Fast repair of deoxythymidine radical anions by two polyphenols: Rutin and quercetin

Article abstract of DOI:10.1016/S0006-2952(03)00196-5

The effects of rutin and quercetin on the repair of the deoxythemindine radical anion (dT^?-) were studied using the technique of pulse radiolysis. The radical anion of dT was formed by the reaction of hydrated electron with dT. After pulse irradiation of nitrogen-saturated aqueous solutions containing dT, 0.2M t-BuOH and either rutin or quercetin, the initially formed dT^?-, detected spectrophotometrically, rapidly decayed with the concurrent formation of the radical anion of rutin or quercetin. The results indicated that dT^?- can be rapidly repaired by rutin or quercetin. The rate constants of the repair reactions were determined to be 3.1 and 4.1×10⁹M^-1s^-1 for rutin and quercetin, respectively. With substitution by glycosyl groups at C₃-OH being neighbor to C₄ keto group, which is the active site for electron transfer, rutin has a lower repair reaction rate constant toward dT^?- than quercetin. Together with findings from our previous studies, the present results demonstrated that nonenzymatic fast repair may be a universal form of repair involving phenolic antioxidants.

Full text of DOI:10.1016/S0006-2952(03)00196-5

Biochemical Pharmacology 65 (2003) 1967–1971
Fast repair of deoxythymidine radical anions by two polyphenols:
rutin and quercetin
Chenyang Zhaoa, Yimin Shia, Wenfeng Wangb, Zhongjian Jiac, Side Yaob,
Botao Fand, Rongliang Zhenga,*
aSchool of life Sciences, Lanzhou University, Lanzhou 730000, China
bLaboratory of Radiation Chemistry, Shanghai Institute of Nuclear Research,
Chinese Academy of Sciences, Shanghai 201800, China
cState Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, China
dInstitute de Topologie et de Dynamique des Systems, University Paris 7, Paris 75005, France
Received 10 October 2002; accepted 26 February 2003
Abstract
The effects of rutin and quercetin on the repair of the deoxythemindine radical anion (dTꢀꢁ) were studied using the technique of pulse
radiolysis. The radical anion of dTwas formed by the reaction of hydrated electron with dT. After pulse irradiation of nitrogen-saturated
aqueous solutions containing dT, 0.2 M t-BuOH and either rutin or quercetin, the initially formed dTꢀꢁ, detected spectrophotome-
trically, rapidly decayed with the concurrent formation of the radical anion of rutin or quercetin. The results indicated that dTꢀꢁ can be
rapidly repaired by rutin or quercetin. The rate constants of the repair reactions were determined to be 3.1 and 4:1 ꢂ 109 Mꢁ1 sꢁ1 for
rutin and quercetin, respectively. With substitution by glycosyl groups at C3–OH being neighbor to C4 keto group, which is the active
site for electron transfer, rutin has a lower repair reaction rate constant toward dTꢀꢁ than quercetin. Together with findings from our
previous studies, the present results demonstrated that nonenzymatic fast repair may be a universal form of repair involving phenolic
antioxidants.
# 2003 Elsevier Science Inc. All rights reserved.
Keywords: DNA damage; Fast repair; dT radical anion; Polyphenol; Electron transfer; Pulse radiolysis
1. Introduction
very high intrinsic reactivity with eaqꢁ [2] and pyrimidine is
a much better electron acceptor than purine. The results of
electron paramagnetic resonance studies demonstrated that
base radicals including radical cations and anions, being
primary and major products of ionizing radiation as well as
eaqꢁ addition, can induce strand breaks [3,4] or form stable
base lesions [2]. The enzymatic repair systems in both
prokaryotic and eukaryotic cells can efficiently repair DNA
damage including DNA strand breaks and stable base
lesions [5], but they are not perfect. It is true that some
DNA damage always escape from enzymatic repair sys-
tems, even existing SOS process allowing misrepaired
products present [6]. Moreover, during the process of aging
or disease, metabolism of xenobiotics, DNA damage may
be exacerbated, most of the repair capacity decreases,
resulting in the accumulation of DNA damage and increase
in mutation frequency. This means that DNA damage
always exists and probably leads to degenerative disease
DNA damage is generally regarded as carcinogenic and
actively participates in the process of tumor initiation,
promotion and progression. DNA damage also can be
induced by ionizing radiation, UV light and a variety of
chemical agents as well as reactive oxygen species pro-
duced in the normal metabolism as byproducts. Ionization
can induce DNA damage through two ways: directly occurs
within the DNA itself and generates base radical cations
and base radical anions [1] or indirectly produces hydrated
ꢁ
electrons (eaq ) and hydroxyl radicals (OHꢀ) through ioni-
zation of water in close vicinity to DNA which easily react
with DNA and induce DNA damage. DNA bases have a
* Corresponding author. Tel.: þ86-931-8912563;
fax: þ86-931-8912561.
