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Fast repair activities of quercetin and rutin toward dGMP hydroxyl radical adducts
Radiation Physics and Chemistry 63 (2002) 137–142
Fast repair activities of quercetin and rutin toward dGMP hydroxyl radical adducts Chenyang Zhaoa, Yimin Shia, Wenfeng Wangb, Weizhen Linb, Botao Fanc, Zhongjian Jiad, Side Yaob, Rongliang Zhenga,* a
b
State Key Laboratory of Arid Agriecology, School of Life Science, Lanzhou University, Lanzhou, China Laboratory of Radiation Chemistry, Shanghai Institute of Nuclear Research, Chinese Academy of Sciences, Shanghai 201800, People’s Republic of China c Institute de Topologie et de Dynamique des Systems, University Paris 7, Paris 75005, France d State Key Laboratory of Applied Organic Chemistry, Lanzhou 730000, People’s Republic of China Received 28 August 2000; accepted 10 March 2001
Abstract The repair activities and mechanisms of both quercetin and rutin towards the oxidizing deoxyguanosine monophosphate (dGMP) hydroxyl radical adduct were investigated with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mM dGMP aqueous solution containing 0.1 mM quercetin, the transient absorption spectrum of the dGMP hydroxyl radical adduct decays with the formation of phenoxyl radical of quercetin within tens of microseconds. It indicates that there is a repair reaction between dGMP hydroxyl radical adduct and quercetin. The repair activity of rutin towards hydroxyl radical adducts of dGMP was also observed. The rate constants of the repair reactions were calculated to be 3.05 108 and 1.31 108 M1 s1 for quercetin and rutin, respectively. This result together with our previous studies demonstrated that non-enzymatic, fast repair is a universal repair mechanism of phenolic antioxidants. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: DNA damage; dGMP hydroxyl radical adducts; Fast repair; Quercetin; Rutin
1. Introduction In living aerobic cells, reactive oxygen species (ROS) are continuously generated by inflammatory, ischemiareperfusion injury, and xenobiotic metabolism as well as physiological mitochondria respiration (Floyd, 1990; Klebanoff, 1988). By escaping the cell’s antioxidant defenses, ROS can damage biomolecules including DNA (Terry and Lawrence, 1998). Oxidative DNA damage generated thereby has been thought to be an important source of mutation leading to aging and a wide range of degenerative diseases, such as cardiovascular disease (Ames et al., 1993), immune-system decline (Bashir et al., *Corresponding author. Tel.: +86-931-8912563: fax: +86931-8911100. E-mail address: [email protected] (R. Zheng).
1993; Lunec et al., 1994), brain dysfunction (Ames et al., 1993), and cataracts (Ames et al., 1993). Generally, cells can efficiently repair most of DNA damage through effective enzymatic repair systems (Wallace, 1997). However, the repair is not perfect, even under existing SOS process allowing misrepaired products to occur. On the other hand, DNA repair capacity decreases and more deleterious oxygen species arise with the progressing of aging. Both changes result in accumulation of DNA damage, and increasing mutation frequency. All this means that DNA damage is produced and always exists prior to DNA replication, and hence the possibility of mutation leading to degenerative diseases always exists. It is important to look for exogenous antioxidants which either can scavenge ROS prior to DNA damage or can repair damaged DNA to supplement the inadequate repair capacity of cells.
