Tyrosine-Dependent Oxidative DNA Damage Induced by

Mie, 514-8507, Japan, and Suzuka UniVersity of Medical Science, Faculty of Health Science, Suzuka,. Mie, 510-0293, Japan. ReceiVed June 20, 2006...
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Chem. Res. Toxicol. 2006, 19, 1379-1385

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Tyrosine-Dependent Oxidative DNA Damage Induced by Carcinogenic Tetranitromethane Mariko Murata,*,† Saori Kurimoto,† and Shosuke Kawanishi†,‡ Department of EnVironmental and Molecular Medicine, Mie UniVersity Graduate School of Medicine, Tsu, Mie, 514-8507, Japan, and Suzuka UniVersity of Medical Science, Faculty of Health Science, Suzuka, Mie, 510-0293, Japan ReceiVed June 20, 2006

Tetranitromethane (TNM) is used as an oxidizer in rocket propellants and explosives and as an additive to increase the cetane number of diesel fuel. TNM was reported to induce pulmonary adenocarcinomas and squamous cell carcinomas in mice and rats. However, the mechanisms underlying carcinogenesis induced by TNM has not yet been clarified. We previously revealed that nitroTyr and nitroTyr-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction. Since TNM is a reagent for nitration of Tyr in proteins and peptides, we have hypothesized that TNM-treated Tyr and Tyr-containing peptides induce DNA damage by the modification of Tyr. We examined DNA damage induced by TNM-treated amino acids or peptides using 32P-5′-endlabeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene. TNM-treated Tyr and Lys-Tyr-Lys induced DNA damage including the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine in the presence of Cu(II) and NADH. DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H2O2 and Cu(I). The cytosine residue of the ACG sequence complementary to codon 273, well-known hotspots of the p53 gene, was cleaved with piperidine and Fpg treatments. On the other hand, nitroTyr and Lys-nitroTyr-Lys did not induce DNA damage in the presence of Cu(II) and NADH. Time-of-flight mass spectrometry confirmed that reactions between Lys-Tyr-Lys and TNM yielded not only Lys-nitroTyr-Lys but also Lys-nitrosoTyrLys. Therefore, it is speculated that the nitrosotyrosine residue can induce oxidative DNA damage in the presence of Cu(II) and NADH. It is concluded that Tyr-dependent DNA damage may play an important role in the carcinogenicity of TNM. TNM is a new type of carcinogen that induces DNA damage not by itself but via Tyr modification. Introduction (TNM)1

Tetranitromethane is used as an oxidizer in rocket propellants and explosives and as an additive to increase the cetane number of diesel fuel (1, 2). TNM is genotoxic in bacteria and cultured mammalian cells (3, 4). Toxicology and carcinogenesis studies were conducted in rats and mice by exposure to TNM vapor. TNM caused mild irritation and hyperplastic lesions in the nasal passages (2, 3, 5). Furthermore, this nitro compound induced pulmonary adenocarcinomas and squamous cell carcinomas in treated mice and rats (1-3, 5, 6). The International Agency for Research on Cancer (IARC) has classified TNM as a group 2B carcinogen, which is possibly carcinogenic to humans (3). Several studies demonstrated that G:C f A:T transitions at the second base of the K-ras codon 12 (GGT f GAT) were the most frequent pattern of K-ras mutations identified in TNM-induced lung neoplasms (1, 7). However, the mechanisms underlying carcinogenesis induced by TNM have not yet been clarified. It is generally accepted that DNA damage, such as adduct formation and oxidative damage, is the first step of initiation pro* Corresponding author. Phone and fax. : +81-59-231-5011; e-mail: [email protected]. † Mie University Graduate School of Medicine. ‡ Suzuka University of Medical Science. 1 Abbreviations: TNM, tetranitromethane; nitroY, nitrotyrosine; nitroso Y, nitrosotyrosine; 8-oxodG, 8-oxo-7,8-dihydro-2′-deoxyguanosine; HPLCECD, HPLC coupled with an electrochemical detector; DTPA, diethylenetriamine-N,N,N′,N′,N′′-pentaacetic acid; SOD, superoxide dismutase.