E-mail address: zhengrl@lzu.edu.cn (R. Zheng).
0006-2952/03/$ – see front matter # 2003 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0006-2952(03)00196-5

1968
C. Zhao et al. / Biochemical Pharmacology 65 (2003) 1967–1971
including cancer and aging. So it is necessary to look for an
approach to supplement the inadequate repair capacity of
cells by scavenging reactive oxygen species or hydrated
electrons prior to damaging DNA or repairing damaged
DNA before DNA replication.
potential to rapidly repair DNA radical anion has not been
investigated.
The current study focuses on the repair of dT radical
anion (dTꢀꢁ) by employing two representative flavonoids:
rutin (R) and quercetin (Q).
Based on this understanding, much attention has been
focused on the scavenging activity of antioxidants to reducꢁe
the risk of cancers and other chronic diseases. The eaq
scavenging potential of some compounds has been inves-
tigated. Cai et al. [7] reported that some flavonoids and
phenolic acids can react with eaqꢁ at close diffusion control
2. Materials and methods
2.1. Materials
rate (109 to 1010
M
ꢁ1 sꢁ1) implicating that these com-
Deoxythymidine (dT), rutin and quercetin were pur-
chased from Sigma. All other chemicals were purchased
from Shanghai Biochemical Corporation.
All solutions were prepared with triple distilled water,
saturated with high purity nitrogen by bubbling for
20 min before irradiation and buffered with phosphate
(2 M, pH 7.0). All experiments were carried out at room
temperature.
pounds may act as efficient scavengers of eaqꢁ. Our study
also showed that phenylpropanoid glycosides and their
ꢁ
analogs are potent eaq scavengers (no published data).
With direct effect of radiation, however, because ionization
occurs within the DNA itself, DNA damage caused thereby
cannot be prevented by antioxidants or eaqꢁ scavengers. In
indirect effect, because of very high reactiꢁvity of the
primary products of ionization of water, eaq and OHꢀ
[2] and the much higher concentration of biomolecules
2.2. Pulse radiolysis
ꢁ
than scavengers in cells, reactions of eaq or OHꢀ with
biomolecules are very difficult to prevent in vivo unless the
concentration of scavengers is sufficiently high. Therefore,
a more effective and feasible strategy to prevent mutation
should be elimination or neutralization of DNA radicals
either resulting from ionization of DNA or generated sec-
ondarily by the attack of OHꢀ and eaqꢁ, that is nonenzymatic
repair of DNA damage or fast repair [8].
Pulse radiolysis experiments were conducted using a
linear accelerator providing 8 MeV electron pulse of 8 ns
duration. The thiocyanate dosimeter was used for dose
determination, assuming e
¼ 7600 dm3 molꢁ1 cmꢁ1
ꢁ
ðSCNÞ
at 480 nm in nitrous oxide saturated 10 mM KSCN aqu-
eous solution. The detailed descriptions of the pulse radi-
olysis equipment and experimental conditions were given
elsewhere [25]. In these experiments, the average dose per
pulse is 14 Gy.
