Requirement of Glutathione and Cysteine in Guanine-Specific

Potassium bromate (KBrO3), a food additive, induces renal-cell tumors in rats. KBrO3 induced 8-oxo-7, 8-dihydro-2'-deoxyguanosine (8-oxodG) formation ...
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Chem. Res. Toxicol. 2001, 14, 678-685

Requirement of Glutathione and Cysteine in Guanine-Specific Oxidation of DNA by Carcinogenic Potassium Bromate Mariko Murata,† Yuriko Bansho,† Sumiko Inoue,‡ Kimiko Ito,‡ Shiho Ohnishi,† Kaoru Midorikawa,† and Shosuke Kawanishi*,† Department of Hygiene, Mie University School of Medicine, Tsu, Mie, Japan, Department of Public Health, Graduate School of Medicine, Kyoto University, Kyoto, Kyoto, Japan, Received October 3, 2000

Potassium bromate (KBrO3), a food additive, induces renal-cell tumors in rats. KBrO3 induced 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) formation in human leukemia cell line HL-60 as well as in its H2O2-resistant clone, HP100, suggesting no involvement of H2O2. Depletion of GSH by buthionine sulfoximine (BSO) had a little inhibitory effect on KBrO3-induced 8-oxodG formation. However, the amount of 8-oxodG was still significantly higher than that in control, suggesting that intracellular Cys can affect KBrO3 to oxidize DNA, when GSH decreased. KBrO3 caused 8-oxodG in isolated DNA in the presence of GSH (tripeptide; γ-GluCysGly), γ-GluCys, CysGly, or Cys. Methional completely inhibited 8-oxodG formation induced by KBrO3 plus GSH, but typical hydroxyl radical scavengers, SOD and catalase, had little or no inhibitory effects. When bromine solution (BrO-) was used instead of BrO3-, similar scavenger effects were observed. Experiments with 32P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene suggested that KBrO3 induced 8-oxodG formation at 5′-site guanine of GG and GGG sequences of double-stranded DNA in the presence of GSH and that treatment of formamidopyrimidine-DNA glycosylase led to chain cleavages at the guanine residues. ESR spin-trapping studies showed that 1:2:2:1 quartet DMPO (5,5-dimethyl-1-pyrroline N-oxide) spectrum similar to DMPO/hydroxy radical (•OH) adduct, but the signals were not inhibited by ethanol. Therefore, the signal seemed not to be due to •OH but byproduct due to oxidation of DMPO by the reactive species. The signals were suppressed by the addition of dGMP, but not by other mononucleotides, suggesting the specific reactivity with guanine. On the basis of our results and previous literature, it is speculated that reduction of KBrO3 by SH compounds in renal proximal tubular cells yields bromine oxides and bromine radicals, which are the reactive species that cause guanine oxidation, leading to renal carcinogenesis of KBrO3.

Introduction Potassium bromate (KBrO3)1 has been used as a food additive in bread making processing because of its oxidizing property. KBrO3 is potentially genotoxic in bacterial mutation assays (1) and in chromosome aberration tests using a hamster cell line (1). In a micronucleus assay, KBrO3 induced micronucleated polychromatic erythrocytes dose-dependently in male mice (2, 3). KBrO3 is a well-known renal carcinogen to rats, and also tumors in other organs have been observed after oral administration of KBrO3 (4). KBrO3 can increase the level of 8-oxo-7, 8-dihydro-2′-deoxyguanosine (8-oxodG) and induce its repair enzyme more efficiently in kidney than * To whom request for reprints should be addressed. Phone/Fax: (+81) (59) 231 5011. E-mail: [email protected]. † Mie University School of Medicine. ‡ Kyoto University. 1 Abbreviations: KBrO , potassium bromate; 8-oxodG, 8-oxo-7, 3 8-dihydro-2′-deoxyguanosine; BSO, buthionine sulfoximine; NEM, N-ethylmaleimide; Fpg, formamidopyrimidine-DNA glycosylase; ECD, electrochemical detector; DTPA, diethylenetriamine-N,N,N′,N′′,N′′pentaacetic acid; DMPO, 5,5-dimethyl-1-pyrroline N-oxide; Na2IrCl6, sodium hexachloroiridate(IV) hexahydrate; FBS, fetal bovine serum; H2O2, hydrogen peroxide; SOD, superoxide dismutase; •OH, hydroxyl radical; BrO2, BrO, bromine oxides; Br•, bromine radical; BrO-, oxobromate ion.