cess in chemical carcinogenesis. Either chemical compounds or their metabolites are ultimate carcinogens, which can induce DNA damage. TNM is a nitrating reagent that can be employed under mild condition (2, 8), and it was more effective than other reagents at nitrating Tyr-containing peptides (9). However, Yermilov et al. reported that TNM did not induce the formation of 8-nitroguanine, a representative of nitrative DNA damage (10). In addition, there is no report regarding metabolites of TNM. Therefore, TNM may be another type of carcinogen different from already-known types of carcinogens. We revealed that nitrotyrosine (nitroY) and nitroY-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase, which is considered to yield nitroreduction (11). We have hypothesized that TNM-treated Tyr or Tyr-containing peptides induce DNA damage by the nitration of Tyr. To examine the hypothesis in relation to TNM carcinogenesis, we investigated whether TNMtreated amino acids and peptides could induce DNA damage or not. We utilized 32P-5′-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene and the c-Ha-ras-1 protooncogene as physiologically relevant targets for chemical carcinogens (12). Furthermore, we investigated the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA by using an electrochemical detector coupled to HPLC (HPLC-ECD). In addition, we analyzed the reaction products of TNM and Tyrcontaining peptides by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS).

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Figure 1. Autoradiogram of 32P-5′-end-labeled DNA fragments incubated with TNM-treated amino acids and peptides. (A) The reaction mixtures contained 32P-5′-end-labeled 443 bp DNA fragments (ApaI 14179-EcoRI* 14621), calf thymus DNA (20 µM/base), 20 µM CuCl2, and the indicated concentrations of TNM-treated KYK in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA with or without 100 µM NADH. (B) The reaction mixtures contained the 32P-5′-end-labeled 261 bp DNA fragments (AVaI* 1645-XbaI 1905), calf thymus DNA (20 µM/base), 40 µM CuCl2, 100 µM NADH, 100 µM TNM, and TNM-treated Y, G, K, KYK, GYG, or KWK in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. (C) The reaction mixtures contained the 32P-5′-end-labeled 443 bp DNA fragments, calf thymus DNA (20 M/base), 53.8 µM CuCl2, 100 µM NADH, and 100 µM nitroY, Y, K-nitroY-K, or KYK with or without TNM treatment in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. After the incubation at 37 °C for 1 h, DNA fragments were treated with 1 M piperidine for 20 min at 90 °C and then electrophoresed on an 8% (w/v) polyacrylamide/8 M urea gel. The autoradiogram was obtained by exposing X-ray film to the gel.

Materials and Methods Materials. Restriction enzymes (ApaI, AVaI, EcoRI, and XbaI) and calf intestine phosphatase were purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Restriction enzyme (BamHI) and T4 polynucleotide kinase were obtained from New England Biolabs (Beverly, MA). [γ-32P] ATP (222 TBq/mmol) was acquired from New England Nuclear (Boston, MA). Diethylenetriamine-N,N,N′,N′′,N′′-pentaacetic acid (DTPA) and bathocuproinedisulfonic acid were procured from Dojin Chemical Co. (Kumamoto, Japan). Acrylamide, piperidine, dimethyl sulfoxide (DMSO), bisacrylamide, and iron(III) chloride hexahydrate were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). CuCl2, MnCl2, CoCl2, iron(III) monosodium ethylenediaminetetraacetate (Fe(III)-EDTA), ethanol, D-mannitol, sodium formate, L-tyrosine (Tyr), L-lysine monohdrochloride (Lys), and glycine hydrochloride (Gly) were acquired from Nacalai Tesque (Kyoto, Japan). β-Nicotinamide adenine dinucleotide (reduced form) (NADH), tetranitromethane (TNM), R-cyano-4-hydroxycinnamic acid, and Lys-Tyr-Lys (KYK) were procured from Sigma-Aldrich Japan K.K. (Tokyo, Japan). Calf thymus DNA, superoxide dismutase (SOD, 3000 units/mg from bovine erythrocytes), catalase (45000 units/ mg from bovine liver), 3-nitro-L-Tyr (nitroY) and Lys-Trp-Lys (KWK) were obtained from Sigma Chemical Co. (St. Louis, MO). Lys-nitroTyr-Lys (K-nitroY-K) and Gly-nitroTyr-Gly (G-nitroYG) were obtained from Sigma-Aldrich Japan K.K. Genosys division (Hokkaido, Japan). Nuclease P1 (400 units/mg) was purchased from Yamasa Shoyu Co. (Chiba, Japan). Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) was obtained from Trevigen Inc. (Gaithersburg, MD). Gly-Tyr-Gly (GYG) was obtained from Bachemag AG (Bubendorf, Switzerland). Preparation of 32P-5′-End-Labeled DNA Fragments. DNA fragments were obtained from the human p53 tumor suppressor gene (13) and the c-Ha-ras-1 protooncogene (14). The DNA fragment of the p53 tumor suppressor gene was prepared from the pUC18 plasmid. A singly 32P-5′-end-labeled 443 bp fragment (ApaI 14179-EcoRI* 14621) was obtained according to the method described previously (15). Furthermore, DNA fragments were also