With this consideration, the possible influence of
some chemicals in nonenzymatic repair of DNA damage
have been explored. O’Neill reported the fast repair
effects of endogenous antioxidants, such as thiols and
ascorbate towards oxidizing hydroxyl radical adducts
of dGMP and dG with high rate constants (3:6 ꢂ 107 to
8:4 ꢂ 108 Mꢁ1 sꢁ1) [9,10]. Jiang et al. [11] showed that
hydroxycinnamic acid derivatives can fast repair hydroxyl
radical adduct of dGMP. The rapid electron transfer
reaction between thymidine anion and caffeic acid was
reported by Zou et al. [12]. We found fast repair activities
of polyphenols, phenylpropanoid glycosides and their
analogs, towards hydroxyl radical adducts of dGMP,
dAMP [13–16], poly A, poly G, ssDNA and dsDNA
(doctoral thesis of Shi YM), thymine radical anion
[17,18], TMP radical anion [19], radical cations of dAMP,
dGMP and dCMP [20]. The mechanism of fast repair was
elucidated as either reduction or oxidation of DNA radi-
cals. However, the evidences are still insufficient to estab-
lish the universality of fast repair of DNA damage by
chemicals.
2.3. Generation of hydrated electron
In aqueousꢁsolutions upon pulse radiolysis, hydrated
electrons (eaq ), OHꢀ and hydrogen atoms (Hꢀ) are pro-
duced with G’s (mmol Jꢁ1) of 0.29, 0.29 and 0.06, respec-
tively [26]. OHꢀ is scavenged by t-BuOH:
H2O ! OHꢀ þ eaqꢁ þ Hꢀ
(1)
(2)
t-BuOH þ OHꢀ ! t-BuOHðꢁHÞꢀ þ H2O
3. Results
3.1. Transient optical absorption spectrum
of dT radical anion
On pulse irradiation of 2 mM dT aqueous solution satu-
rated with nitrogen at pH 7.0, a transient optical absorption
ꢁ
The pharmacological significance of polyphenols was
recognized very early. These polyphenols are known to
scavenge free radicals [21], have beneficial action in cardi-
ovascular disorders [22], inhibit H2O2-induced V79 cell
death, prevent DNA single strand breakage [23] and repair
the radical cations of dCMP and poly C [24]. However, their
spectrum arising from reaction of eaq with dT was
observed, and was characterized by an optical absorption
maximum at 340 nm (Fig. 1). This transient absorption
spectrum was assigned to dT radical anion (dTꢀꢁ) [17].
The transient optical absorption reached a maximum at 1 ms
after the pulse irradiation (Fig. 1, inset).

C. Zhao et al. / Biochemical Pharmacology 65 (2003) 1967–1971
1969
Fig. 1. Transient absorption spectrum upon pulse radiolysis of 2 mM dT
(at 4 ms) aqueous solution containing 200 mM t-BuOH and saturated with
N2 at pH 7.0. Inset: the buildup trace of absorption at 340 nm.
3.2. Transient absorption spectra of radical anions
of the tested polyphenols
In the pulse radiolysis of 0.1 mM rutin aqueous solution
containing 200 mM t-BuOH and saturated with nitrogen, a
transient absorption spectrum appeared and was character-
ized by a maximum absorption at 380 nm (Fig. 2A). The
optical absorption reached a maximum after 5 ms (Fig. 2A,
inset).
The transient absorption spectrum of product of reaction
between hydrated electron and quercetin was also observed
with similar condition (Fig. 2B).
Fig. 2. Transient absorption spectra upon pulse radiolysis of 0.1 mM
polyphenols aqueous solution containing 200 mM t-BuOH and saturated
with N2 at pH 7.0. (A) Rutin, at 4 ms; (B) quercetin, at 2 ms. Inset: the
buildup trace of absorption at (A) 380 nm and (B) 370 nm.
ꢁ
With regard to the reactions of polyphenols with eaq
,
the benzoyl or the styryl keto group of polyphenols is the
ꢁ
site on which eaq attacked and resulting in ketyl radical
ions, which are radical anions of polyphenols [7,27].
Therefore, the transient absorption spectra shown in
Fig. 2 shouldꢁbe assigned to radical anion of rutin and
quercetin (Rꢀ and Qꢀꢁ), respectively.
tin, 200 mM t-BuOH and saturated with nitrogen at pH 7.0
(Fig. 3B).