liver of treated rats (5-7). Formation of 8-oxodG is known to induce G f T transversion, and this mutation is thought to contribute to activation of oncogenes and/or inactivation of tumor suppressor genes, thereby leading to carcinogenesis (8, 9). There is a report that exposure of cultured renal cells to KBrO3 resulted in the induction of p53 gene mutation (10). Furthermore, KBrO3 has been shown to induce DNA strand breaks and poly(ADPribosyl)ation (11) and proliferative response (12) in the kidney as tumor promoting potential. However, the mechanism of 8-oxodG formation and renal carcinogenesis by KBrO3 remains to be clarified. Intracellular GSH is an essential component of cellular defense against oxidants and various types of toxic stress. GSH is a tripeptide, γ-glutamyl-cysteinyl-glycine (γGluCysGly), and distributed widely in animal and plant tissues. Conjugation with GSH represents an important detoxification reaction (phase II) after xenobiotic biotransformation (phase I). In certain tissues, the intracellular concentration of GSH is extremely high (2-10 mM), so conjugation of xenobiotics with GSH can be also significant as detoxification (13). Ballmaier and Epe (14) showed that KBrO3-induced DNA damage was partially prevented by depletion of intracellular GSH with diethylmaleate, suggesting an important role of GSH. In

10.1021/tx000209q CCC: $20.00 © 2001 American Chemical Society Published on Web 05/19/2001

Guanine-Specific Oxidation by Potassium Bromate

addition to the activation by GSH, high Cys levels at the proximal tubule brush border may have some association with its nephrotoxicity (15) as regarding GSH metabolism in kidney. As a method for approaching the mechanism of carcinogenesis, the extent of oxidative DNA damage by KBrO3 was investigated using HPLC equipped with an electrochemical detector (ECD). We measured the level of 8-oxodG in human leukemia cell line HL-60 and its H2O2-resistant clone HP100 after the addition of KBrO3. The effect of GSH depletion by buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis, on 8-oxodG formation was examined in HL-60 cells treated with KBrO3. In addition, N-ethylmaleimide (NEM), a lipophilic sulfhydryl-alkylating agent, was used for depletion of intracellular SH compounds. We analyzed 8-oxodG formation in calf thymus DNA treated with KBrO3 in the presence of GSH and its constituent compounds. We compared the intensity of DNA damage by KBrO3 with that by bromine solution which contains oxobromate ion (BrO-). We also detected DNA damage by KBrO3 using 32P-5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. Furthermore, we examined reactive species by the ESR spin-trapping method.

Materials and Methods Materials. KBrO3, bromine solution, L-cysteine (Cys), ethanol, D-mannitol, dCMP and dTMP were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Catalase (45 000 units/mg from bovine liver), superoxide dismutase (SOD, 3000 units/mg from bovine erythrocytes), methional, bacterial alkaline phosphatase, RNase A, dAMP, dGMP, cysteinyl-glycine (CysGly), l-buthionine-[S,R]-sulfoximine (BSO) and N-ethylmaleimide (NEM) were from Sigma Chemical Co. (St. Louis, MO). GSH and γ-glutamylcysteine (γ-GluCys) was from Kohjin Co. (Tokyo Japan). Nuclease P1 was from Yamasa Shoyu Co. (Chiba Japan). Restriction enzymes (EcoRI and Apa I), proteinase K and calf intestine phosphatase were from Boehringer Mannheim G.m.b.H. (Mannheim, Germany). Restriction enzymes (HindIII, AvaI, XbaI, and PstI) and T4 polynucleotide kinase were purchased from New England Biolabs. [γ-32P]ATP (222 TBq/mmol) was from New England Nuclear (Boston, MA). Diethylenetriamine-N,N,N′,N′′,N"-pentaacetic acid (DTPA) was from Dojin Chemicals Co. (Kumamoto, Japan). Trifluoroacetic acid and piperidine were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 5,5Dimethyl-1-pyrroline N-oxide (DMPO) was from Labotec, Co. Ltd. (Tokyo, Japan). Formamidopyrimidine-DNA glycosylase (Fpg, 20 000 units/mg from Escherichia coli) was from Trevigen Inc. (Gaithersburg, MD). Sodium hexachloroiridate (IV) hexahydrate (Na2IrCl6) from Strem Chemicals, Inc. (Newburybort, MA). A lysis buffer for DNA extraction (model 340A) was from Applied Biosystems (Foster City, CA). Measurement of 8-oxodG in Cultured Cells. Human leukemia HL-60 cells and its H2O2-resistant clone HP100 cells were grown in RPMI 1640 supplemented with 6% fetal bovine serum (FBS) at 37 °C under 5% CO2 in a humidified atmosphere. Catalase activity of HP100 cells was 18 times higher than that of parent HL-60 cells (16). When H2O2-related oxidative DNA damage was induced, 8-oxodG formation in HL-60 cells should be higher than that in HP100 cells (17). Cells (106 cells/mL) were incubated with KBrO3 for the indicated time at 37 °C and immediately washed three times with phosphate-buffered saline (PBS, pH 7.4), and DNA was extracted by using RNase A, proteinase K and a lysis buffer for DNA extraction. In certain experiments, BSO or NEM was pretreated, and then KBrO3 was added, followed by additional incubation. The DNA was enzymatically digested by nuclease P1 and bacterial alkaline phosphatase to the nucleosides and analyzed by the HPLC-ECD method described previously (18, 19).

