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In the present communication, we find that R-tocopherol can act as a ... model. R-Tocopherol was found to promote copper-dependent reactive oxygen spe...
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Chem. Res. Toxicol. 1998, 11, 855-862

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r-Tocopherol Induces Oxidative Damage to DNA in the Presence of Copper(II) Ions Naruto Yamashita,† Mariko Murata,† Sumiko Inoue,‡ Mark J. Burkitt,†,⊥ Lesley Milne,§ and Shosuke Kawanishi*,† Department of Hygiene, Mie University School of Medicine, Tsu, Mie 514, Japan, Department of Public Health, Graduate School of Medicine, Kyoto University, Kyoto 606, Japan, and Division of Biochemical Sciences, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, U.K. Received July 25, 1997

There is currently much interest in the possibility that dietary antioxidants may confer protection from certain diseases, such as atherosclerosis and cancer. The importance of R-tocopherol (vitamin E) as a biological antioxidant is widely recognized. However, pro-oxidant properties of R-tocopherol have been observed in chemical systems, and it has been reported that the vitamin can induce tumor formation and act as a complete tumor promotor in laboratory animals. In the present communication, we find that R-tocopherol can act as a potent DNA-damaging agent in the presence of copper(II) ions, using a simplified, in vitro model. R-Tocopherol was found to promote copper-dependent reactive oxygen species formation from molecular oxygen, resulting in DNA base oxidation and backbone cleavage. Neither R-tocopherol nor Cu(II) alone induced DNA damage. Bathocuproine, a Cu(I)-specific chelator, and catalase inhibited the DNA damage, whereas free hydroxyl radical scavengers did not. The order of DNA cleavage sites was thymine, cytosine > guanine residues. Examinations using an oxygen electrode and cytochrome c indicate that molecular oxygen was consumed in the reaction of R-tocopherol and Cu(II) and that superoxide was formed. Stoichiometry studies showed that two Cu(II) ions could be reduced by each R-tocopherol molecule. Electron spin resonance spin-trapping investigations were then used to demonstrate that hydrogen peroxide interacts with Cu(I) to generate the reactive species responsible for DNA damage, which is either the hydroxyl radical or a species of similar reactivity. These findings may be of relevance to the tumorigenic properties of the vitamin reported in the literature. However, further studies are required to establish the significance of these reactions under in vivo conditions.

Introduction Although there is considerable interest in the possibility that vitamin E (R-tocopherol) may be protective against cancer (1-4), several studies have demonstrated that the vitamin can act as a carcinogen, at both the initiation and promotion stages (5-11): the chronic subcutaneous administration of vitamin E in soya oil induces fibrosarcoma formation (5, 6); the vitamin can act as a complete tumor promoter (7) and is reported to enhance intestinal tumorigenesis induction by 1,2-dimethylhydrazine (8, 9); similarly, tumorigenesis in chickens challenged with virus-transformed tumor cells is increased by vitamin E (10); additionally vitamin E significantly elevated liver cancer incidence by continuous oral administration (11). * To whom requests for reprints should be addressed. E-mail: [email protected]. † Mie University School of Medicine. ‡ Kyoto University. § Rowett Research Institute. ⊥ On leave from the Rowett Research Institute, Aberdeen AB21 9SB, Scotland, U.K. Present address: Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-22100 Lund, Sweden. 1 Abbreviations: •OH, hydroxyl radical; H O , hydrogen peroxide; 2 2 DTPA, diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid; MNP, 2-methyl-2-nitrosopropane; SOD, superoxide dismutase; 8-oxodG, 8-oxo-7,8dihydro-2′-deoxyguanosine; HPLC-ECD, electrochemical detector coupled to a high-pressure liquid chromatograph; O2•-, superoxide; MOPS, 3-(N-morpholino)propanesulfonic acid.