0969-806X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 0 1 ) 0 0 2 2 4 - 9
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C. Zhao et al. / Radiation Physics and Chemistry 63 (2002) 137–142
Much attention has been focused on the scavenging activity of antioxidants against DNA damage from ROS attacking. The concentrations of biomolecules in cells are much higher than those of antioxidants, thus, the reaction of hydroxyl radical which has very high reactivity (Steenken, 1989) with biomolecules is very difficult to prevent even by the most reactive hydroxyl radical scavengers in vivo unless the concentration of antioxidants is high enough. Therefore, the strategies for prevention of DNA damage induced by hydroxyl radical, should concentrate on the elimination or neutralization of the secondary DNA radicals resulting from the hydroxyl radical reactions, that is non-enzymatic repair of DNA damage or fast repair (Simic, 1989). The natural antioxidants, such as thiols and ascorbate, can react with oxidizing hydroxyl radical adducts of deoxyguanosine monophosphate (dGMP) and deoxyguanosine (dG) with high rate constants (3.6 1078.4 108 M1 s1) (O’Neill, 1983), hydroxycinnamic acid derivatives can fast repair hydroxyl adducts of dGMP (Jiang et al., 1997). In our laboratory, the fast repair activities of phenylpropanoid glycosides (PPGs) and their analogs towards hydroxyl radical adducts of dGMP, deoxyadenosine monophosphate (dAMP), polyadenylic acid (poly A), polyguanylic acid (poly G) and single strand or double strand DNA(Li et al., 1996a, 1997b; Shi et al., 1999b, c), thymine radical anion (Li, et al., 1997a, 1996b), thymidine monophosphate (TMP) radical anion (Shi et al., 2000), radical cations of dGMP and dAMP (Shi et al., 1999a) have been found. However, more evidence is required to prove the universality of fast repair of DNA damage by antioxidants. Flavonoids are the representatives of a large and complex group of phenolic compounds that occur throughout the plant kingdom and are synthesized in most plant tissues, providing colour, flavour, antifungal and anti-bacterial activities, and contributing to many aspects of plant physiology (Bors and Saran, 1987; Bors et al., 1990; Jovanovic et al., 1994; Gee et al., 1998). These polyphenols known as free radical scavengers (Bors et al., 1994) have beneficial action in cardiovascular disorders (Jovanovic et al., 1996), inhibit H2O2induced V79 cell death, and prevent DNA single strand breakage (Piero et al., 1998). However, their potential to fast repair DNA damage has not been investigated. The present study focuses the attention on the repair of dGMP hydroxyl radical adduct by two representative flavonoids: quercetin and rutin.
All the other reagents were obtained from Shanghai Biochemical Co. (Shanghai, China). All solutions were freshly prepared with triple distilled water before each experiment and were used immediately. The solutions were buffered with phosphate (2 mM, pH 7.0), and were saturated with high purity nitrous oxide (>99.99%) by bubbling for 20 min before irradiation to enable conversion of e aq to the hydroxyl radical. All experiments were carried out in room temperature. 2.2. Pulse radiolysis Pulse radiolysis experiment was conducted by using a linear accelerator providing 8 MeV electron pulse with a duration of 8 ns. A 2 cm Suprasil quartz cell was used for sample irradiation. Thiocyanate dosimetry was used for 3 1 dose determination, assuming e ðSCNÞ ¼ 7600 dm mol 1 cm at 480 nm in nitrous oxide saturated 10 mM KSCN aqueous solution. The average pulse dose was 14 Gy. The source of analyzing light was a 500 W xenon lamp and its intensity was increased about 100 times during the detection of transient absorption. The transmitted light entered a 44 W monochromator equipped with a IP28 photomultiplier. The signals were gathered by a 100 MHz transient recorder, and processed with a computer. The rate constants of one-electron oxidation of flavonoids were determined by analysis of the buildup trace of the absorption of phenoxyl radical. 2.3. Generation of dGMP hydroxyl radical adduct On pulse irradiation of 2 mM dGMP aqueous solutions saturated with N2O, hydrated electrons (e aq ), hydroxyl radical ( OH) and hydrogen atoms (H) are produced with G’s (mM J1) of 0.29, 0.29 and 0.06, respectively (Asmus, 1983). e aq was scavenged by saturation of N2O to form OH, and then OH reacts with dGMP to generate hydroxyl radical adduct of dGMP (dGMP–OH ) (Eqs. 1–3). H2 O ! OH þ e aq þH;
ð1Þ
e aq þN2 O ! OH þ OH þ N2 ;
ð2Þ
dGMP þ OH ! dGMP2OH :
ð3Þ
3. Results 2. Materials and methods 3.1. The transient absorption spectrum of dGMP–OH 2.1. Materials Quercetin (Q), Rutin (R) and 20 -deoxyguanosine-50 monophosphate (dGMP) were purchased from Sigma.
On pulse radiolysis of 2 mM dGMP aqueous solution saturated with N2O at pH 7.0, an optical transient absorption spectrum with lmax ¼ 320 nm was measured
C. Zhao et al. / Radiation Physics and Chemistry 63 (2002) 137–142
139
Fig. 1. Transient absorption spectrum (at 5 ms) upon pulse radiolysis of 2 mM dGMP aqueous solution saturated with N2O at pH 7.0. Inset: the buildup trace of absorption at 330 nm.
at 3 ms after pulse irradiation (Fig. 1), which reached its maximum at 5 ms after pulse irradiation (Fig. 1, inset). This transient absorption spectrum was assigned to dGMP–OH . 3.2. Transient absorption spectra of quercetin and rutin A transient absorption spectrum with lmax ¼ 450 nm appeared at 5 ms after pulse irradiation on 0.1 mM quercetin aqueous solution saturated with nitrous oxide (Fig. 2B). This transient absorption spectrum was very similar to the transient absorption spectrum observed on pulse radiolysis of 0.1 mM quercetin aqueous solution containing 20 mM K2S2O8 and 200 mM t-BuOH saturated with N2. In the latter system, OH is scavenged by t-BuOH to form the t-BuOH(–H) radical, while H 2 and e (Eqs. 4–6). eq react with S2O8 to produce SO4 t-BuOH þ OH !t-BuOHð2HÞ þH2 O;
ð4Þ
2 e eq þS2 O8 !
