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1990 Oxford University Press 1217. Free-radical reactions induced by OH-radical attack on cytosine-related compounds: a study by a method combining ESR ...
© 1990 Oxford University Press 1217

Nucleic Acids Research, Vol. 18, No. 5

Free-radical reactions induced by OH-radical attack on cytosine-related compounds: a study by a method combining ESR, spin trapping and HPLC Wakako Hiraoka, Mikinori Kuwabara*, Fumiaki Sato, Akira Matsuda1 and Tohru Ueda1 Department of Radiation Biology, Faculty of Veterinary Medicine and 1 Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan Received November 14, 1989, Revised and Accepted January 30, 1990

Free-radical reactions induced by OH-radical attack on cytosine-related compounds were investigated by a method combining ESR, spin trapping with 2-methyl-2-nitrosopropane and high-performance liquid chromatography (HPLC). Cytidine, 2'-deoxycytidine, cytidine 3'-monophosphate, cytidine 5'-monophosphate, 2'-deoxycytidine 5'-monophosphate and their derivatives, of which 5,6-protons at the base moiety were replaced by deuterons, and polycytidylic acid (poly(C)) were employed as samples. OH radicals were generated by X-irradiating an N2O-saturated aqueous solution. Five spin adducts were separated by HPLC. Examination of them by ESR spectroscopy and UV photospectrometry showed that spin adducts assigned to C5 and C6 radicals due to OH addition to the 5,6 double-bond, a deaminated form of the spin adduct derived from a C5 radical due to the cyclization reaction between C5' of the sugar and C6 of the base, and a spin adduct assigned to the C4' radical due to H abstraction by OH radicals were produced. From these results the sites of OH-radlcal attack and the subsequent radical reactions in cytosine-related compounds were clarified. INTRODUCTION Oxidative damage inflicted by reactive oxygen species is associated with the induction of carcinogenesis, aging, inflammation, radiation damage, and photobiological effects (1). The reactions of oxygen species in diverse biological molecules have been studied by biochemical and radiation-chemical methods, and OH attack on DNA has been shown to have a serious effect on living organisms (2). Therefore, the characterization of final products through OH-induced intermediates in DNA is important for elucidating oxidative stress in biology. It is well-known that when DNA is exposed to OH radicals, a deamination reaction occurs at the cytosine base moiety and results in the formation of a uracil base, and uracil glycol and urea as oxidative base damage. Furthermore, as a result of

* To whom correspondence should be addressed

OH-radical attack on the cytosine base, its inherent products such as 5-hydroxycytosine and 3-carbamoyl-4-hydroxyhydantoin have also been reported (3). Recent studies have elucidated the presence of some enzymes which serve in the repair processes of these modified cytosine residues (4-7), and radiation-induced mutagenicity at cytosine residues has been mentioned (8). This report aims to get information about the chemical processes in the cytidine moiety of DNA starting with OH attack to produce free radicals and ending with the formation of stable products. For this purpose, we employed both spin trapping and highperformance liquid chromatography (HPLC), because we have successfully applied this method to studies on OH-induced free radical reactions in uracil-, thymine-, adenine- and guaninerelated compounds (9-15). In the present study, to further confirm the identification of free radicals which can be analyzed by ESR spectra, we synthesized 5,6-deuterated derivatives and subjected them to spin trapping experiments. Furthermore, to estimate whether the information obtained from free radical reactions in nucleoside or nucleotide levels can be justified to that from polymerized forms, we employed polycytidylic acid (poly(C)). In this case poly(C) was digested by RNase A prior to the separation of the spin adducts by the HPLC system. The results showed that OH radicals induced four base radicals and one sugar radical. From the results, the sites of OH-radicaJ attack and the subsequent radical reactions in cytosine-related compounds were elucidated. MATERIALS AND METHODS Chemicals and Enzyme. Cytidine, 2'-deoxycytidine.HCl, cytidine 5'-monophosphate.2Na, and 2'-deoxycytidine 5'-monophosphate.2Na were purchased from Sigma Chemical Company. Their derivatives, of which 5,6-protons at the base moiety were replaced by deuterons, were prepared according to the method of Maeda and Kawazoe (16). 'H-NMR analysis with a JEOL GX-270 confirmed that the purities of deuterated samples were almost 100%. Polycytidylic acid sodium salt (poly(Q, S20, W=9.2, M r =4xl0 5 ) was purchased from Seikagaku Kogyo Company, Ltd., Japan and was desalted by dialysis against triply