Chem. Res. Toxicol., Vol. 14, No. 6, 2001 679 Measurement of GSH and Cys in Cultured Cells. HL60 cells (106 cells/mL) were treated with KBrO3 and/or BSO for the indicated time and immediately washed twice with PBS. The cells were suspended in 1 mL of cytoplasm extraction buffer [10 mM Tris (pH 7.5), 150 mM NaCl, and 5 mM MgCl2 in 0.5% Triton X] and centrifuged. Trifluoroacetic acid of 1/50 volume was added to the supernatant and centrifuged. The supernatant was analyzed by the HPLC-ECD (Eicom Corp., Kyoto, Japan) with gold disk electrode that can detect thiol group, according to a modified method of Lakritz et al. (20). Analysis of 8-oxodG Formation in Calf Thymus DNA by Reaction of KBrO3 and Bromine Solution in the Presence of GSH. The standard reaction mixture in a microtube (2.0 mL, Eppendorf) contained various concentrations of KBrO3, GSH and calf thymus DNA (100 µM/base) in 400 µL of 4 mM sodium phosphate buffer (pH 7.8) containing 1.0 µM DTPA. DNA fragments were incubated with KBrO3 and GSH for the indicated duration at 37 °C. In certain experiments, bromine solution which contains BrO- (21) was used instead of KBrO3. For the experiment with denatured DNA, calf thymus DNA was treated at 90 °C for 5 min and quickly chilled before incubation. After ethanol precipitation, DNA was enzymatically digested by nuclease P1 and calf intestine phosphatase to the nucleosides and analyzed by the HPLC-ECD, as described previously (18). Preparation of 32P-5′-End-Labeled DNA Fragments. DNA fragments were obtained from the human p53 tumor suppressor gene (22). The 5′-end-labeled 460-bp fragment (HindIII *13038-EcoRI *13507) was obtained by dephosphorylation with calf intestine phosphatase and rephosphorylation with [γ-32P]ATP and T4 polynucleotide kinase (The asterisk indicates 32P-labeling.). The 460-bp fragment was further digested with StyI to obtain a singly labeled 348-bp fragment (StyI 13160-EcoRI* 13507) as described previously (23). DNA fragment was obtained from the human c-Ha-ras-1 protooncogene (24). DNA fragments were prepared from plasmid pbcNI, which carries a 6.6-kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 protooncogene. The singly labeled 337-bp fragment (PstI 2345-AvaI *2681), 261-bp fragment (AvaI* 1645-XbaI 1905) and 341-bp fragment (XbaI 1906-AvaI* 2246) were obtained according to the method described previously (25). Nucleotide numbering starts with the BamHI site (24). Detection of Damage to Isolated DNA Induced by KBrO3 in the Presence of GSH. The standard reaction mixture in a microtube (1.5 mL, Eppendorf) contained various concentrations of KBrO3, GSH, 32P-labeled DNA fragment, and calf thymus DNA in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 2.5 µM DTPA. After incubation at 37 °C for the indicated duration, the DNA fragment was precipitated by ethanol. And then, the DNA fragments were treated in three ways. That is, one was piperidine treatment, in which the DNA was heated at 90 °C in 1 M piperidine for 20 min as described previously (25). In the second experiment, the DNA was treated with 100 µM Na2IrCl6 at 37 °C for 1 h, followed by piperidine treatment. Na2IrCl6 treatment was performed for further oxidizing 8-oxo-7, 8-dihydroguanine lesion to guanidinohydantoin (26) that is piperidine-labile (27) in order to visualize in autoradiogram. In the third experiment, the DNA was treated with 10 units of Fpg protein in 10 µL of the reaction buffer [10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA, and 0.1 mg/mL BSA] at 37 °C for 2 h. Fpg protein catalyzes the excision of 8-oxo-7,8-dihydroguanine (28). The preferred cleavage sites were determined by direct comparison of the positions of the oligonucleotides with those produced by the chemical reactions of the Maxam-Gilbert procedure (29) using a DNA-sequencing system (LKB 2010 Macrophor, Pharmacia Biotech, Uppsala, Sweden). A laser densitometer (LKB 2222 UltroScan XL, Pharmacia Biotech, Uppsala, Sweden) was used for the measurement of the relative amounts of oligonucleotides from treated DNA fragments.