Fig. 3A inset showed the growth of Rꢀ on pulse
radiolysis of 2 mM dT aqueous solution containing
ꢁ
3.3. Repair reaction of dT radical anion by tested
polyphenols
0.1 mM rutin, 200 mM t-BuOH and saturated with nitro-
ꢁ
gen. The curve represents the change of absorption of Rꢀ
at 440 nm with time after the pulse radiation. The growth of
At 1 ms after pulse radiolysis of 2 mM dT aqueous
solution containing 0.1 mM rutin, 200 mM t-BuOH and
saturated with nitrogen at pH 7.0, a transient absorption
spectrum wasꢁobserved (Fig. 3A(a)). This spectrum is same
absorbance follows first order kinetics, from which the
ꢁ
apparent rate constant for the formation of Rꢀ by the
repair reaction, kapp was determined. From the linear
dependence of kapp on the concentration of rutin (0.02–
0.1 mM), the rate constant (k) for electron transfer from
dTꢀꢁ to R was determined. The rate constants of reactions
of tested polyphenols with dTꢀꢁ were deduced and shown
in Table 1.
ꢁ
as that of dTꢀ , therefore the spectrum is assigned to dTꢀ
.
At 15 ms after the pulse irradiation, a new optical absorp-
tion with lmax ¼ 380 nm grows in concurrence with the
ꢁ
disappearance of that of dTꢀ (Fig. 3A(b)). Based on its
similarity with rutin radical anion (Rꢀꢁ), the new transient
absorption spectrum is assigned to Rꢀꢁ. This change of
transient absorption spectrum is duꢁe to one electron trans-
Table 1
The rate constants of repair reaction of dT radical anion by polyphenols
ꢁ
fer from dTꢀ to R by which dTꢀ is restored to dT and
k/108 Mꢁ1 sꢁ1
ꢁ
then Rꢀ is formed.
Rutin
Quercetin
3.1
4.1
The analogous results were observed on pulse radiolysis
of 2 mM dT aqueous solution containing 0.1 mM querce-

1970
C. Zhao et al. / Biochemical Pharmacology 65 (2003) 1967–1971
they may act as electron acceptors, can also react with dT
radical anion (Eq. (6)).
ꢁ
ꢁ
dTꢀ þ R ðQÞ ! dT þ R ðQÞꢀ
However, it is generally suggested that in aqueous solu-
(6)
tion dT radical anion can undergo reversible protonation at
M
ꢁ1 sꢁ1
O4 whose rate constant was kþH ¼ 2:6 ꢂ 1010
þ
andkꢁH ¼ 1 ꢂ 1010
M
ꢁ1 sꢁ1, andcanundergoirreversible
þ
protonation at C6 at rate constants of 5 ꢂ 103 Mꢁ1 sꢁ1
(Eq. (7)) [28].
(7)
Obviously, the rate constants of reversible protonation
are higher than that of repair reaction of dTꢀꢁ by rutin and
quercetin. Thus, in the above repair system the protonation
reaction would compete with repair reaction. Fortunately,
the protonation rꢁeaction of dTꢀꢁ is reversible, and on the
other hand, dTꢀ is a weak base and only partly was
protonated in neutral aqueous solution. Therefore, the
reaction of dT radical anion with rutin and quercetin
can destroy the reversibility, and leads to the equilibrium
(Eq. (6)) toward dT radical anion, and finally reaction 2 is
advantageous in competition between reactions 1 and 2.
Therefore, it is necessary to suppress the protonation at C6,
which would result in generation of stable dihydrothymine.
It is proposed that rutin and quercetin are able to inhibit
protonation that can cause further damage to cell, impli-
cating a potential repair activity.
Fig. 3. Transient absorption spectra upon pulse radiolysis of saturated with
N2 2 mM dT aqueous solution containing 200 mM t-BuOH and (A)
0.1 mM rutin, a: 1 ms, b: 15 ms; (B) 0.1 mM quercetin, a: 1 ms, b: 35 ms.
Inset: the buildup trace of absorption at (A) 440 nm and (B) 460 nm.
For polyphenols acting as electron acceptors, their ben-
zoyl or styryl keto group is the active site, whichever bulky
group being neighbor to the keto would have negative
effects on their electron reception capacities. With glyco-
sylation at C3–OH, rutin has a lower electron reception
capacity than quercetin [7]. The current study also shows
that the rate constant of fast repair reaction of dT radical
anion by rutin is lower than that by quercetin (Table 1).