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ABSTRACT

1218 Nucleic Acids Research distilled water before use. Ribonuclease A (RNase A) from bovine pancreas (Type HI-A, EC 3.1.27.5) was purchased from Sigma Chemical Company. 2-Methyl-2-nitrosopropane (MNP) was obtained from Aldnch Chemical Company. Spin trapping of OH-induced free radicals Aqueous solutions of cytosine-related compounds (20mM) other than poly(C) were prepared with triply distilled water. Two milligrams of MNP powder was put into 1 ml of the solution, which was then bubbled with N2O gas for 20 min and sealed in a Pyrex tube. In the case of poly(C), 24 mg of the compound and 2 mg of MNP powder were put into 4 ml of triply distilled water, after which the same procedure was followed. The successful dissolution of MNP powder was effected by stirring the solution overnight at 28 °C in the dark. To generate OH radicals, the N2O-saturated solutions were X-irradiated to a dose of 2.7 kGy with a Toshiba KXC-18 X-ray source, operating at 170 kVp and 25 mA.

O.SmT

Separation of spin adducts by HPLC. The irradiated solutions were applied to HPLC to separate the spin adducts. HPLC was carried out using a Tosoh CCP&8000 system equipped with a TSK gel ODS-80TM reverse-phase column (4.6 mm diameter, 250 mm long) (Tosoh 10^m). In the case of nucleosides, the analysis was accomplished by a 15-rrun linear-gradient from water to water/methanol (95:5 v/v), followed by a 40-rrun lineargradient from water/methanol (95:5 v/v) to methanol, at a flow rate of 1 ml/min. In the case of nucleotides or digested poly(C) the analysis was performed with a 60-min linear-gradient from 0.02 M triethylammonium acetate (pH 6.5) to 0.02 M triethylammonium acetate/methanol (1:1 v/v) at a flow rate of 1 ml/min. The eluted solution was monitored for UV absorbance at 260 run with 1.28 absorbance units full scale. The presence of the spin adduct in each fraction was ascertained by ESR spectroscopy. ESR and UV absorbance measurements The separated spin adducts were characterized by ESR spectroscopy and UV spectrophotometry. ESR measurements were made on a JEOL ME-IX X-band spectrometer. The ESR spectra were recorded as first derivatives at room temperature. The ESR scans were traced with a 100-kHz field modulation of 0.02 mT amplitude and the microwave power level was maintained at 10 mW. UV absorbance spectra were recorded with a Hitachi 340 spectrophotometer. Prior to examination of the spin adducts by UV spectrophotometry, the solution was freeze-dried to remove methanol or volatile triethylammonium acetate, and again dissolved in 0.6 ml of triply distilled water. RESULTS The ESR spectra of spin-trapped radicals show a primary triplet splitting due to the I4N nucleus and secondary splittings that arise from the magnetic nuclei of the trapped radical. These primary and secondary splittings are utilized to identify the chemical structure of the spin adduct (17). The a, /3 and y

K)