þSO2 4 ;
ð5Þ
2 H þ S2 O2 8 ! SO4 þSO4 :
ð6Þ
SO4
Then, the SO4 reacts with quercetin to form phenoxyl radicals (PhO ) (Eq. 7): þ Q þ SO4 ! R2PhO þSO2 4 þH :
ð7Þ
Bansal and Fessenden (1978) have shown that phenoxyl radicals will be formed if hydroxyl radical attacks on phenolic hydroxyl groups, and therefore, the transient absorption spectrum shown in Fig. 2B should be assigned to quercetin phenoxyl radical (Q–PhO ). The transient absorption spectrum of rutin phenoxyl radical (R–PhO ) was observed by the same process (Fig. 2A).
Fig. 2. Transient absorption spectra upon pulse radiolysis of rutin and quercetin aqueous solution saturated with N2O at pH 7.0. (A) 0.1 mM rutin, at 25 ms; (B) 0.1 mM quercetin, at 75 ms.
3.3. Fast repair activity of quercetin and rutin to dGMP–OH Upon pulse radiolysis of 2 mM dGMP aqueous solution containing 0.1 mM quercetin and saturated with N2O, the transient absorption spectrum of dGMP–OH arose firstly at 1 ms after pulse irradiation (Fig. 3B-a). At 60 ms after pulse irradiation, the transient absorption of Q–PhO ascended, and at the same time the transient absorption spectrum of dGMP–OH disppeared (Fig. 3B-b). This change of transient absorption spectrum indicated that a reaction takes place between dGMP–OH and Q. With a similar process, the repair activity of rutin towards dGMP–OH was also observed (Fig. 3A). 3.4. The rate constants for repair reaction The inset in Fig. 3 shows the change of absorption of R–PhO and Q–PhO at 460 nm with time after the
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4. Discussion The repair system for dGMP–OH was designed according to the principle of competing reactions. This repair system consisted of 2 mM dGMP nitrous oxide saturated aqueous solution containing 0.1 mM either quercetin or rutin and 2 mM phosphate buffer (pH 7.0). The following reactions (Eqs. 8 and 9) must occur simultaneously in the repair system upon pulse irradiation: dGMP þ OH ! dGMP2OH
ðk1 Þ;
ð8Þ
QðorRÞ þ OH ! QðRÞ2PhO
ðk2 Þ:
ð9Þ
The reaction rate constants at pH 7.0 of OH with dGMP and quercetin are 4.7 109 (Buxton et al., 1988) and 4.3 109 M1 s1 (Bors et al., 1990), respectively. According to the mechanism of competing reactions, the reaction probability (P) of dGMP with OH is calculated by the following formula: P ¼ k1 ½dGMP =ðk1 ½dGMP þk2 ½Q Þ ¼0:966:
Fig. 3. Transient absorption spectra upon pulse radiolysis of N2O saturated 2 mM dGMP aqueous solution and containing (A) 0.1 mM rutin, at 2 and 60 ms and (B) 0.1 mM quercetin, at 1 and 60 ms (pH 7.0). Inset: the buildup trace of absorption at (A) 460 nm, (B) 460 nm.