Concerning cellular DNA, a two-component hypothesis
has been developed. According to this hypothesis, the
electron loss centers (radical cations) end up with the
purines, particularly with the guanine moiety, whereas the
final site of deposition of the ejected electron is with the
pyrimidines, particularly with thymine [2]. In other words,
thetwo-componenthypothesisimpliesthatinDNA thereare
mechanisms of electron and positive hole transfer by which
the initially generated and randomly distributed electron
gain and loss centers are tunneled into the T and G ‘‘traps,’’
respectively. Therefore, it is reasonable to say that by
repairing dT radical anion, rutin and quercetin can repair
indirectly other base radical anions produced in cellular
DNA by radiation, protecting DNA from strand breaks.
As the product of protonation, dihydrothimine present in
DNA is generally considered to be relatively innocuous in
termsofconstitutinga replicative block. However, itappears
4. Discussion
In principle, there are two parallel reactions competing
in the above repair system:
dT þ eaq ! dTꢀꢁ . . . k1
(3)
(4)
ꢁ
R ðor QÞ þ eaq ! R ðor QÞꢀꢁ . . . k2
ꢁ
However, according to the mechanism of competiꢁtion
reaction, the reaction probability (P) of dT with eaq is
calculated by P ¼ k1½dTꢃ=ðk1½dTꢃ þ k2½RꢃÞ to be 0.97
ꢁ
(97%). Therefore, almost all eaq predominantly reacts
with dT to form dT radical anion.
Concerning the reaction of hydrated electron with dT,
the addition of hydrated electron to C4 produces dT radical
anion (Eq. (5)). The product is a reducing radical.
(5)
Although rutin and quercetin have long been known as
efficient natural antioxidants, with benzoyl or styryl keto,

C. Zhao et al. / Biochemical Pharmacology 65 (2003) 1967–1971
1971
[9] O’Neill P. Pulse radiolytic study of the interaction of thiols and
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that accumulation of a large number of dihydrothymines in
DNA may still be a disadvantage for cells since they would
generate a significantly detectable level of termination of
polymerization in vivo. Furthermore, the N-glycosylic bond
of dihydrothymine is more susceptible to hydrolysis than
thymine, therefore apurine/aprymidine (AP) sites would be
formed more frequently from dihydrothymine than from
thymine. It is more important for repair enzymes to recog-
nize and remove dihydrothymine [29].
Cai et al. [7] reported that some flavonoids and phenolic
acids including rutin and quercetin can react with hydrated
electron at close to diffusion control rate meaning they are
efficient scavengers of hydrated electrons. So, rutin and
quercetin not only fast repair DNA damage caused by
radiation, but also scavenge hydrated electron, thereby,
protecting DNA from hydrated electron attack. Therefore,
rutin and quercetin may act as efficient radioprotectors and
therapeutical agents for the diseases related with DNA
damage.
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The result of the current study together with that of our
previous studies demonstrate that nonenzymatic fast repair
may be a universal form of repair involving phenolic
compounds.
[16] Shi YM, Wang WF, Shi YP, Zheng RL, Jia ZJ. Fast repair of dAMP
hydroxyl adducts by verbasicoside via electron transfer. Sci China
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[17] Li WY, Zheng RL, Zhao SL, Jiang Y, Lin NY. Repair effect of thymine
radical anion by echinocoside using pulse radiolysis. Sci China (Series
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Acknowledgments
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phenylpropanoid glycosides on thymine radical anion induced by
pulse radiolysis. Biophys Chem 1997;67:281–6.
This project was supported in part by the National
Natural Science Foundation of China (No. 39870902),
the Doctoral Programme of the Ministry of Education of
China and the China–France Co-operative Research spon-
sored by the Ministry of Education of China.
[19] Shi YM, Lin WZ, Kang JH, Zheng RL, Jia ZJ. Fast repair of TMP
radical anion by phenylpropenoid glycosides and their analogs. Radiat
Phys Chem 2000;58:131–8.
[20] Shi YM, Kang JH, Lin WZ, Zheng RL, Jia ZJ. Fast repair of
deoxynucleotide radical cations by phenylpropanoid glycosides and
their analogs. Biochimica et Biophysica Acta 1999;1472:279–89.
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flavonoid and model phenoyl radicals. Which ring in flavonoids is
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