15

20

25

30

40

Retention Time (min) Figure 1. (a) ESR spectrum of spin-trapped radicals obtained from an aqueous solution containing cytidine, MNP and N2O immediately after X irradiation (b) Elution profile of X-irradiated aqueous solution containing cytidine, MNP and N2O separated by reverse-phase HPLC Separation conditions are described in the text

positions of the magnetic nuclei are defined with respect to the unpaired electron on the nitrogen of the nitroxide group (18). Figure la shows a typical ESR spectrum of spin-trapped radicals obtained from an X-irradiated aqueous solution containing cytidine, MNP and N2O. The overlapping of several signals was observed. Therefore, the next step was to separate the spin adducts by HPLC. The elution profile is presented in Figure lb When each peak was examined by ESR spectroscopy, signals were detected from the four fractions denoted a - d . The ESR and UV absorbance spectra obtained from each fraction containing a spin adduct are shown in Figure 2. The hyperfine splittings (hfs) of ESR spectra are summarized in Table 1. The analysis of the separated spin adduct with UV absorbance was performed based on the presence of maximum absorbances at 235 and 270 nm derived from two chromophores of the cytosine base (shown in Figure 2a: dotted line). The spin adduct recovered in fraction a showed a loss of maximum absorbances at both 235 and 270 nm in the UV-absorbance spectrum (Figure 2a). The spin adducts recovered in fractions b and c showed that one chromophore was preserved (Figures 2b and 2c), whereas the spin adduct recovered in fraction d had an intact UVabsorbance spectrum (Figure 2d). These results suggested that the sites forming the spin adducts were the base moiety for spin adducts in fractions a, b and c, and the sugar moiety for the spin adduct in fraction d. The ESR spectra in Figures 2a and 2b consisted of a primary triplet hfs which further split into a doublet

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Enzymic digestion of spin adducts derived from poly(C). RNase A was dissolved in 10 mM acetate buffer (pH 6.5) The Xirradiated poly(C) solution was dialyzed against the buffer for 4 h. Then the solution was incubated with 5 kilounits of enzyme at 37°C for 3 h. The digestion of poly(C) by RNase A is known to usually give the 3'-monophosphate form of cytidine.

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Figure 2. ESR spectra (left) and UV-absorbance spectra (right) obtained from peaks a - d in Figure lb The hyperfine structures are shown at the bottom of each ESR spectrum. The UV-absorbance spectrum drawn with a dotted line is that of undamaged cytidine

hfs, suggesting that the spin adducts had structures in which a proton at the /3-position interacts with a spin of the nitroxide group. The ESR spectrum in Figure 2c consisted of a primary triplet hfs which further split into 3x2 lines. This means that a proton and a nitrogen are present at the ^-position in the spin adduct and interact with a spin of the nitroxide group. The ESR spectrum in Figure 2d consisted of only a primary triplet, suggesting that no protons or no nitrogens are present at the /3position. Figures 2a and 2b seem to be assigned to spin adducts at the C5 position of the cytosine base moiety because a proton is situated in the j3-position, which permits the interaction with a spin. Replacement of the C5 proton by a deuteron affected the shapes of ESR spectra as shown in Figures 3a and 3b. This is due to the fact that the magnitude of hfs decreases to 0.154 when a proton is replaced by a deuteron (18). This result strongly supports the conclusion that the spin adducts were formed at the C5 position of the base moiety.

Spectra similar to those in Figures 2a and 2b were obtained in all cytosine-related compounds as shown in Table 1. Here, the untrapped forms of radicals denoted (I) in Table 1 correspond to the ESR spectrum shown in Figure 2a and the untrapped forms of radicals denoted (II) correspond to the ESR spectrum shown in Figure 2b. Careful examination of the hfs values denoted (I) and (IT) in Table 1 teaches us that for spin adducts corresponding to radical (I), the phosphate group at the C5' position gives relatively large values (0.5 mT) for the secondary doublet hfs, as shown in cytidine 5'-monophosphate and 2'-deoxycytidine 5'-monophosphate, but cytidine 3'-monophosphate and poly(C), in which the phosphate group is presented at the C3' position, gave values (0.36—0.40 mT) similar to those of cytidine and 2'-deoxycytidine (0.36 mT). For spin adducts corresponding to the radical denoted (H), values similar to that of the secondary doublet due to a /3-proton were obtained for all samples, irrespective of the position of the phosphate group. These results suggest that the negative charge of the phosphate group at the

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Wavelength (nm)

1220 Nucleic Acids Research Table 1. Hyperfme coupling constants of spin-trapped radicals formed by the reactions of OH radicals with cytosine-related compounds

compound

Primary I 4 N splitting(mT)