Table 1 The rate constants of repair reaction of dGMP–OH with quercetin and rutin
dGMP–OH
Quercetin /108 M1 s1
Rutin /108 M1 s1
1.31
3.05
This calculated result means that 96.6% of OH produced react with dGMP to form dGMP–OH . The P of dGMP reacting with OH in repair system containing 0.1 mM rutin was calculated to be 97.4%. Therefore, it may be assumed that in the present repair system OH reacts with dGMP predominantly to generate dGMP–OH . O’Neill and Chapman (1985) showed that addition of OH to dGMP produced two types of OH adducts with respect to their redox properties, oxidizing OH adduct (50%) and reducing OH adduct (50%). The oxidizing dGMP–OH is formed mainly by the addition of OH to C4 (dGMP-4-OH ) of the purine ring. With unpaired spin density located on O atom, dGMP-4-OH is oxidizing, and hence can react with the effective antioxidants, quercetin and rutin. It reverts to dGMP or hydrated dGMP (Eqs. 10 and 11), and quercetin and rutin convert to their phenoxyl radical. This situation is shown by the result of the present study, where the transient absorption spectrum of dGMP–OH was substituted for that of Q–PhO or R–PhO . dGMP2C4 2OH þQðorRÞ ! ðdGMP2OHÞ þQðorRÞ2PhOþHþ ;
ð10Þ
ðdGMP2OHÞ þHþ ! dGMP þ H2 O or hydrated dGMP: pulse irradiation. By varying the concentration of the tested flavonoids in the repair system and analyzing the kinetics curves, the rate constants of the repair reaction of dGMP–OH by the tested flavonoids were deduced and shown in Table 1.
ð11Þ
The additions of hydroxyl radical on C5 and C8 of purine ring forming the mainly reducing radicals dGMP–C5–OH and dGMP–C8–OH . These adducts cannot be repaired by quercetin and rutin. Because dGMP–C4–OH is oxidizing and Q or R is reducing, the reaction between them is a redox reaction,
C. Zhao et al. / Radiation Physics and Chemistry 63 (2002) 137–142
which is completed through transfer of an electron from Q or R to dGMP–C4–OH . Thus, the fast repair process of oxidizing dGMP hydroxyl radical adducts may be regarded as a procedure of electron transfer. It may be inferred theoretically that a chemical that has adequate reduction potentials would have possibility of being a good antioxidant and be able to repair the oxidizing dGMP–OH . The result of present study fits well with this assumption. Reacting with ROS at very high rate constants and forming quite stable phenoxyl radical, quercetin and rutin are well known as potent antioxidants. These properties are also important for fast repair of oxidizing dGMP–OH . On the one hand, the adequate reduction potential enables quercetin and rutin to fast reduce the oxidizing dGMP–OH , and on the other hand, especially high stability of Q–PhO and R–PhO prevent these phenoxyl radicals reacting with other biomolecules. Our previous study showed that the values of rate constants of fast repair reaction by PPGs toward oxidizing dGMP–OH are positively related to the number of phenolic hydroxyl groups (Shi et al., 1999c). However, by comparison of values of the rate constants in Table 1 with those of repair reactions of oxidizing dGMP–OH by PPGs, it can be found that the rate constants of repair reaction of oxidizing dGMP–OH by quercetin and rutin are lower than that by PPGs by one order, although the number of phenolic hydroxyl group in both flavonoids is higher than that of PPGs. This fact demonstrated that the number of phenolic hydroxyl groups is an important factor, but not the sole factor.
5. Conclusion Owing to the generation of ROS being continuous in normal cell metabolism and the scavenging activity not great enough, damage to biomolecules is inevitable. Damaged DNA can be repaired by enzymatic systems, but enzymatic repair of steady DNA damage occurs in a time scale of hours (Yakes and van Houten, 1997). The repair enzymes are proteins that are sensitive to ROS, and the fast repair reactions are initiated and completed in the time scale of microseconds. As a result, they prevent these transient products from reacting with other biological macromolecules. With the understanding of the general redox reaction of fast repair processes, it may be possible to find much more potent antioxidants to protect DNA against ROS. It can also enrich our knowledge of traditional medicines and improve the therapy for free radical related diseases. In summary, the result of this study together with our previous studies demonstrated that non-enzymatic, fast repair is a universal repair mechanism exerted by phenolic antioxidants.
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Acknowledgements This project was supported partly by the National Natural Science Foundation of China, the Doctoral Programme of the Ministry of Education of China, and the China-France Cooperative Research Sponsored by the Ministry of Education of China.