prepared from plasmid pbcNI, which carries a 6.6-kb BamHI chromosomal DNA segment containing the human c-Ha-ras-1 protooncogene (16, 17). A singly 32P-5′-end-labeled 261 bp fragment (AVaI*1645-XbaI 1905) was obtained according to the method described previously (16, 17). The asterisk indicates 32P-labeling. Treatment of Amino Acid or Peptide with TNM. The reaction was started by the addition of TNM (0.8 M stock solution in ethanol) to the amino acid and peptide solutions (0.8 M stock solution in water or NaOH). The standard reaction mixtures (in a 1.5 mL Eppendorf microtube) containing amino acids and peptides treated with TNM (0.4 M amino acid or peptide: 0.4 M TNM ) 1:1) were preincubated at 37 °C for 3 h. Detection of DNA Damage Induced by TNM-Treated Amino Acid or Peptide. Standard reaction mixtures (in a 1.5 mL Eppendorf microtube) containing the indicated concentrations of TNM-treated amino acid or peptide, 32P-5′-end-labeled DNA fragments, 20 µM/base calf thymus DNA, CuCl2, and NADH in 200 µL of 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37 °C for 1 h. Then, the DNA fragments were treated in 10% (v/v) piperidine at 90 °C for 20 min or treated with 6 units of Fpg protein in 20 µL of 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. The treated DNA was electrophoresed on an 8% (w/v) polyacrylamide/8 M urea gel. The autoradiogram was obtained by exposing X-ray film to the gel (16). 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 (18) using a DNA-sequencing system (LKB 2010 Macrophor). A laser densitometer (LKB 2222 UltroScan XL) was used for the measurement of the relative amounts of oligonucleotides from the treated DNA fragments. Measurement of 8-oxodG Formation in Calf Thymus DNA. DNA fragments (100 µM/base) from calf thymus were incubated with TNM-treated amino acid or peptide, CuCl2, and NADH in 400 µL of 4 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA at 37 °C for 1 h. After ethanol precipitation, DNA was digested to nucleosides with nuclease P1 and calf intestine phos-