Secondary splitting aj(mT)

Untrapped* radical

0 36{H) 0.4404) 0440*) 0 24{N)

0) 0D

cytidine

1 50 1 48 1 49

0V)

1 50

2'-deoxy-cytidine

1 48 1 48 1 49

0 3601) 04401) 04801) 0 1204)

(D OD

on OV)

1 48 1 53 1 53 1 53

03601) 0 44(H) 0 5501) 0 1001)

0) OD

1 53

044O0

on)

0 24(N)

01')

cytidine 5'monophosphate

1 54 1 54

0 500D 0 43O0

0)

2'-deoxycytidine 5'monophosphate

1 54 1 53

0 5001) 0 38OD

0) OD

1 54 1 53 1 49

0400D 0 3601) 0 4501) 0 1401) 0 5001) 0 27(N)

0) OD

poly(C) 1 49 1 52

OD

01')

(in) OV)

'Untrapped radicals denoted (I) —(IV) correspond to those shown in Diagram I

C5' position of the sugar moiety can interact with a spin of the nitroxide group at the C5 position of the base moiety to give relatively large hfs values. This interaction is possible when a covalent bond is formed between the C5' position and the C6 position by intramolecular attack of the C5' radical on the 5,6-double bond of the base moiety. Therefore, combining these results with the fact that the spin adduct of this ESR spectrum lost the maximum UV absorbances at both 235 and 270 nm, the deaminated form of the spin adduct derived from the 5',6-cyclo-6-hydrocytidine C5 radical (Diagram I) seems to be consistent with the ESR spectrum in Figure 2a. A complete loss of the chromophore indicates that a deaminadon reaction occurred at the C4' position of the base moiety to alter the cytosine base to the uracil base. In general, it is well known that the hydrolyticdeamination reaction progresses in 64iydroxy-5,6-dihydrccytidine and 5,6-dihydrocytidine (19). The possible pathway leading to this spin adduct shown in diagram II is one is which the hydrogen is abstracted at C5' by an OH radical [1], the C5' radical undergoes intramolecular attack at the C6 position producing a 5,6-cyclo-6-hydrocytidine C5 radical [2], this radical is trapped by MNP [3], and the produced spin adduct is then deaminated at the C4 site [4]. When uridine 3'-monophosphate was subjected to spin-trapping experiments, the formation of the spin adduct at the C5 position gave a UV-absorbance spectrum similar to that in Figure 2a (12). The ESR spectrum in Figure 2b, which corresponds to untrapped radical (II) in Table 1, was assigned to the spin adduct

U

***A/*^^ I

I

J

Figure 3. ESR spectra of spin-trapped radicals obtained from an X-irradiated aqueous solution containing 5,6-deuterated cytidine, MNP and N 2 O separated by reverse-phase HPLC. Separation conditions were identical to those of Figure lb The spin adducts marked a - d were those recovered at the retention times corresponding to a—d in Figure lb.

due to the C5 radical formed by OH-addition to the C6 position of the double bond (see Diagram I), since no phosphate effects were observed on the ESR spectra. Maximum UV-absorbance at 235 nm indicates the presence of an amino group at the C4 position. The ESR spectrum in Figure 2c was assigned to the spin adduct between the C6 radical (TO) and MNP (Diagram I), because a proton and a nitrogen were situated in the /3-position, which permits interaction with the unpaired electron. This assignment was confirmed by the fact that when 5,6-deuterated cytidine was used, an ESR spectrum consisting only of a secondary triplet due to a nitrogen was observed (Figure 3c). The hfs due to a proton at the C6 position disappeared by being replaced by a deuteron. Since the maximum UV-absorbance at 235 nm was preserved in this adduct, an amino group was thought to be present at the C4 position.

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cytidine 3'monophosphate

(UI)

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NH,

HOCH

C5 Radical OH OH

(II) (ID

5',6-Cyclocytidine C5 Radical

(1)

H

H

N

HO-CH^,

OH OH C6 Radical

C4' Radical

(in)

(IV)

Diagram I.