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Shi, Y.M., Lin, W.Z., Kong, J.H., Zheng, R.L., Jia, Z.J., 2000. Fast repair of TMP radical anion by phenylpropanoid glycosides and their analogs. Radiat. Phys. Chem. 58, 131–138. Shi, Y.M., Wang, W.F., Shi, Y.P., Zheng, R.L., Jia, Z.J., 1999b. Fast repair of dAMP hydroxyl adducts by verbasicoside via electron transfer. Sci. China C 42, 621–627. Shi, Y.M., Wang, W.F., Zheng, R.L., Jia, Z.J., 1999c. Fast repair of dAMP and dGMP hydroxyl adducts by phenylpropanoid glycosides and their analogs. Biochim. Biophys. Acta 1472, 115–127. Simic, G., 1989. Mechanisms of inhibition of free-radical processes in mutagenesis and carcinogenesis. Mutat. Res. 202, 377–386. Steenken, J., 1989. Purine bases, nucleosides, and nucleotides: aqueous solution redox chemistry and transformation reaction of their radical cations and e and OH adducts. Chem. Rev. 89, 503–520. Terry, G.N., Lawrence, A.L., 1998. Oxidative DNA damage and mutagenesis. In: Nickoloff, J.A., hoekstra, M.F. (Eds.), DNA Damage and Repair. Vol. 1. DNA Repair in Prokaryotes and Lower Eukaryotes. Humana Press Inc., Totowa, NJ, pp. 65–84. Wallace, S.S., 1997. Oxidative damage to DNA and its repair. In: Scandalios, J.G. (Ed.), Oxidative Stress and the Molocular Biology of Antioxidant Defenses. Harbor Laboratory Press, Cold Spring, pp. 49–90. Yakes, F.M., van Houten, B., 1997. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Natl. Acad. Sci. USA. 94, 514–519.
Fast repair activities of quercetin and rutin toward dGMP hydroxyl radical adducts Chenyang Zhaoa, Yimin Shia, Wenfeng Wangb, Weizhen Linb, Botao Fanc, Zhongjian Jiad, Side Yaob, Rongliang Zhenga,* a
b
State Key Laboratory of Arid Agriecology, School of Life Science, Lanzhou University, Lanzhou, China Laboratory of Radiation Chemistry, Shanghai Institute of Nuclear Research, Chinese Academy of Sciences, Shanghai 201800, People’s Republic of China c Institute de Topologie et de Dynamique des Systems, University Paris 7, Paris 75005, France d State Key Laboratory of Applied Organic Chemistry, Lanzhou 730000, People’s Republic of China Received 28 August 2000; accepted 10 March 2001
Abstract The repair activities and mechanisms of both quercetin and rutin towards the oxidizing deoxyguanosine monophosphate (dGMP) hydroxyl radical adduct were investigated with pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mM dGMP aqueous solution containing 0.1 mM quercetin, the transient absorption spectrum of the dGMP hydroxyl radical adduct decays with the formation of phenoxyl radical of quercetin within tens of microseconds. It indicates that there is a repair reaction between dGMP hydroxyl radical adduct and quercetin. The repair activity of rutin towards hydroxyl radical adducts of dGMP was also observed. The rate constants of the repair reactions were calculated to be 3.05 108 and 1.31 108 M1 s1 for quercetin and rutin, respectively. This result together with our previous studies demonstrated that non-enzymatic, fast repair is a universal repair mechanism of phenolic antioxidants. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: DNA damage; dGMP hydroxyl radical adducts; Fast repair; Quercetin; Rutin
1. Introduction In living aerobic cells, reactive oxygen species (ROS) are continuously generated by inflammatory, ischemiareperfusion injury, and xenobiotic metabolism as well as physiological mitochondria respiration (Floyd, 1990; Klebanoff, 1988). By escaping the cell’s antioxidant defenses, ROS can damage biomolecules including DNA (Terry and Lawrence, 1998). Oxidative DNA damage generated thereby has been thought to be an important source of mutation leading to aging and a wide range of degenerative diseases, such as cardiovascular disease (Ames et al., 1993), immune-system decline (Bashir et al., *Corresponding author. Tel.: +86-931-8912563: fax: +86931-8911100. E-mail address: [email protected] (R. Zheng).
1993; Lunec et al., 1994), brain dysfunction (Ames et al., 1993), and cataracts (Ames et al., 1993). Generally, cells can efficiently repair most of DNA damage through effective enzymatic repair systems (Wallace, 1997). However, the repair is not perfect, even under existing SOS process allowing misrepaired products to occur. On the other hand, DNA repair capacity decreases and more deleterious oxygen species arise with the progressing of aging. Both changes result in accumulation of DNA damage, and increasing mutation frequency. All this means that DNA damage is produced and always exists prior to DNA replication, and hence the possibility of mutation leading to degenerative diseases always exists. It is important to look for exogenous antioxidants which either can scavenge ROS prior to DNA damage or can repair damaged DNA to supplement the inadequate repair capacity of cells.