R-Tocopherol is widely recognized as being the most important biological antioxidant of the lipid phase (12, 13). Upon the chemical repair of, for example, a chaincarrying lipid peroxyl radical, R-tocopherol undergoes a one-electron oxidation to form the R-tocopheroxyl radical (12). Although the R-tocopheroxyl radical is generally considered to be poorly reactive and to serve as a radical sink, under certain in vitro conditions the radical can abstract a hydrogen atom from the bisallylic methylene groups of polyunsaturated fatty acids, albeit rather slowly (14). Consequently, as well as being an antioxidant, R-tocopherol can also be considered to possess pro-oxidant properties, which has been confirmed experimentally (15-20). Whereas the bisallylic methylene groups of polyunsaturated fatty acids can be oxidized relatively easily, it is considered unlikely that DNA damage by the R-tocopheroxyl radical plays any significant role in tumorigenesis and other disease processes that are associated with pro-oxidative conditions. Indeed, most investigations into the pro-oxidant properties of R-tocopherol have been concerned with low-density lipoprotein oxidation, including those in which the R-tocopheroxyl radical is generated from R-tocopherol using Cu(II) (14-20). Recently, however, it has been suggested that, in the presence of Cu(II) ions (and in the absence of preformed hydroperoxides), the pro-oxidant properties of vitamin E can be accounted for by the formation of hydroxyl radicals (•OH), generated following the interaction of Cu(I) with

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hydrogen peroxide (H2O2) (21). Since •OH is capable of reacting with all biomolecules at essentially diffusioncontrolled rates, formation of the radical and its reaction with DNA following the interaction of R-tocopherol with copper ions may also be responsible for the reported tumorigenic properties of the vitamin (5-11). Indeed, Cu(II) ions are known to bind tightly to DNA, where they can interact with reducing agents (ascorbic acid, glutathione, phenolics, or NADH) and H2O2 (which may be generated in situ from molecular oxygen), resulting in oxidative damage to the nucleic acid, including base modification and strand breakage (22-29). The present investigation was therefore undertaken to explore the possibility that R-tocopherol can promote copper-dependent damage to DNA in a simplified, in vitro model.

Materials and Methods Reagents. The restriction enzymes EcoRI and ApaI were purchased from Boehringer Mannheim GmbH. HindIII, StyI, and T4 polynucleotide kinase were from New England Biolabs, and the human p53 Amplimer Panel was from Clontech Lab, CA. [γ-32P]ATP (222 TBq/mmol) was from New England Nuclear. R-Tocopherol was from Nacalai Tesque Co. (Kyoto, Japan). DTPA and bathocuproinedisulfonic acid were from Dojin Chemical Co. (Kumamoto, Japan). Calf thymus DNA, 2-methyl-2-nitrosopropane (MNP) dimer, catalase (from bovine liver, 45 000 units mg-1), cytochrome c, superoxide dismutase (SOD) (from bovine erythrocytes, 3000 units mg-1), liposome kit for negatively charged liposomes, and the R-tocopherol used in the ESR experiments were from Sigma Chemical Co. Liposome kit for negatively charged liposomes contains 63 µmol of L-Rphosphatidylcholine (egg yolk), 18 µmol of dicetyl phosphate and 9 µmol of cholesterol in 5 mL of chloroform. Preparation of 32P-5′-End-Labeled DNA Fragments from the p53 Gene. Human p53 tumor suppressor genecontaining fragments (30) were amplified by the polymerase chain reaction process (PCR) using the primers provided (Clontech Lab, CA). The PCR products were digested with SmaI and ligated into SmaI-cleaved pUC 18 plasmid, which was then transferred to Escherichia coli MC 1061. The pUC plasmid was digested with EcoRI and HindIII, and the resulting DNA fragments were separated by electrophoresis in 2% agarose gels. 5′-End-labeled 654-base pair fragment (HindIII*13972-EcoRI* 14621) and 461-base pair fragment (HindIII*13038-EcoRI*13507) were prepared by dephosphorylation with calf intestine phosphatase and rephosphorylation with T4 polynucleotide kinase using [γ-32P]ATP. The 654-base pair fragment was further digested using ApaI to obtain a singly labeled 443-base pair fragment (ApaI14179-EcoRI*14621) and a 211-base pair fragment (ApaI13972-HindIII*14182), and the 461-base pair fragment was further digested with StyI to obtain a singly labeled 343-base pair fragment (StyI13160-EcoRI*13507) and a 118base pair fragment (HindIII*13038-StyI13155), as described previously (31). The asterisk indicates 32P-labeling. Detection of Damage to 32P-5′-End-Labeled DNA Fragments from the p53 Gene. The standard reaction mixture (prepared in a 1.5-mL Eppendorf tube) contained 20 µM CuCl2, 50 µM/base calf thymus DNA and 32P-5′-end-labeled DNA fragments, and R-tocopherol in liposome or Me2SO (at the concentrations indicated) in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. When indicated, heatdenatured DNA was used. Liposomes were prepared by the method of Fukuzawa et al. (32) with a minor modification. R-Tocopherol was dissolved in a liposome kit, and the solution was placed into a flask. Liposome kit containing R-tocopherol was evaporated to dryness under nitrogen and dispersed in 20 mM phosphate buffer, pH 7.8. The suspensions were vortexed tightly for 10 min. Liposomes contained 7.9 mol % R-tocopherol (mol of R-tocopherol/mol of phospholipid), and experiments with