Tyrosine-Dependent DNA Damage by Tetranitromethane phatase and analyzed by HPLC-ECD, as described previously (19). The resulting deoxynucleoside mixture was injected into an HPLC apparatus (LC-10A, Shimadzu, Kyoto, Japan) equipped with both a UV detector (SPD-10A, Shimadzu) and an electrochemical detector (Coulochem model-5200-2, ESA, MA): column, Shiseido Fine Chemicals ODS (0.46 × 15 cm); eluent, 10 mM sodium dihydrogenphosphate dihydrate/methanol (92:8, v/v); and flow rate 1 mL/min. The molar ratio of 8-oxodG to deoxyguanosine (dG) in each DNA sample was measured based on the peak height of authentic 8-oxodG with the electrochemical detector and the UV absorbance at 254 nm of dG. Measurement of Nitrated Amino Acid or Peptide by HPLCPhotodiode Array (PDA). The standard reaction mixtures (in a 1.5 mL Eppendorf microtube) containing Tyr, KYK, or GYG treated with TNM (0.4 M amino acid or peptide: 0.4 M TNM ) 1:1) were incubated at 37 °C for 3 h. The NitroY, K-nitroY-K, or G-nitroY-G amount was analyzed by HPLC with a Shimadzu photodiode array UV detector (SPD-M10A, Kyoto, Japan) at 360 nm, in which nitroY, K-nitroY-K, or G-nitroY-G had maximum absorption at pH 6, with Shiseido Fine Chemicals ODS (0.46 × 15 cm) in the mobile phase containing 100 mM potassium phosphate buffer (pH 6)/methanol (98:2, v/v) at flow rate of 1 mL/ min. The nitration efficiency of TNM-treated Tyr, KYK, or GYG was calculated based on the UV absorbance at 360 nm of authentic nitroY, K-nitroY-K, or G-nitroY-G. MALDI-TOFMS Analysis. MALDI-TOFMS analysis was performed on a Voyager B-RP (PerSeptive Biosystems, Framingham, MA) equipped with a nitrogen laser (337 nm, 3 ns pulse) to determine the molecular weight of the products from the reaction of KYK and TNM (0.4 M KYK/0.4 M TNM ) 1:1) incubated at 37 °C for 3 h. Matrix solution (saturated R-cyano-4-hydroxycinnamic acid : 0.1% TFA/50% acetonitrile ) 1:1) was added to the sample and then air-dried on a stainless-steel probe tip. Statistical Analysis. The data with agents and concentrations as two independent variables were analyzed statistically using a two-way analysis of variance (ANOVA). The differences among reagents at one concentration were computed by using a one-way ANOVA. Multiple comparisons were made with the Scheffe test. P-values less than 0.05 were considered statistically significant.

Results Damage to 32P-Labeled DNA Fragments Induced by TNM-Treated Amino Acid or Peptide. Figure 1A shows an autoradiogram of Cu(II)-mediated DNA damage by TNMtreated KYK in the presence and absence of NADH. TNMtreated KYK induced DNA damage in the presence of Cu(II) and NADH but did not in the absence of NADH. The intensity of DNA damage increased with increasing concentrations of TNM-treated KYK. Furthermore, hot piperidine treatment increased DNA damage as compared with no treatment (data not shown). When Fe(III), Fe(III)-EDTA, Mn(II), and Co(II) were used instead of Cu(II), TNM-treated KYK induced no DNA damage under the conditions used (data not shown). As shown in Figure 1B, TNM alone induced no DNA damage even in the presence of Cu(II) and NADH under the conditions used. Also, TNM-treated Gly, Lys, GYG, and KWK induced little or no DNA damage. TNM-treated Tyr and KYK induced DNA damage in the presence of Cu(II) and NADH (Figure 1B). TNMtreated KYK can induce DNA damage more efficiently than TNM-treated Tyr. However, regardless of TNM treatment, nitroY and K-nitroY-K induced no DNA damage in the presence of Cu(II) and NADH (Figure 1C). Effects of Scavengers and a Metal Chelator on DNA Damage Induced by TNM-Treated KYK. Figure 2 shows the effects of scavengers and bathocuproine. Catalase and bathocuproine inhibited Cu(II)/NADH-mediated DNA damage induced by TNM-treated KYK, indicating the involvement of

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Figure 2. Effects of scavengers and a metal chelator on DNA damage induced by TNM-treated KYK in the presence of Cu(II) and NADH. The reaction mixtures containing 32P-5′-end-labeled 443 bp DNA fragments, calf thymus DNA (20 µM/base), 20 µM CuCl2, 100 µM NADH, and 200 µM TNM-treated KYK in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA were incubated at 37 °C for 1 h. Ethanol (5% (v/v)), mannitol (0.1 M), sodium formate (0.1 M), DMSO (5% (v/v)), catalase (150 units/mL), heat-inactivated catalase (150 units/mL), SOD (150 units/mL), or bathocuproine (50 µM) were added before the incubation. DNA fragment was treated as described in the Figure 1 caption.