0969-806X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 6 X ( 0 1 ) 0 0 2 2 4 - 9
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C. Zhao et al. / Radiation Physics and Chemistry 63 (2002) 137–142
Much attention has been focused on the scavenging activity of antioxidants against DNA damage from ROS attacking. The concentrations of biomolecules in cells are much higher than those of antioxidants, thus, the reaction of hydroxyl radical which has very high reactivity (Steenken, 1989) with biomolecules is very difficult to prevent even by the most reactive hydroxyl radical scavengers in vivo unless the concentration of antioxidants is high enough. Therefore, the strategies for prevention of DNA damage induced by hydroxyl radical, should concentrate on the elimination or neutralization of the secondary DNA radicals resulting from the hydroxyl radical reactions, that is non-enzymatic repair of DNA damage or fast repair (Simic, 1989). The natural antioxidants, such as thiols and ascorbate, can react with oxidizing hydroxyl radical adducts of deoxyguanosine monophosphate (dGMP) and deoxyguanosine (dG) with high rate constants (3.6 1078.4 108 M1 s1) (O’Neill, 1983), hydroxycinnamic acid derivatives can fast repair hydroxyl adducts of dGMP (Jiang et al., 1997). In our laboratory, the fast repair activities of phenylpropanoid glycosides (PPGs) and their analogs towards hydroxyl radical adducts of dGMP, deoxyadenosine monophosphate (dAMP), polyadenylic acid (poly A), polyguanylic acid (poly G) and single strand or double strand DNA(Li et al., 1996a, 1997b; Shi et al., 1999b, c), thymine radical anion (Li, et al., 1997a, 1996b), thymidine monophosphate (TMP) radical anion (Shi et al., 2000), radical cations of dGMP and dAMP (Shi et al., 1999a) have been found. However, more evidence is required to prove the universality of fast repair of DNA damage by antioxidants. Flavonoids are the representatives of a large and complex group of phenolic compounds that occur throughout the plant kingdom and are synthesized in most plant tissues, providing colour, flavour, antifungal and anti-bacterial activities, and contributing to many aspects of plant physiology (Bors and Saran, 1987; Bors et al., 1990; Jovanovic et al., 1994; Gee et al., 1998). These polyphenols known as free radical scavengers (Bors et al., 1994) have beneficial action in cardiovascular disorders (Jovanovic et al., 1996), inhibit H2O2induced V79 cell death, and prevent DNA single strand breakage (Piero et al., 1998). However, their potential to fast repair DNA damage has not been investigated. The present study focuses the attention on the repair of dGMP hydroxyl radical adduct by two representative flavonoids: quercetin and rutin.
All the other reagents were obtained from Shanghai Biochemical Co. (Shanghai, China). All solutions were freshly prepared with triple distilled water before each experiment and were used immediately. The solutions were buffered with phosphate (2 mM, pH 7.0), and were saturated with high purity nitrous oxide (>99.99%) by bubbling for 20 min before irradiation to enable conversion of e aq to the hydroxyl radical. All experiments were carried out in room temperature. 2.2. Pulse radiolysis Pulse radiolysis experiment was conducted by using a linear accelerator providing 8 MeV electron pulse with a duration of 8 ns. A 2 cm Suprasil quartz cell was used for sample irradiation. Thiocyanate dosimetry was used for 3 1 dose determination, assuming e ðSCNÞ ¼ 7600 dm mol 1 cm at 480 nm in nitrous oxide saturated 10 mM KSCN aqueous solution. The average pulse dose was 14 Gy. The source of analyzing light was a 500 W xenon lamp and its intensity was increased about 100 times during the detection of transient absorption. The transmitted light entered a 44 W monochromator equipped with a IP28 photomultiplier. The signals were gathered by a 100 MHz transient recorder, and processed with a computer. The rate constants of one-electron oxidation of flavonoids were determined by analysis of the buildup trace of the absorption of phenoxyl radical. 2.3. Generation of dGMP hydroxyl radical adduct On pulse irradiation of 2 mM dGMP aqueous solutions saturated with N2O, hydrated electrons (e aq ), hydroxyl radical ( OH) and hydrogen atoms (H) are produced with G’s (mM J1) of 0.29, 0.29 and 0.06, respectively (Asmus, 1983). e aq was scavenged by saturation of N2O to form OH, and then OH reacts with dGMP to generate hydroxyl radical adduct of dGMP (dGMP–OH ) (Eqs. 1–3). H2 O ! OH þ e aq þH;
ð1Þ
e aq þN2 O ! OH þ OH þ N2 ;
ð2Þ
dGMP þ OH ! dGMP2OH :
ð3Þ
3. Results 2. Materials and methods 3.1. The transient absorption spectrum of dGMP–OH 2.1. Materials Quercetin (Q), Rutin (R) and 20 -deoxyguanosine-50 monophosphate (dGMP) were purchased from Sigma.