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Figure 1. Content of 8-oxodG in DNA of human cultured cells treated with KBrO3. The HL-60 (open bar) and HP100 (hatched bar) cells (1.0 × 106 cells/mL) were incubated with various concentrations of KBrO3 for 4 h. DNA was extracted and subjected to enzyme digestion and analyzed by an HPLC-ECD as described in the Materials and Methods. Results are expressed as means and SD of values obtained from three independent experiments. Asterisks indicate significant differences compared with control by t-test (*p < 0.01).

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Figure 2. Content of endogenous GSH and Cys in HL-60 cells treated with BSO and KBrO3. HL-60 cells (1.0 × 106 cells/mL) were preincubated with 5 mM BSO and then incubated with 1 mM KBrO3 for 4 h. Cytosol was extracted and analyzed by an HPLC-ECD as described in the Materials and Methods. Results are expressed as means and SD of values obtained from three independent experiments. Asterisks indicate significant differences compared with control (*p < 0.01) by t-test.

Electron Spin Resonance Spectroscopic Studies. The generation of radicals in the reaction system of KBrO3/GSH trapped by DMPO was detected, using an ESR (model JESTE100, JEOL, Tokyo, Japan) with 100-kHz field modulation at room temperature (25 °C). To clarify the reactivity with DNA bases, mononucleotides were added to the reaction system of KBrO3/GSH in certain experiments. Spectra were recorded immediately after mixture with a microwave power of 16 mW, a modulation amplitude of 0.1 mT, and a receiver gain of 1000.

Results Formation of 8-oxodG in Human Cultured Cells by KBrO3. We measured the content of 8-oxodG, a relevant indicator of oxidative base damage, in human cultured cells by HPLC-ECD. Although artifactual oxidation generated during DNA extraction step may be responsible for overestimated level of 8-oxodG, the yield of 8-oxodG measured by HPLC-ECD was correlated with the value in a comet assay (30-32). To investigate cellular induction of oxidative DNA damage, we measured the content of 8-oxodG, a relevant indicator of oxidative base damage, in HL-60 and HP100 cells treated with KBrO3 (Figure 1). HP100 cells were used to assess whether H2O2 participates in KBrO3-induced oxidative DNA lesion. The basal level of 8-oxodG in the extracted DNA from the untreated cells was about 0.3 per 105 dG. The contents of 8-oxodG of DNA in HL-60 and HP100 cells treated with KBrO3 were significantly increased in comparison with no treated cells. However, there were no significant differences between HL-60 cells and HP100 cells treated with the same concentration of KBrO3. It was reported that HP100 cells were approximately 340fold more resistant to H2O2 than the parent HL-60 cells (16). These results suggest that KBrO3 can cause oxidative DNA damage in human cultured cells and that H2O2 does not participate in KBrO3-induced oxidative DNA lesion. GSH- and Sulfhydryls-Depletion and the Effects on 8-oxodG Formation. Intracellular levels of GSH and Cys were detected by the HPLC-ECD with a unique gold disk electrode. Figure 2 shows the contents of endogenous GSH and Cys in HL-60 cells treated with BSO and/or KBrO3. KBrO3 alone did not affect both GSH and Cys

