynamically possible for electron transfer to take place to radical sites on DNA which are known to be of higher E(1), i.e. >1.0 V (53,54). It may be that the negative charges borne by vitamin C, Trolox and the thiolate ion are repelled by the negatively charged phosphate backbone of DNA inhibiting electron transfer. (This argument would not apply to neutral vitamin E, but, as it is mainly sequestered into membranes, it is not likely to be available to reduce oxidative damage on DNA.) The much higher concentrations of vitamin C and glutathione than catechins in the cell means that these antioxidants could still possibly function as scavengers of a proportion of the ROS diffusing to the DNA.
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Notes
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3 To whom correspondence should be addressedEmail: r.anderson{at}auckland.ac.nz 
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Acknowledgments
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This research was financially supported by grants from the Health Research Council of New Zealand and the Lottery Grants Board.
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Received February 19, 2001;
revised April 17, 2001;
accepted April 17, 2001.