hydrogen peroxide (H2O2) and Cu(I). On the other hand, heatinactivated catalase had no inhibitory effect. SOD and typical free hydoxyl radical (•OH) scavengers, such as ethanol, mannitol, sodium formate, and DMSO, showed little or no inhibitory effects on DNA damage. Site Specificity of DNA Damage by TNM-Treated KYK in the Presence of Cu(II) and NADH. An autoradiogram was obtained and scanned with a laser densitometer to measure the relative intensity of DNA cleavage in the human c-Ha-ras-1 protooncogene (Figure 3A,B) and the human p53 tumor suppressor gene (Figure 3C,D), as shown in Figure 3. TNMtreated KYK induced piperidine-labile sites preferentially at guanine, cytosine, and thymine residues (Figure 3A,C). With Fpg treatment, the DNA cleavage occurred frequently at guanine and cytosine residues (Figure 3B,D). The cytosine residue of the ACG sequence complementary to codon 273, a well-known hotspot of the p53 gene (20, 21), was cleaved with piperidine and Fpg treatments (Figure 3C,D). In addition, the guanine residue of the CCG sequence complementary to hotspot codon 282 of the p53 gene (20, 21) was cleaved with Fpg treatment. Formation of 8-oxodG by TNM-Treated KYK in the Presence of Cu(II) and NADH. By using the HPLC-ECD, we measured the 8-oxodG content in calf thymus DNA by the reaction of TNM-treated Tyr, KYK, and GYG (Figure 4A). Two-way ANOVA showed the significant differences with agent

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Figure 3. Site specificity of DNA damage induced by TNM-treated KYK in the presence of Cu(II) and NADH. The reaction mixtures contained the 32P-5′-end-labeled 261 bp (AVaI* 1645-XbaI 1905) DNA fragment of the human c-Ha-ras-1 protooncogene (A and B) or 443 bp (ApaI 14179EcoRI* 14621) DNA fragment of the p53 tumor suppressor gene (C and D), calf thymus DNA (20 µM/base), 20 µM CuCl2, 100 µM NADH, and 200 µM TNM-treated KYK in 10 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. Reaction mixtures were incubated at 37 °C for 1 h. After piperidine (A and C) or Fpg (B and D) treatment, the DNA fragments were analyzed as described in the Materials and Methods.

Figure 4. Formation of 8-oxodG in calf thymus DNA induced by TNM-treated Tyr, KYK, or GYG in the presence of Cu(II) and NADH. (A) The reaction mixtures contained calf thymus DNA (100 µM/base), 53.8 µM CuCl2, 100 µM NADH, and the indicated concentrations of TNM-treated Tyr (circles), KYK (squares), or GYG (triangles) in 4 mM sodium phosphate buffer (pH 7.8) containing 5 µM DTPA. (B) The reaction mixtures contained calf thymus DNA (100 µM/base), 53.8 µM CuCl2, 100 µM NADH, 100 µM TNM-treated Tyr, and catalase (150 units/mL) or heatinactivated catalase (150 units/mL) in 4 mM sodium phosphate buffer (pH 7.8) containing 5 M DTPA. After incubation at 37 °C for 1 h, the DNA fragment was enzymatically digested into the nucleoside, and 8-oxodG formation was analyzed by using the HPLC-ECD, as described in the Materials and Methods. Results are expressed as means and SD of values obtained from three independent experiments. *P < 0.05 and **P < 0.01, significant differences as compared with the control, and ##P < 0.01, significant differences as compared with the TNM-treated KYK + catalase condition by using Scheffe’s pairwise multiple comparison after significant ANOVA results.

and concentration of agent as two independent variables. Scheffe’s pairwise multiple comparison revealed significant differences between either two agents. TNM-treated Tyr and KYK significantly increased 8-oxodG formation in a dosedependent manner in the presence of Cu(II) and NADH. In the case of TNM-treated GYG, no significant increase of 8-oxodG formation was observed even in the presence of Cu(II) and NADH. Catalase significantly inhibited the formation of 8-oxodG induced by TNM-treated KYK, but heat-inactivated catalase had no inhibitory effect (Figure 4B), indicating that H2O2 plays an important role in oxidative DNA damage.