Yamashita et al. liposomes containing different concentrations of R-tocopherol were done by maintaining the tocopherol to liposomal lipid ratio. Where indicated, either 5% ethanol, 0.1 M mannitol, 0.1 M sodium formate, 30 units of catalase, 30 units of SOD, or 50 µM bathocuproine was included in reactions. The mixtures were then incubated at 37 oC for 2 h and were incubated for 20 min at 90 °C in 1 M piperidine. DNA fragments were electrophoresed on an 8% polyacrylamide/8 M urea gel followed by autoradiography. DNA cleavage sites were determined by direct comparison with those produced by the Maxam-Gilbert procedure (33) using a DNA sequencing system (LKB 2010 Macrophor). Laser densitometry (LKB 2222 UltroScan XL) was used to quantitate the DNA fragments generated. Determination of 8-OxodG Formation in Calf Thymus DNA. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) formation was determined by a modification of a reported method (34). The reaction mixture (prepared in a 2-mL Eppendorf tube) contained 5 µM DTPA, 20 µM CuCl2, 50 µM/base calf thymus DNA, and R-tocopherol (at the concentrations indicated) in 400 µL of 4 mM sodium phosphate buffer (pH 7.8) containing 1% Me2SO. Calf thymus DNA was incubated with R-tocopherol and CuCl2 at 37 °C for 2 h. When indicated, heat-denatured DNA was used. Following incubation, the DNA was precipitated using ethanol, digested with nuclease P1 and calf intestine phosphatase, and then analyzed by HPLC-ECD, as described (35). Stoichiometric Study of Cu(II) Reduction by R-Tocopherol. The stoichiometry for the reduction of Cu(II) to Cu(I) by R-tocopherol using bathocuproine as a Cu(I)-specific chelator was determined as described previously (36). The reaction mixture, which contained 40 µM bathocuproine, 20 µM CuCl2, and R-tocopherol in 10 mM sodium phosphate buffer (pH 7.8) containing 1% Me2SO, was kept at 37 °C. An increase of absorption at 480 nm of the Cu(I)-bathocuproine complex was measured with a UV-visible spectrophotometer (Shimadzu UV2500PC). Spectral tracing was started by the addition of R-tocopherol and recorded every 1 min. Measurement of Oxygen Consumption and Superoxide Formation. Oxygen consumption by the reactions of R-tocopherol with Cu(II) was measured for 30 min using a Clarke oxygen electrode (electronic stirrer model 300, Rank Brothers Ltd., Bottisham, Cambridge, England). The reaction mixture contained 100 µM R-tocopherol incorporated into liposomes or in Me2SO and 5 µM DTPA in 2 mL of 10 mM sodium phosphate buffer (pH 7.8), and the reaction was started by the addition of 20 µM CuCl2 into the chamber of the oxygen electrode at 37 °C using the magnetic stirrer. When indicated, R-tocopherol or CuCl2 was omitted from incubations. The amounts of superoxide formation by the reactions of R-tocopherol with Cu(II) were determined by measuring cytochrome c reduction. The mixtures containing 200 µM ferricytochrome c, 100 µM R-tocopherol incorporated into liposomes or in Me2SO, 20 µM CuCl2, and 5 µM DTPA in 1 mL of 10 mM sodium phosphate buffer, pH 7.8, were then incubated at 37 °C. The absorption at 550 nm was recorded every 2 min for 20 min using a UV-visible spectrophotometer. When indicated, CuCl2 was omitted from incubations and SOD was included (90 units/mL). Electron Spin Resonance Spin-Trapping Studies. The complete reaction system contained 25 mM R-tocopherol, 12 mM 2-methyl-2-nitrosopropane (MNP) monomer (added from a 75 mM stock solution of the dimer in Me2SO), and 0.1 mM CuCl2 in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. When indicated, CuCl2 and R-tocopherol were omitted from reactions. Reaction mixtures were then transferred to a quartz flat cell and spectra recorded after 5, 15, and 30 min using a Bruker ECS 106 ESR spectrometer operating with the following instrument settings: modulation frequency, 100 kHz; sweep width, 100 G; modulation amplitude, 1.27 G; receiver gain, 2.5 × 104; time constant, 163.84 ms; sweep time, 335.54 s. The spectra shown are those recorded after 30 min (identical, but