Figure 3. Effect of GSH depletion on 8-oxodG formation in HL-60 cells treated with KBrO3. (A) The HL-60 cells (1.0 × 106 cells/mL) were preincubated with 5 mM BSO for 2 h and then incubated with 1 mM KBrO3 for 4 h. (B) The cells (1.0 × 106 cells/mL) were preincubated with 10 µM NEM for 30 min and then incubated with 1 mM KBrO3 for 1 h. DNA was extracted and subjected to enzyme digestion and analyzed by an HPLCECD as described in the Materials and Methods. Results are expressed as means and SD of values obtained from three independent experiments. Symbols indicate significant differences compared with control (#p < 0.05, *p < 0.01).

level. BSO, an inhibitor of GSH biosynthesis, made the intracellular GSH level lower at 20%, but did not affect Cys level. Treatment with both BSO and KBrO3 significantly decreased the intracellular level of GSH and Cys. Figure 3A shows the 8-oxodG formation in cells treated with BSO and/or KBrO3. There was no significant difference in 8-oxodG formation between BSO alone and the control. Although BSO had a little inhibitory effect on KBrO3-induced 8-oxodG formation, the amount of 8oxodG was still significantly higher than the control. These results may be explained by assuming that Cys can affect KBrO3-induced 8-oxodG formation instead of GSH, when GSH was depleted by BSO. Figure 3B shows the 8-oxodG formation in cells treated with NEM and/or KBrO3. It was reported that NEM (10 µM at 37 °C for 30 min) decreased the total cellular thiols by 50% (33). Although incubation time must be shorter than that in the case of BSO because of low viability of cells treated with NEM, NEM had a small inhibitory effect on KBrO3induced 8-oxodG formation. It is suggested that KBrO3 requires intracellular SH compounds in causing DNA damage.

Guanine-Specific Oxidation by Potassium Bromate

Figure 4. Formation of 8-oxodG in calf thymus DNA by KBrO3 in the presence of GSH, and its constituent compounds. Calf thymus DNA (100 µM/base) was incubated with the indicated concentrations of KBrO3 and 2 mM GSH, γ-GluCys, CysGly, Cys, γ-Glu, or Gly in 4 mM phosphate buffer (pH 7.8) containing 1.0 µM DTPA at 37 °C for 2 h. The DNA fragment was enzymatically digested into nucleotides, and 8-oxodG formation was analyzed by the HPLC-ECD, as described in the Materials and Methods.

Formation of 8-oxodG in Calf Thymus DNA by KBrO3 in the Presence of GSH and Its Constituent Amino Acids/Dipeptides. Using HPLC-ECD, we measured 8-oxodG content in calf thymus DNA treated with KBrO3 in the presence of GSH and its constituent compounds. KBrO3 caused increases of 8-oxodG formation in the presence of GSH, γ-GluCys, CysGly and Cys, whereas no increase was observed in the cases of Gly and γ-Glu (Figure 4). KBrO3/GSH more efficiently induced 8-oxodG formation in denatured single-stranded DNA than that in native double-stranded DNA (data not shown). Since GSH most effectively acts on KBrO3 among these compounds containing cysteine residue, GSH was used in the following experiments. Comparison of 8-oxodG Formation by KBrO3 and Bromine Solution in the Presence of GSH. KBrO3 or bromine solution (BrO-) caused no increase of 8-oxodG formation. Although both KBrO3 and BrO- can induce 8-oxodG formation in the presence of GSH, KBrO3 induced higher yield of 8-oxodG formation than BrO- did (Figure 5). Under the experimental condition used (2 mM GSH, 2 h incubation), BrO- induced 8-oxodG formation in dose-dependent manner. On the other hand, KBrO3 increased 8-oxodG to the dose of 0.5 mM, but the increase in 8-oxodG formation reached a plateau at the dose of 1 mM KBrO3. Although 8-oxodG formation increased with increasing both concentration KBrO3/GSH and incubation time under mild condition, high concentrations of KBrO3/GSH and the longer incubation made the content of 8-oxodG decrease (data not shown). It is speculated that 8-oxodG lesions were further oxidized to degraded form of guanine in severe experimental conditions. In any way, it is noteworthy that KBrO3 induced three times higher yield of 8-oxodG than BrO- did at the dose of 0.2 mM. Effects of Scavengers on 8-oxodG Formation by KBrO3 in the Presence of GSH. Figure 6 shows the effects of scavengers on GSH-mediated 8-oxodG formation by KBrO3. Methional completely inhibited 8-oxodG formation. However, SOD, catalase, and typical free hydroxyl radical (•OH) scavengers such as ethanol and mannitol, showed little or no inhibitory effects on the formation of 8-oxodG. The similar effects of scavengers were observed in the case of bromine solution.