Nitration Efficiency of Amino Acid and Peptide Treated with TNM. Nitration efficiency of Tyr, KYK, and GYG by TNM was analyzed by HPLC with photodiode array detection (Figure 5). Tyr and KYK were nitrated by TNM, whereas GYG was not nitrated under the conditions used. There were significant differences among agents by one-way ANOVA, and each two agents had significant differences by using pairwise comparison of the Scheffe test. TNM nitrated KYK more efficiently than Tyr. Nitration efficiency may depend on amino acid sequence. Determination of the Products from Reaction of KYK and TNM. Figure 6 shows the mass spectrum of reaction products

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Figure 5. Nitration efficiency of Tyr, KYK, or GYG treated with TNM. The reaction mixture containing 0.4 M Tyr, KYK, or GYG and 0.4 M TNM was incubated at 37 °C for 3 h and then was analyzed by HPLC with a photodiode array UV detector (360 nm), as described in the Materials and Methods. Figure 7. Possible mechanism of oxidative DNA damage induced by TNM-treated YKY in the presence of Cu(II) and NADH.

Figure 6. MALDI-TOFMS spectra of the products from the reaction of KYK and TNM. The reaction mixture containing 0.4 M KYK and 0.4 M TNM was incubated at 37 °C for 3 h. A matrix solution was added to the sample and then air-dried on a stainless-steel probe tip.

of KYK reacted with TNM. The molecule ion at m/z 438 (M+) was assigned to KYK, and m/z 483 (M+) was assigned to K-nitroY-K. In addition, the mass spectrum with a molecule ion at m/z 467 (M+) appeared (an arrow). We speculate that the molecule ion at m/z 467 (M+) corresponds to K-nitrosoYK, nitrosated form of Tyr.

Discussion 32P-labeled

Experiments with DNA revealed that TNM-treated Tyr and KYK caused DNA damage in the presence of Cu(II) and NADH. TNM-treated Tyr and KYK significantly increased the formation of 8-oxodG, which is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells (22-24). TNM-treated Tyr and KYK did not cause Cu(II)-mediated DNA damage in the absence of NADH, indicating that the reduction of TNM-treated Tyr and KYK by NADH was essential. Regardless of TNM treatment, nitrotyrosine and K-nitroY-K induced no DNA damage in the presence of Cu(II) and NADH. Our previous study demonstrated that nitrotyrosine and nitrotyrosine-containing peptides caused Cu(II)-dependent DNA damage in the presence of P450 reductase (11). These nitro compounds required the enzymatic nitroreduction to cause DNA damage. Therefore, we proposed that the TNM-modified compound other than nitrotyrosine or K-nitroY-K in TNM-treated products induced oxidative DNA damage. MALDI-TOFMS detected the product with a molecular ion at m/z 467 (M+), which can be