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Figure 1. Detection of damage to denatured and native DNA fragments from the human p53 tumor suppressor gene following incubation with R-tocopherol in the presence of Cu(II) ions (A, B) and determination of the effects of •OH scavengers, catalase, SOD, and bathocuproine (C). The standard reaction mixture contained 32P-5′-end-labeled DNA fragments, 20 µM/base (A) or 50 µM/base (B, C) calf thymus DNA, 20 µM CuCl2 and R-tocopherol (at the concentrations indicated) in 10 mM sodium phosphate buffer, pH 7.8. Where indicated, scavengers or bathocuproine was included in reactions. The mixtures were then incubated at 37 oC for 2 h. After the mixtures were incubated for 20 min at 90 °C in 1 M piperidine, DNA fragments were electrophoresed on an 8% polyacrylamide/8 M urea gel and visualized by autoradiography: (A) R-tocopherol incorporated into liposomes; (B) R-tocopherol in 1% Me2SO; (C) lane 1, Cu(II) alone as control; lane 2, 50 µM R-tocopherol alone; lane 3, 50 µM R-tocopherol with Cu(II); lane 4, 5% (v/v) ethanol; lane 5, 0.1 M mannitol; lane 6, 0.1 M sodium formate; lane 7, 30 units of catalase; lane 8, 30 units of SOD; lane 9, 50 µM bathocuproine. slightly weaker, signals were observed after 5 and 15 min). Hyperfine coupling constants were obtained from computer simulations of spectra using the SIMEPR program (37).

Results Oxidative DNA Damage Induced by r-Tocopherol and Cu(II). Incubation of 32P-labeled DNA fragments from the p53 suppressor gene with R-tocopherol in the presence of Cu(II) resulted in extensive damage, as revealed by polyacrylamide gel electrophoresis and autoradiography following piperidine treatment (Figure 1A,B). R-Tocopherol in Me2SO induced damage in a dosedependent manner, but R-tocopherol incorporated into liposomes induced damage in a different manner (Figure 1A,B). Neither reagent alone nor Cu(II) was able to induce detectable DNA damage (Figure 1A,C). Liposomes in the presence and absence of Cu(II) did not induce DNA damage (Figure 1A). Denatured DNA was more sensitive to damage than native DNA (data not shown). DNA fragmentation was also observed without piperidine treatment, indicating that R-tocopherol and Cu(II) can induce fragmentation directly (data not shown). However, the finding that fragmentation was enhanced following piperidine treatment suggests that the reagents also induced base damage. Ethanol, mannitol, and sodium formate, which are expected to compete for reaction with •OH, did not protect the DNA from damage by R-tocopherol and Cu(II) (Figure 1C). However, protection was afforded by catalase and bathocuproine, a Cu(I)-specific chelator (Figure 1C). When using R-tocopherol incorporated into liposomes instead of Me2SO, almost the same protection was observed. Sequencing of the DNA fragments generated following incubation with R-tocopherol and Cu(II) (followed by piperidine treatment)

revealed that thymine and cytosine sites were particularly sensitive to damage (Figure 2 A,B). Using denatured DNA, preferential damage occurred at guanine and adenine sites (Figure 2C). Site specificity of the DNA damage using R-tocopherol incorporated into liposomes instead of Me2SO was also specific for thymine and cytosine sites (Figure 3). Guanine residues in DNA are often found to undergo hydroxylation in response to an oxidative insult. Since this modification is believed to cause mismatch replication of the DNA, resulting in mutation (38), 8-oxodG was measured in DNA following incubation with R-tocopherol and Cu(II). As shown in Figure 4, in the presence of Cu(II), R-tocopherol induced dose-dependent 8-oxodG formation in calf thymus DNA. Higher levels of 8-oxodG were detected in denatured DNA than in native DNA following the treatment (Figure 4). Stoichiometric Study of Cu(II) Reduction by r-Tocopherol. After the incubation of R-tocopherol with Cu(II) and bathocuproine for 2 min, the increase in absorption at 480 nm reached a plateau. This indicated that R-tocopherol reduced Cu(II) to produce the Cu(I) chelate immediately. Cu(II) alone slightly interfered with the absorption maximum. As the absorbance of the Cu(I) chelate was not masked by less than 30 µM R-tocopherol, it was possible to calculate the absorbance of the Cu(I) chelate in the presence of R-tocopherol from the molar extinction coefficient of the Cu(I)-bathocuproine complex ( ) 12 250 M-1 cm-1) at 480 nm (39). The ratio of Cu(II)/R-tocopherol, at which maximum conversion of Cu(II) to Cu(I) was achieved, was deduced from the intersection of two dotted lines as shown in Figure 5. This implies that each R-tocopherol molecule