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Figure 5. Formation of 8-oxodG in calf thymus DNA by KBrO3 and bromine solution in the presence of GSH. Calf thymus DNA (100 µM/base) was incubated with the indicated concentrations of KBrO3 or bromine solution, and 2 mM GSH in 4 mM phosphate buffer (pH 7.8) containing 1.0 µM DTPA at 37 °C for 2 h. The DNA fragment was treated as described in the legend to Figure 4.

Figure 6. Effects of scavengers on 8-oxodG formation induced by KBrO3 in the presence of GSH. Calf thymus DNA (100 µM/ base) was incubated with 1 mM KBrO3 and 2mM GSH in 4 mM phosphate buffer (pH 7.8) containing 1.0 µM DTPA at 37 °C for 2 h. Ethanol (5%), mannitol (0.1 M), sodium formate (0.1 M), methional (0.1 M), SOD (150 units/mL) or catalase (150 units/ mL) was added as indicated. The DNA fragment was treated as described in the legend to Figure 4.

Damage to 32P-Labeled DNA Fragments by KBrO3 in the Presence of GSH. Although KBrO3/GSH induced 8-oxodG formation for 2 h incubation, experiments with 32P-labeled DNA, followed by piperidine treatment, resulted in no oligonucleotides detected on the autoradiogram (Figure 7A). This result can be reasonably explained by the piperidine-resistant property of 8-oxodG (27). On the other hand, it is known that piperidine treatment can visualize the base liberation on the autoradiogram. Piperidine-labile lesions may contain several oxidized bases including thymine glycols, cytosine glycols, 5-formyluracil, oxazolone derivative and its precursor. Therefore, the result that KBrO3/GSH induced no release of free bases from DNA followed by hot piperidine. This result suggests no involvement of •OH as pointed out by Ballmaier and Epe (14). When Na2IrCl6 treatment was performed before piperidine treatment, DNA damage

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Figure 7. Autoradiogram of 32P-labeled DNA fragments incubated with KBrO3 and GSH. The reaction mixture contained 32P-5′-end-labeled DNA fragment [348-bp (A, B) or 261-bp (C)], calf thymus DNA [50 µM/base (A, B)], the indicated concentrations of KBrO3 and 2 mM GSH in 10 mM sodium phosphate buffer (pH 7.8) containing 2.5 µM DTPA. The mixture was incubated at 37 °C for 18 h (A, B) or 2 h (C), and then DNA was precipitated by ethanol. DNA (A) was treated with 1 M piperidine at 90 °C for 20 min. DNA (B) was treated with 100 µM Na2IrCl6 at 37 °C for 1 h, followed by piperidine treatment. DNA (C) was treated with 10 units of Fpg protein in the reaction buffer at 37 °C for 2 h. Then, the DNA fragments were electrophoresed on an 8% polyacrylamide-8 M urea gel and the autoradiogram was obtained by exposing X-ray film to the gel.