On pulse radiolysis of 2 mM dGMP aqueous solution saturated with N2O at pH 7.0, an optical transient absorption spectrum with lmax ¼ 320 nm was measured
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Fig. 1. Transient absorption spectrum (at 5 ms) upon pulse radiolysis of 2 mM dGMP aqueous solution saturated with N2O at pH 7.0. Inset: the buildup trace of absorption at 330 nm.
at 3 ms after pulse irradiation (Fig. 1), which reached its maximum at 5 ms after pulse irradiation (Fig. 1, inset). This transient absorption spectrum was assigned to dGMP–OH . 3.2. Transient absorption spectra of quercetin and rutin A transient absorption spectrum with lmax ¼ 450 nm appeared at 5 ms after pulse irradiation on 0.1 mM quercetin aqueous solution saturated with nitrous oxide (Fig. 2B). This transient absorption spectrum was very similar to the transient absorption spectrum observed on pulse radiolysis of 0.1 mM quercetin aqueous solution containing 20 mM K2S2O8 and 200 mM t-BuOH saturated with N2. In the latter system, OH is scavenged by t-BuOH to form the t-BuOH(–H) radical, while H 2 and e (Eqs. 4–6). eq react with S2O8 to produce SO4 t-BuOH þ OH !t-BuOHð2HÞ þH2 O;
ð4Þ
2 e eq þS2 O8 !
þSO2 4 ;
ð5Þ
2 H þ S2 O2 8 ! SO4 þSO4 :
ð6Þ
SO4
Then, the SO4 reacts with quercetin to form phenoxyl radicals (PhO ) (Eq. 7): þ Q þ SO4 ! R2PhO þSO2 4 þH :
ð7Þ
Bansal and Fessenden (1978) have shown that phenoxyl radicals will be formed if hydroxyl radical attacks on phenolic hydroxyl groups, and therefore, the transient absorption spectrum shown in Fig. 2B should be assigned to quercetin phenoxyl radical (Q–PhO ). The transient absorption spectrum of rutin phenoxyl radical (R–PhO ) was observed by the same process (Fig. 2A).
Fig. 2. Transient absorption spectra upon pulse radiolysis of rutin and quercetin aqueous solution saturated with N2O at pH 7.0. (A) 0.1 mM rutin, at 25 ms; (B) 0.1 mM quercetin, at 75 ms.
3.3. Fast repair activity of quercetin and rutin to dGMP–OH Upon pulse radiolysis of 2 mM dGMP aqueous solution containing 0.1 mM quercetin and saturated with N2O, the transient absorption spectrum of dGMP–OH arose firstly at 1 ms after pulse irradiation (Fig. 3B-a). At 60 ms after pulse irradiation, the transient absorption of Q–PhO ascended, and at the same time the transient absorption spectrum of dGMP–OH disppeared (Fig. 3B-b). This change of transient absorption spectrum indicated that a reaction takes place between dGMP–OH and Q. With a similar process, the repair activity of rutin towards dGMP–OH was also observed (Fig. 3A). 3.4. The rate constants for repair reaction The inset in Fig. 3 shows the change of absorption of R–PhO and Q–PhO at 460 nm with time after the
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4. Discussion The repair system for dGMP–OH was designed according to the principle of competing reactions. This repair system consisted of 2 mM dGMP nitrous oxide saturated aqueous solution containing 0.1 mM either quercetin or rutin and 2 mM phosphate buffer (pH 7.0). The following reactions (Eqs. 8 and 9) must occur simultaneously in the repair system upon pulse irradiation: dGMP þ OH ! dGMP2OH
ðk1 Þ;
ð8Þ
QðorRÞ þ OH ! QðRÞ2PhO
ðk2 Þ:
ð9Þ
The reaction rate constants at pH 7.0 of OH with dGMP and quercetin are 4.7 109 (Buxton et al., 1988) and 4.3 109 M1 s1 (Bors et al., 1990), respectively. According to the mechanism of competing reactions, the reaction probability (P) of dGMP with OH is calculated by the following formula: P ¼ k1 ½dGMP =ðk1 ½dGMP þk2 ½Q Þ ¼0:966:
Fig. 3. Transient absorption spectra upon pulse radiolysis of N2O saturated 2 mM dGMP aqueous solution and containing (A) 0.1 mM rutin, at 2 and 60 ms and (B) 0.1 mM quercetin, at 1 and 60 ms (pH 7.0). Inset: the buildup trace of absorption at (A) 460 nm, (B) 460 nm.