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Figure 2. Site specificity of damage induction in native (A, B) and denatured (C) DNA from the human p53 tumor suppressor gene by R-tocopherol in the presence of Cu(II) ions. The reaction mixtures contained 32P-5′-end-labeled 443-base pair fragment (ApaI14179EcoRI*14621) and 211-base pair fragment (ApaI13972-Hind III*14182), 20 µM CuCl2, 50 µM/base calf thymus DNA, and 50 µM R-tocopherol in 10 mM sodium phosphate buffer containing 1% Me2SO, pH 7.8 (*, 32P-labeling). For the experiment with denatured DNA, the 5′-end-labeled DNA fragment and calf thymus DNA were heated at 90 °C for 10 min and quickly chilled before the reaction. The mixtures were then incubated at 37 oC for 2 h. Mixtures were then incubated for 20 min at 90 °C in 1 M piperidine. DNA cleavage sites were determined by direct comparison with those produced by the Maxam-Gilbert procedure, as described under Materials and Methods. The horizontal axis shows the nucleotide number of the human p53 tumor suppressor gene.

can support the reduction of two Cu(II) ions due to formation of a stable Cu(I)-bathocuproine complex. UV-visible spectrophotometric studies indicated the formation of R-tocopherol quinone during the autoxidation of 200 µM R-tocopherol in a buffer solution containing 4% Me2SO. R-Tocopherol showed a maximum absorption at 294 nm in sodium phosphate buffer at pH 7.8. The decrease of absorbance at 294 nm was observed in the presence of Cu(II). Subsequently, the appearance of a bicuspid peak at 275 and 281 nm, which was characteristic of R-tocopherol quinone absorption (40), was observed (data not shown). The reaction mixture became turbid gradually, suggesting micelle formation of R-tocopherol. Detection of Oxygen Consumption and Superoxide Formation. Oxygen consumption by the interaction of R-tocopherol incorporated into liposomes with Cu(II) increased time dependently. No oxygen was consumed in the absence of Cu(II) or R-tocopherol, and liposomes

in the presence of Cu(II) consumed a little oxygen (data not shown). The incubation of ferricytochome c in the presence of Cu(II) and R-tocopherol incorporated into liposomes led to a time-dependent increase in the absorption at 550 nm (data not shown). Although the cytochome c reduction was inhibited by SOD, indicating the generation of O2•-, this inhibition was only partial, suggesting the operation of additional, superoxideindependent mechanisms of cytochome c reduction, probably mediated by Cu(I). Liposomes in the presence of Cu(II) reduced smaller amount of cytochome c and generated a little O2•- (data not shown). When using R-tocopherol in Me2SO, almost the same results were obtained in these experiments (data not shown). These findings suggest that R-tocopherol reduces Cu(II), giving Cu(I) and R-tocopheroxyl radical (18), followed by oxygen reduction to O2•- by Cu(I) (41). Electron Spin Resonance Spin-Trapping Studies. To provide evidence for the formation of •OH (or species

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Figure 3. Site specificity of damage induction in native DNA from the human p53 tumor suppressor gene using R-tocopherol incorporated into liposomes. The reaction mixtures contained 32P-5′-end-labeled 118-base pair fragment (Hind III*13038-StyI13155) and 343-base pair fragment (StyI13160-EcoRI*13507), 20 µM CuCl2, 20 µM/base calf thymus DNA, and 100 µM R-tocopherol incorporated into liposomes in 10 mM sodium phosphate buffer, pH 7.8 (*, 32P-labeling). The mixtures were then incubated at 37 oC for 2 h. After piperidine treatment, DNA cleavage sites were determined as described under Materials and Methods. The horizontal axis shows the nucleotide number of the human p53 tumor suppressor gene.