could be detected (Figure 7B). Fpg treatment also could result in oligonucleotides detected on the autoradiogram (Figure 7C). These results were consistent with the facts that Na2IrCl6 treatment converted piperidine-resistant 8-oxo-G lesion (27) to guanidinohydantoin that is piperidine-labile (26, 27), and Fpg catalyzed the excision of 8-oxo-G and released the bases (28). Site Specificity of GSH-Mediated DNA Damage by KBrO3. Scanning the autoradiogram with a laser densitometer gave the results as shown in Figure 8. KBrO3 plus GSH caused preferential DNA damage at the 5′-site of guanines, particularly at the 5′-GG-3′, 5′-GGG3′, and 5′-GGGG-3′ sequences in double-stranded DNA by both Na2IrCl6 treatment (Figure 8A) and Fpg treatment (Figure 8B). Electron Spin Resonance Spectroscopic Studies with DMPO. Figure 9A shows an ESR spectrum of a spin adduct obtained when DMPO was added to a buffer solution containing KBrO3 and GSH. Similar radicals were observed in the case of bromine solution and GSH. The spectrum (aN ) 1.49 mT, aH ) 1.49 mT) is resembled to the 1:2:2:1 quartet DMPO/•OH adducts by referring to the reported constant (34). However, ethanol did not inhibit the signals (data not shown), suggesting that the radical adduct is not due to the trapping of •OH. The reactivity of KBrO3 with mononucleotides was examined with ESR spectrometry. Addition of dGMP induced a decrease in the signal (Figure 9B), whereas dAMP, dTMP, and dCMP had no effect on the intensity of the signal (Figure 9, panels C-E).

Discussion In the present study, we showed that KBrO3 induced 8-oxodG formation equivalently in HL-60 and HP100 cells, H2O2-resistant clone of HL-60 cells. KBrO3/GSH induced no release of free bases from DNA, and typical •OH scavengers, SOD and catalase showed no or little inhibitory effects on damage to isolated DNA, suggesting

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no involvement of •OH, O2-, and H2O2. These results support the idea that H2O2 is not responsible to the DNA damage. Methional inhibited the formation of 8-oxodG, since it can scavenge not only •OH but also species with weaker reactivity than •OH (35). Therefore, it is suggested that the reactive species is not •OH itself but has less reactivity. Although ESR signal from reaction mixture of KBrO3 and GSH trapped by DMPO was resembled to that of DMPO/•OH, ethanol, a typical •OH scavenger, had no inhibitory effects on ESR signal trapped by DMPO, suggesting that the reactive species are not •OH. The 1:2:2:1 quartet DMPO spectrum would be obtained when DMPO was directly oxidized by reactive species, followed by the addition of a water molecule, resulting in formation of OH adduct to DMPO. This mechanism is supported by several reports (36, 37). Therefore, it is reasonably considered that the DMPO spectrum observed in this study is not •OH but the signal of byproduct due to one-electron oxidation of DMPO by the reactive species. In ESR spin-trapping studies, addition of dGMP induced a decrease in the signal whereas dAMP, dTMP, and dCMP had no effect. Furthermore, we revealed that KBrO3/GSH caused preferential DNA damage at the 5′site of guanines, particularly at the 5′-GG-3′, 5′-GGG-3′, and 5′-GGGG-3′ sequences in double-stranded DNA. When denatured single-stranded DNA was used, DNA cleavage occurred frequently at single guanine residues. It is considered that KBrO3 reacts with guanine residues in specific manner and that the radicals in the reaction systems of KBrO3/GSH have less oxidizing power than free •OH, which can cause DNA cleavage at every nucleotide with no marked site specificity (38). Recently, molecular orbital calculations have revealed that a large part of highest occupied molecular orbital (HOMO) is located on the 5′-G of GG and GGG sequences in doublestranded DNA (39, 40), and therefore, this guanine is likely to be oxidized. The site specificity of DNA damage induced by reactive species less reactive than •OH could be explained by HOMO distribution. Figure 10 shows a possible mechanism of guanine oxidation induced by KBrO3 in the presence of GSH/Cys. GSH/Cys reduces KBrO3 (BrO3-) to BrO2, and then BrO2 abstracts one electron from guanine, and itself is reduced to BrO2-. The one-electron oxidation of guanine yields cation radicals at the 5′-site guanine, and the radicals react with a water molecule to form C-8 OH adduct radicals, followed by oxidation, leading to 8-oxo-G formation (41, 42). In the same way, GSH/Cys can reduce BrO2and BrO- to BrO and Br•, which may participate in 8-oxo-G formation. We observed that KBrO3 induced about 3-fold 8-oxodG formation than bromine solution did with the sufficient amount of GSH. The content of 8-oxodG was higher in the buffer of pH 6.4 than that in the buffer of pH 7.8 (data not shown), since KBrO3 is easy to be reduced in acid solution. In other words, efficient formation of bromine oxides and Br• should result in high yield of 8-oxodG formation. Therefore, it is speculated that bromine oxides and Br• participate in formation of 8-oxo-7, 8-dihydro-2′-deoxyguanine, although we could not directly provide the evidence for these radical formation. Relevantly, it has been reported that piperidinelabile oxazolone and imidazolone are major guanine oxidation products (43, 44). But we could not detect piperidine-labile residues in DNA damage induced by KBrO3/GSH. This can be explained by assuming that SH