NH2

OH OH

NH2

OH OH

OH OH

Diagram II.

DISCUSSION

The ESR spectrum in Figure 2d was assigned to the spin adduct between the C4' radical (IV) and MNP (Diagram I). This spin adduct had no protons and nitrogens at the /3-position. The fact that the replacement of 5,6-protons by deuterons had no effect on this ESR spectrum, as shown in Figure 3d, further supported this assignment. As other candidates, the C2' and C3' radicals were considered, but the C2' radical was excluded because a

This study was performed to analyze the OH-induced free radicals in cytosine-related compounds. Until now OH-induced free radicals in cytosine-related compounds have been poorly characterized compared with the other nucleic-acid constituents because of the complex reactivity of cytosine base. Nevertheless, E. coli endonuclease HI and mammalian DNA glycosylase/endonuclease, which can recognize modified cytosine residues, have recently been reported (3-7). Therefore, it seems

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VH2

similar spectrum was obtained from 2'-deoxycytidine. Our previous study of TMP proved that the possibility that the spin adduct was formed at the C3' position is less likely (15). Figures 4a and 4b show the ESR spectra obtained from 2'-deoxycytidine and its 5,6-deuterated derivative, respectively. An ESR spectrum consisting of a secondary doublet due to a /3proton that further split into a doublet due to a 7-proton was newly found in this sample, in addition to the ESR spectra described above. When 5,6-deuterated 2'-deoxycytidine was used instead of ordinary 2'-deoxycytidine, the ESR spectrum obtained from the corresponding spin adduct showed the lack of a secondary hyperfine structure. From this result it was concluded that the spin adduct assigned to the C5 radical of the base was consistent with this ESR spectrum (denoted II' in Diagram I and Table 1), because this adduct had protons at the /3- and 7-positions. The spin adducts derived from one radical species such as the C5 radical (II or II') must be used to explain the two ESR spectra shown in Figures 2a and 4a. This may be due to the formation of two stereoisomers. Similar observations were obtained from 3'-UMP (12) and TMP (15). The ESR spectrum shown in Figure 4b consisted of a somewhat broad signal. This may be explained by the interaction of a spin with two deuterons at me C5 and C6 positions. The UV-absorbance spectrum shows the presence of maximum absorbance at 235 nm, suggesting that the amino group is present at the C4 position. X irradiation of an aqueous solution containing cytidine 3'-monophosphate, MNP and N2O produced the spin adducts (ESR spectra not shown). A total of four spin adducts were identified (Table 1). These adducts were the same as those of cytidine and deoxycytidine except for the spin adduct of the C4' radical. In the case of nucleoside monophosphates, no spin adducts arising from the C4' radical were detected, probably due to the instability of the corresponding spin adducts. X irradiation of an aqueous solution containing poly(C), MNP and N2O produced the ESR spectrum. The ESR spectrum consisted of a broad line due to slow tumbling of the nitroxide group in the polymer (ESR spectra not shown). The digestion of poly(C) with RNase A made the signal sharp. The separation of spin adducts in the digested poly(C) by HPLC with the ionsuppression mode proved the presence of five spin adducts (Table 1). AJI adducts were the same as those obtained from nucleosides and nucleotides. The hfs due to the 5-proton in the deaminated spin adduct derived from the 5',6-cyclo-6-hydrocytidine C5 radical produced in poly(C) was identical to that of cytidine 3'-monophosphate. This was shown by the fact that poly(C) was digested into a 3'-nucleotide form by RNase A. However, HPLC retention times of the spin adducts in poly(Q were different from those of cytidine 3'-monophosphate. This may be explained by the production of altered hydrolysates at the sugar moiety in Xirradiated poly(C) or changes in the susceptibility of X-irradiated poly(Q to digestion by RNase A.