assigned to K-nitrosoY-K. The isotopic distribution of m/z 467 (M+) appeared to be different from others such as m/z 438 (M+) of KYK and m/z 483 (M+) of K-nitro-K. That is, the cluster looks like it contains two substances with m/z 466 and 467. TNM produces not only the nitronium ion but also nitric oxide (25), and the reaction between nitric oxide and tyrosine can form nitrosotyrosine as an intermediate (26). And thereafter, nitrosotyrosine becomes nitrotyrosine via the tyrosine iminoxyl radical (m/z 466) (25). This may explain the different isotopic distribution. To clarify what kinds of reactive oxygen species cause DNA damage, we examined the effects of scavengers on DNA damage induced by TNM-treated KYK. Both catalase and bathocuproine inhibited DNA damage, indicating the involvement of H2O2 and Cu(I). Typical •OH scavengers had little or no inhibitory effect on DNA damage, indicating that •OH might not be involved. We previously revealed that nitroso compounds, such as nitrosobenzene (27), nitrosopyrene (28), and nitrosonaphthol (29), caused oxdative DNA damage in the presence of Cu(II) and NADH via the generation of H2O2 and Cu(I). Therefore, it is suggested that TNM induces DNA damage via formation of nitrosotyrosine. On the basis of these results and references, we propose the following possible mechanism (Figure 7). TNM reacts with tyrosine residues to form not only nitrotyrosine but also nitrosotyrosine. Electron spin resonance (ESR) studies (30, 31) have indicated that nitroso aromatic compounds can be nonenzymatically reduced to aminoxyl radical by NADH. Therefore, the nitrosotyrosine residue may be also reduced to aminoxyl radical by NADH. NADH itself is oxidized to NAD•, which reacts with O2 to form O2•- and NAD+. We measured NADH consumption as the decrease of absorbance at 340 nm ( ) 6.22 × 103 M-1 cm-1) by using a UV-vis spectrophotometer. The decreased amount of NADH (100 µM) during the reaction with 100 µM TNM-treated KYK, Tyr, and GYG was 51.8, 14.5, and 2.4 µM, respectively, at 37 °C for 1 h in the presence of Cu(II). This suggests that the redox cycle between NADH and reactive intermediate is formed, although we cannot provide actual amounts of the peptide altered. Auto-oxidation of the aminoxyl radical to the nitroso compound occurs with simultaneous generation of O2•-, which is dismutated to H2O2. O2•mediates the reduction of Cu(II) to Cu(I). Finally, H2O2 interacts with Cu(I) to form a metal-oxygen complex such as Cu(I)OOH, which can cause oxidative DNA damage.

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G:C f A:T transitions were the most frequent pattern of K-ras mutations identified in TNM-induced lung neoplasms (7). Belinsky et al. (6) suggested that the TNM-induced G:C f A:T transition could have been derived from the deamination of cytosine. We showed that TNM-treated Tyr and KYK induced DNA cleavage frequently at piperidine-labile guanine, cytosine, and thymine residues and Fpg-sensitive guanine and cytosine residues. It is known that cytosine oxidation followed by deamination leads to the G:C f A:T transition (32). In addition, TNM-treated Tyr and KYK efficiently induced 8-oxodG formation in the presence of Cu(II) and NADH. 8-oxodG has attracted much attention in relation to mutagenesis and carcinogenesis (22-24). Although the source of the G:C f A:T transitions is not obvious, the guanine and cytosine lesions observed in this study may be attributable to the mutation. Sodum et al. (33) reported the increased level of protein nitrotyrosine in the lung cytosols of rats treated with TNM. The present study showed the formation of both nitrotyrosine and nitrosotyrosine in the reaction between TNM and Tyr-containing peptide. Therefore, TNM may yield not only nitrotyrosine but also nitrosotyrosine in vivo. We previously reported that nitrotyrosine induced oxidative DNA damage via enzymatic reduction and might participate in the carcinogenesis (11). The present study has demonstrated that TNM can cause oxidative DNA damage by the formation of nitrosotyrosine without enzymatic activation. Therefore, Tyr-dependent DNA damage may play an important role in the carcinogenicity of TNM via nitration and nitrosation. In addition, TNM has irritant properties leading to cell proliferation (1). Reactive oxygen species is a cause of irritation and induces cell proliferation. In general, DNA damage may play a role in tumor initiation, and cell proliferation may induce tumor promotion and/or progression, leading to carcinogenesis. TNM may exert carcinogenic action by both DNA damage and stimulation of cell proliferation though Tyrdependent oxidative stress. Moreover, it is reported that the nitration of the enzyme with TNM resulted in the loss of the enzyme activity (34). Inhibition of the modified base excision DNA repair processes would be expected to increase the rate of mutagenic DNA lesions (35). If TNM nitrates the DNA repair enzyme, it may lead to mutagenesis and carcinogenesis. The present study and our previous one indicate that TNM yields nitrotyrosine and nitrosotyrosine, both of which mediate oxidative DNA damage with or without metabolic activation. In other words, TNM is a new type of carcinogen that induced DNA damage not by itself but via Tyr modification. Finding DNA damage by TNM as a new type may provide an insight into the mechanism of carcinogenesis, in addition to already-known types of carcinogens. Acknowledgment. This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.

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