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Figure 8. Site specificity of DNA damage induced by KBrO3 in the presence of GSH. The reaction mixture contained 32P-5′-endlabeled 261-bp fragment (AvaI* 1645-XbaI 1905), calf thymus DNA [50 µM/base (A)], 1 mM KBrO3 and 2 mM GSH in 10 mM sodium phosphate buffer (pH 7.8). The mixture was incubated at 37 °C for 18 h (A) or 2 h (B), and then DNA was precipitated by ethanol. The DNA (A) was treated with 100 µM Na2IrCl6 at 37 °C for 1 h, followed by piperidine treatment. The DNA (B) was treated with 10 units of Fpg protein at 37 °C for 2 h. DNA fragments were electrophoresed and the autoradiograms were scanned with a laser densitometer.

compounds are able to efficiently reduce the highly oxidizing guanine radical which has been shown to be the precursor of both imidazolone and oxazolone decomposition products (45).

Although KBrO3 is a renal carcinogen to rats, the tumor incidence is higher (46) and the elevation of 8-oxodG is earlier (47) in males than in females. The GSH level in male rats is higher than that in female rats (48),

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Figure 10. A possible mechanism of guanine oxidation induced by KBrO3 in the presence of GSH/Cys.

(14). We considered that GSH can most efficiently activate KBrO3 and that high level Cys at proximal tubule brush border may also activate KBrO3. Other SH compounds including γ-GluCys, CysGly, and protein sulfhydryls have some possibilities of activating KBrO3 to cause DNA damage. It is concluded that endogenous SH compounds are required for the guanine-specific oxidation and that the oxidative DNA damage may be relevant for expression of renal carcinogenicity of KBrO3.

Acknowledgment. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and a Grant-in-Aid 2000 from the Mie Medical Research Foundation.

References Figure 9. ESR spectra of KBrO3/GSH trapped by DMPO. The sample (100 µL) contained 20 mM KBrO3, 5 mM GSH, 300 mM DMPO and 25 mM mononucleotides in 10 mM sodium phosphate buffer (pH 7.8) containing 2.5 µM DTPA. Spectra were recorded immediately after mixture by ESR spectroscopy. (A) KBrO3 + GSH, (B) KBrO3 + GSH + dGMP, (C) KBrO3 + GSH + dAMP, (D) KBrO3 + GSH + dTMP, (E) KBrO3 + GSH + dCMP.

and this difference of GSH level may have some relation with sex difference in KBrO3-induced carcinogenesis. GSH in plasma undergoes glomerular filtration, enters the lumen, and reaches the brush-border membrane of proximal tubules. Filtered GSH is rapidly hydrolyzed by the brush-border enzymes, γ-glutamyltransferase and dipeptidase, to γ-Glu, Cys and Gly. These amino acids are reabsorbed in proximal tubular cells. It is known that degradation of GSH in kidney produces mM quantities of Cys at the proximal tubule brush border (49). Sai et al. (50) also reported that 8-oxodG formation was coupled to SH-group reduction in renal proximal tubules treated with KBrO3. In this study, we have demonstrated that KBrO3 has the ability to cause guanine-specific oxidation in the presence of GSH, Cys, γ-GluCys, and CysGly, and that the depletion of GSH by BSO still significantly higher formation of 8-oxodG by KBrO3. In addition, formation of 8-oxodG was partially prevented by depletion of intracellular SH compounds by NEM, and this result is consisted with the finding of Ballmaier and Epe

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