of similar reactivity to •OH) generated from R-tocopherol and Cu(II), ESR spin-trapping was employed. In the previous study using Trolox, a secondary spin-trapping approach was employed in which •OH radicals are converted to methyl radicals (•CH3) via reaction with Me2SO. The methyl radicals are then detected by ESR as their relatively stable adduct to the spin trap N-tertbutyl-R-phenylnitrone (21). Under the reaction conditions employed, the methyl radical adduct of a different spin trap, 2-methyl-2-nitrosopropane (MNP), was also detected: it was suggested that MNP is generated via the copper-catalyzed decomposition of •OH adduct of N-tert-butyl-R-phenylnitrone (21). Consequently, in the present study, N-tert-butyl-R-phenylnitrone was simply replaced with MNP and detection of the MNP methyl radical adduct was taken as evidence for the generation of •OH (or similar species). When R-tocopherol and Cu(II) were incubated in the presence of Me2SO and MNP, the distinctive 12-line ESR signal from the MNP/•CH3 radical adduct was observed (Figure 6). The weak 3-line signal also present in this spectrum is from the MNP decomposition product, di-tert-butyl nitroxide (Figure 6) (42). No signals were observed in the absence of R-toco-

pherol (Figure 6). In the absence of added Cu(II), only very weak signals were detected (mainly from di-tertbutyl nitroxide), which are believed to reflect interactions involving traces of contaminating metal ions (Figure 6). No ESR signals were observed in the absence of both R-tocopherol and Cu(II) (Figure 6). No methyl radical generation occurred when either catalase or bathocuproine was included in incubations containing Cu(II) and R-tocopherol (data not shown), which reflects previous findings using Trolox in place of R-tocopherol (21). Additional ESR study without spin-trapping revealed formation of R-tocopheroxyl radical (data not shown) (43).

Discussion In the present investigation we have demonstrated that R-tocopherol in the presence of Cu(II) can induce extensive DNA damage, including base modification and strand breakage. The predominant DNA cleavage sites were thymine and cytosine residues. Inhibitory effects of catalase and bathocuproine on DNA damage suggest that H2O2 and Cu(I) are required for the DNA damage.

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Figure 4. 8-OxodG formation in calf thymus DNA following incubation with R-tocopherol in the presence of Cu(II). The reaction mixtures contained 20 µM CuCl2, 50 µM/base calf thymus DNA, and R-tocopherol (at the concentrations indicated) in 4 mM sodium phosphate buffer, pH 7.8. Native or denatured calf thymus DNA (50 µM/base) was incubated with R-tocopherol and CuCl2 for 2 h at 37 °C. Following incubation, the DNA was analyzed for 8-oxodG using HPLC-ECD, as described under Materials and Methods. Points, mean level of 8-oxodG/105 dG in denatured DNA and native DNA present in duplicate and four experiments SD, respectively. Figure 6. Detection of •OH or species of similar reactivity by ESR spin-trapping during the interaction of R-tocopherol with Cu(II). The complete reaction system contained 25 mM R-tocopherol, 12 mM MNP monomer, and 0.1 mM CuCl2 in 50 mM MOPS buffer. Reaction mixtures were then transferred to a quartz flat cell and spectra recorded after 30 min using a Bruker ECS 106 ESR spectrometer, as described under Materials and Methods: (A) complete reaction system; (B) computer simulation of the spectrum shown under panel A, consisting of signals from MNP/•CH3 [aN ) 17.3 G; aβH(3H) ) 14.2 G] and di-tert-butyl nitroxide (aN ) 16.9 G) with relative concentrations of 94.4 and 5.6, respectively; (C) complete system minus R-tocopherol; (D) complete system minus Cu(II); (E) complete system minus R-tocopherol and Cu(II). Figure 5. Stoichiometric study of Cu(II) reduction by R-tocopherol. Sodium phosphate buffer (10 mM) at pH 7.8 containing 40 µM bathocuproine and 20 µM CuCl2 was kept at 37 °C, and the spectral tracing was started by the addition of R-tocopherol. The absorbances at 480 nm after 2 min were recorded, and Cu(I) chelate concentrations were calculated. Concentration ) (A480(x) - A480(0))/12250, where A480(x) and A480(0) are the absorbances at 480 nm of the sample with and without Cu(II) and 12250 is the molar absorptivity of the chromophore.