1222 Nucleic Acids Research

1.0

©

o (0 .Q k.

o

200

250

300

Figure 4. (a) ESR and UV-absorbance spectra of the spin adduct recovered in one fraction of an X-irradiated aqueous solution containing 2'-deoxycytidine, MNP and N 2 0 separated by reverse-phase HPLC Separation conditions were identical to those of Figure lb (b) ESR spectrum of spin adduct recovered in one fraction of an X-irradoated aqueous solution containing 5,6-deuterated 2'-deoxycyudine, MNP and N2O separated by reverse-phase HPLC Separation conditions were identical to those of Figure lb The retention times of a and b were approximately identical

that the OH-induced chemical reactions in the cytosine moiety of DNA leading to end-products should be elucidated. Since, in our previous studies, a method combining spin trapping and chromatography was shown to be a powerful approach for the radical-mediated degradation processes in nucleic acid constituents (9—15), we applied this method to elucidate the OHinduced radical processes in cytosine-related compounds. First, free radicals derived from OH addition at the C5 or C6 position, which can be regarded as the precursors for main end-products such as 3-carbamoyl-4,5-dihydroxy-2-oxo-imidazolidine, urea, uracil glycol, 5-hydroxycytosine and dimers (20-24), were detected. Second, the spin adduct assigned to the C4' radical of the sugar moiety was detected. The C4' radical was thought to be the precursor of 2-deoxypentos-4-ulose and 2-deoxypentos-4-ulose-5-phosphate, which were shown in 7 radiolysis of 2'-deoxycytidine 5'-monophosphate. These products in DNA are thought to be connected to the chain scission of DNA. Finally, the deaminated form of the spin adduct derived from the 5',6-cyclo-6-hydrocytidine C5 radical was detected. We assigned this spin adduct for the following reasons. [1] The UVabsorbance spectrum of this spin adduct indicated the possibility that the base radical was trapped, and also indicated that the deamination reaction occurred at the C4-NH2 group of the cytosine base moiety. [2] The effects of proton-deuteron exchange at the 5,6-positions on the ESR spectrum suggested the site at which the radical was induced was the C5 or C6 position. [3] The negative charge of the phosphate group at the C5' position affected hfs values of the ESR spectrum, suggesting that the C5' and C6 positions were linked by an intramolecular cyclization reaction. [4] The ESR spectrum showed no hfs due to the /3nitrogen, suggesting that the only candidate for the trapped site was the C5 position. [5] No ESR spectrum consistent with the C5' radical was detected in the cytosine-related compounds, while the ESR spectra arising from the C5' radical were observable in other pyrimidine nucleosides and nucleotides (9,11,12,15) as well as punne nucleosides and nucleotides (10,14). This

discrepancy can be interpreted by assuming that the 5',6-cyclo-6-hydrocytidine C5 radical was formed by the intramolecular attack of the C5' radical on the C6 of the base in the case of cytosine-related compounds. [6] Shaw and Cadet reported the formation of 5',6-cyclo-5,6-dihydro-2'-deoxyuridine when 2'-deoxycytidine was irradiated with 7-rays in the frozen state (25). Although the present study was carried out in the aqueous state, both direct and indirect effects of irradiation on DNA constituents have been known to produce the C5' radical (9,11,12,14,15,26). The spin adduct assigned to the C4' radical of the sugar moiety was detected. The C4' radical was thought to be the precursor of 2'-deoxypentos-4-ulose and 2'-deoxypentos-4-ulose-5phosphate, which were shown in 7 radiolysis of 2'-deoxycytidine 5'-monophosphate (27). These products in DNA are thought to be connected to the chain scission of DNA. It is important to note that the spin adducts on polynucleotides were stable enough for hard treatments such as enzymic digestion and HPLC. In the experiment with poly(C) it was demonstrated that the reactivities of OH radicals with nucleosides and nucleotides were identical to those in macromolecules. We identified the precursor radicals for various oxidized base alterations as well as the precursor radical for strand breaks in cytosine-related compounds. All radicals except for the 5',6-cyclo-6-hydrocytidine C5 radical could be regarded as precursors for OH-induced degradation products of cytosine residue in DNA. It is noteworthy that [1] the C5' radical can attack the 5,6-double bond to make cyclization compounds and result in a change inducing deamination at the C4 to alter cytosine to uracil, and [2] OH addition at C5 or C6 of the base moiety causes no deamination, while the next radical step is probably needed for deamination. The finding of the deaminated form of the spin adduct derived from the 5',6-cyclo-6-hydrocytidine C5 radical in the present study suggests the possibility that a 5',6-cyclo-compound, which has already been reported in the direct effects of ionizing radiation on cytosine-related compounds