Table 1 The rate constants of repair reaction of dGMP–OH with quercetin and rutin
dGMP–OH
Quercetin /108 M1 s1
Rutin /108 M1 s1
1.31
3.05
This calculated result means that 96.6% of OH produced react with dGMP to form dGMP–OH . The P of dGMP reacting with OH in repair system containing 0.1 mM rutin was calculated to be 97.4%. Therefore, it may be assumed that in the present repair system OH reacts with dGMP predominantly to generate dGMP–OH . O’Neill and Chapman (1985) showed that addition of OH to dGMP produced two types of OH adducts with respect to their redox properties, oxidizing OH adduct (50%) and reducing OH adduct (50%). The oxidizing dGMP–OH is formed mainly by the addition of OH to C4 (dGMP-4-OH ) of the purine ring. With unpaired spin density located on O atom, dGMP-4-OH is oxidizing, and hence can react with the effective antioxidants, quercetin and rutin. It reverts to dGMP or hydrated dGMP (Eqs. 10 and 11), and quercetin and rutin convert to their phenoxyl radical. This situation is shown by the result of the present study, where the transient absorption spectrum of dGMP–OH was substituted for that of Q–PhO or R–PhO . dGMP2C4 2OH þQðorRÞ ! ðdGMP2OHÞ þQðorRÞ2PhOþHþ ;
ð10Þ
ðdGMP2OHÞ þHþ ! dGMP þ H2 O or hydrated dGMP: pulse irradiation. By varying the concentration of the tested flavonoids in the repair system and analyzing the kinetics curves, the rate constants of the repair reaction of dGMP–OH by the tested flavonoids were deduced and shown in Table 1.
ð11Þ
The additions of hydroxyl radical on C5 and C8 of purine ring forming the mainly reducing radicals dGMP–C5–OH and dGMP–C8–OH . These adducts cannot be repaired by quercetin and rutin. Because dGMP–C4–OH is oxidizing and Q or R is reducing, the reaction between them is a redox reaction,
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which is completed through transfer of an electron from Q or R to dGMP–C4–OH . Thus, the fast repair process of oxidizing dGMP hydroxyl radical adducts may be regarded as a procedure of electron transfer. It may be inferred theoretically that a chemical that has adequate reduction potentials would have possibility of being a good antioxidant and be able to repair the oxidizing dGMP–OH . The result of present study fits well with this assumption. Reacting with ROS at very high rate constants and forming quite stable phenoxyl radical, quercetin and rutin are well known as potent antioxidants. These properties are also important for fast repair of oxidizing dGMP–OH . On the one hand, the adequate reduction potential enables quercetin and rutin to fast reduce the oxidizing dGMP–OH , and on the other hand, especially high stability of Q–PhO and R–PhO prevent these phenoxyl radicals reacting with other biomolecules. Our previous study showed that the values of rate constants of fast repair reaction by PPGs toward oxidizing dGMP–OH are positively related to the number of phenolic hydroxyl groups (Shi et al., 1999c). However, by comparison of values of the rate constants in Table 1 with those of repair reactions of oxidizing dGMP–OH by PPGs, it can be found that the rate constants of repair reaction of oxidizing dGMP–OH by quercetin and rutin are lower than that by PPGs by one order, although the number of phenolic hydroxyl group in both flavonoids is higher than that of PPGs. This fact demonstrated that the number of phenolic hydroxyl groups is an important factor, but not the sole factor.
5. Conclusion Owing to the generation of ROS being continuous in normal cell metabolism and the scavenging activity not great enough, damage to biomolecules is inevitable. Damaged DNA can be repaired by enzymatic systems, but enzymatic repair of steady DNA damage occurs in a time scale of hours (Yakes and van Houten, 1997). The repair enzymes are proteins that are sensitive to ROS, and the fast repair reactions are initiated and completed in the time scale of microseconds. As a result, they prevent these transient products from reacting with other biological macromolecules. With the understanding of the general redox reaction of fast repair processes, it may be possible to find much more potent antioxidants to protect DNA against ROS. It can also enrich our knowledge of traditional medicines and improve the therapy for free radical related diseases. In summary, the result of this study together with our previous studies demonstrated that non-enzymatic, fast repair is a universal repair mechanism exerted by phenolic antioxidants.
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Acknowledgements This project was supported partly by the National Natural Science Foundation of China, the Doctoral Programme of the Ministry of Education of China, and the China-France Cooperative Research Sponsored by the Ministry of Education of China.
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