Formation of 8-oxodG, a relevant indicator of oxidative base damage (33, 34, 37), increased with concentration of R-tocopherol in the presence of Cu(II). Our observations can be accounted for by a mechanism of DNA damage involving the interaction of R-tocopherol with DNA-associated Cu(II) ions. R-Tocopherol is first oxidized to the R-tocopheroxyl radical by Cu(II), followed by R-tocopherol quinone formation by disproportionation of R-tocopheroxyl radical (44) or by their further oxidation (45). The Cu(I) formed during the oxidation of R-tocopherol reacts with molecular oxygen to generate O2•- (41), which then forms H2O2 by dismutation. The Cu(I) complexed to DNA then interacts with the H2O2 to generate the reactive species responsible for DNA dam-

age, which is either the hydroxyl radical or a species of similar reactivity. ESR spin-trapping study showed the formation of •OH generated from R-tocopherol and Cu(II). This suggests that •OH, generated via the reaction of Cu(I) with H2O2, is responsible for the induction of DNA damage. However, although Cu(I) ions appear to generate free •OH upon reaction with H2O2 in free solution (21, 46, 47), it has been suggested that DNA-associated Cu(I) ions generate other oxidants, including a copper-peroxo intermediate and “bound” hydroxyl radicals (28, 29, 48, 49). While the observation that typical •OH “scavengers” did not offer DNA protection from R-tocopherol and Cu(II) could be taken to suggest that free •OH is not involved in damage, this observation is also consistent with DNA damage induction by free •OH generated in very close proximity to the nucleic acid by the bound metal ion: it is widely recognized that it is very difficult to intercept •OH generated by such a mechanism (25-27). Similarly, the site specificity of copper-dependent DNA damage observed here has also been used to support the suggestion that oxidants other than free •OH are responsible for damage (29, 48). However, because the binding of

R-Tocopherol Induces Cu(II)-Dependent DNA Damage

copper ions to DNA is site-specific (23), it might also be expected that DNA damage by free •OH generated by bound copper ions is also site-specific, which has been confirmed experimentally (26). The identity of the oxidant responsible for copper-dependent DNA damage remains therefore a matter of some controversy. Additionally, it is necessary to consider the possibility of excessive R-tocopherol within the nuclear membrane and its accessibility to DNA-associated copper ions (which occur naturally in the cell nucleus), although it has been reported that R-tocopherol is predominantly associated with non-histone proteins having a high affinity for DNA (50). Our experiments have shown that R-tocopherol incorporated into liposomes is able to induce DNA damage in the presence of Cu(II) to an extent similar to that observed using R-tocopherol dispersed in Me2SO. These findings suggest that R-tocopherol incorporated within the nuclear membrane may also be accessible to DNA-associated copper, as are aqueous-phase reductants, such as GSH, ascorbic acid, and NADH (24, 25, 27-29). However, since the cell nucleus contains high levels of GSH (27), which prevents radical formation and DNA damage by its stabilization of Cu(I), it is expected that DNA damage by endogenous copper ions occurs to a significant extent only during oxidative stress. Therefore, it is important in future studies to investigate whether excessive vitamin E can promote oxidative stress, resulting in DNA damage in cellular systems. Our findings may have important implications regarding the increased scientific and public interest that has emerged over the past decade concerning the possibility that antioxidants, such as R-tocopherol and β-carotene, might protect against cancer. It has been suggested that DNA damage, associated with cancer, might be prevented via the scavenging of reactive oxygen species that are generated by chemical carcinogens, during inflammation, and as byproducts of many normal metabolic processes (1, 3). However, a recent epidemiological investigation in Finland failed to confirm this hypothesis: the incidence of lung cancer in male smokers was unaffected by R-tocopherol supplementation and was unexpectedly increased by β-carotene supplementation (4). Several studies employing laboratory animals have also demonstrated that vitamin E can induce or promote tumor formation (5-11). Additionally, the present study showed that R-tocopherol induced oxidative DNA damage in the presence of Cu(II), although it was in a simplified, in vitro model. Therefore, excessive vitamin E may have the potential to increase DNA damage, and in the future it will be necessary to confirm the biological significance of our finding using cellular systems and laboratory animals. Consequently, not only should the nutritional and antioxidant aspects of vitamin E be considered, but also its toxicological properties, as mediated by reactive oxygen species generation (51).

Acknowledgment. M.J.B. is grateful to the Japan Society for the Promotion of Science (JSPS) for the provision of a JSPS Fellowship for Research in Japan and to David R. Duling (Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, NC) for the gift of the ESR simulation software.

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