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Wavelength (nm)

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(25), will be found even in the case of indirect effects of radiation. It is also important to look for enzymes to repair the products formed from the intermediates reported here. ACKNOWLEDGMENTS This work was supported in part by the Grants-In-Aid (No. 01790487 for W.H. and No. 01560333 for M.K.) from the Ministry of Education, Science and Culture of Japan. REFERENCES 1 2 3 4 5

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

23 24 25 26 27

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Sies, H (1985) Oxidative Stress Academic Press, London Chapman, D and Gillespie, C J (1981) Adv Radial Biol , 9 , 143-198 Teoule, R (1987) Int. J Radiat Biol , 51, 573-589 Teebor, G W and Frenkel, K (1983) Adv. Cancer Res , 38, 23-59 Doetsch, P W , Helland, D E and Haseltine, W E. (\9S6) Biochemistry, 25, 2212-2220 Weiss, R B and Duker, N J (1986) Nucleic Acids Res, 14, 6621-6631 Weiss, R B , Gallagher, P E , Brent, T P and Duker, N J (1989) Biochemistry, 28, 1488-1492 Ayaki, H , Higo, K. and Yamamoto, O (1986) Nucleic Acids Res , 14, 5013-5018 Kuwabara, M , Zhang, Z -Y and Yoshn, G (1982) Int J Radial Biol , 41, 241-259 Kuwabara, M , Inanami, O and Sato, F (1986) Int J Radiat Biol , 49, 829-844 Inanami, O , Kuwabara, M , Endoh, D and Sato, F (1986) Radiat Res , 108, 1-11 Inanami, O , Kuwabara M and Sato, F (1987) Radiat Res , 112, 36-44 Kuwabara, M , Inanami O , Endoh, D and Sato, F (1987) Biochemistry, 26, 2458-2465 Hiraoka, W , Kuwabara, M and Sato, F (1989) Int J Radiat Biol , 55, 51-58 Kuwabara, M , Hiraoka, W and Sato, F Biochemistry, 28, 9625-9632 Maeda, M and Kawazoe, Y (1975) Tetrahedron Letters, 19, 1643-1646 Janzen, E G (1971) Ace Chem. Res , 4, 31-40 Riesz, P and Rustgi, S (1979) Radiat. Phys Chem , 13, 21-40 Ueda, T (1988) In Townsend, L B (ed ), Chemistry of Nucleosides and Nucelotides Plenum Press, New York and London, 1, 1-112 von Sonntag, C and Schuchmann, H -P (1986) Int J Radiat Biol , 49, 1-34 Teebor, G W , Boorstein, R. J and Cadet, J (1988) Int J Radiat. Biol , 54, 131-150 Teoule, R , Borucel, A , Managgi, N and Polverelli, M (1983) In Broerse, J J , Barendsen, G W , Kal, H B and van der Kogel, A J (ed ), Proceedings of the Seventh International Congress of Radiation ResearchChemistry and Physics Martinus Nijhoff Publishers, Sessions A, A3 - 4 3 Polverclh, M , Bomcel, A and Teoule, R (1976) J Radial Res., 17, 127-134 Schuchmann, H -P , Wagner, R and von Sonntag, C. (1983) 2 Nanuforsch , 38b, 1213-1220 Shaw, A A and Cadet, J (1988) Int. J Radiat Biol, 54, 987-997 Zhang Z -Y , Kuwabara, M and Yoshn, G (1983) Radiat Res , 93 213-231. Dizdaroglu, M and Sirruc, M G. (1984) Radial Res , 100, 41-46