Document N2Jx5bkJOv0pO2j8zn5EDQmyE

984 Chem. Res. Toxicol. 2009, 22, 984-989 DNA Methylation at the C-5 Position of Cytosine by Methyl Radicals: A Possible Role for Epigenetic Change during Carcinogenesis by Environmental Agents Hiroshi Kasai* and Kazuaki Kawai Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1, Iseigaoka, Yahatanishi-ku, Kitakyushu, 807-8555, Japan Received March 12, 2009 During carcinogenesis, methylation of the C-5 position of cytosines in the promoter region of tumor suppressor genes is often observed. Enzymatic DNA methylation is a widely accepted mechanism for this phenomenon. It is interesting to propose a free radical mechanism for 5-methyldeoxycytidine (m5dC) production, because the C-5 position of cytosine is an active site for free radical reactions. When deoxycytidine (dC) and cumene hydroperoxide (CuOOH), a tumor promoter and a methyl radical producer, were reacted in the presence of ferrous ion at pH 7.4, the formation of m5dC was observed. The same reaction also proceeded with t-butyl hydroperoxide (BuOOH). The formation of m5dC was also observed in DNA by the CuOOH treatment. This is the first report of chemical DNA methylation at cytosine C-5 by environmental tumor promoters. We propose here that this reaction is one of the important mechanisms of de novo DNA methylation during carcinogenesis, because methyl radicals are produced by the biotransformation of various endogenous and exogenous compounds. Introduction DNA methylation is an important epigenetic mechanism of transcriptional control and plays an essential role in maintaining normal cellular function. During carcinogenesis, the methylation of CpG islands in the promoter regions of tumor suppressor genes occurs, and this can lead to a loss of the gene function or to gene silencing (1, 2). The hypermethylation of the promoter regions of these genes is frequently observed in human cancer (3) . Therefore, during multistage carcinogenesis, both mutation and hypermethylation can lead to an inactive tumor suppressor gene. It is generally accepted that methylation occurs enzymati cally by de novo DNA methyl transferases, such as DNMT3b (4) . However, the exact mechanisms of hypermethylation, particularly in relation to environmental factors during carcino genesis, are not clear. Valinluck and Sowers reported that one possible mechanism of DNA hypermethylation is the formation of inflammationinduced 5-halogenated dC, such as 5-chloro-dC in DNA, which mimics m5dC and induces inappropriate methylation by the maintenance DNMT1 enzyme within the CpG sequence (5). They also reported that a form of inflammation-induced oxida tive DNA damage, 5-hydroxymethyldeoxycytidine, prevents DNMT1 methylation within CpG sequences and induces hypomethylation. They proposed that the chemical modification of DNA could cause heritable changes in cytosine methylation patterns, resulting in human tumor formation. The formation of a mutagenic methyl radical-deoxyguanosine adduct, 8-methyl-2'-deoxyguanosine, has been detected in DNA after a treatment with BuOOH and ferrous ion in vitro or after the administration of 1,2-dimethylhydrazine to rats (6-8). We proposed a free radical mechanism to produce m5dC in DNA or the nucleotide pool, because the C-5 position of cytosine is * To whom correspondence should be addressed. Tel: +81-93-691-7469. Fax: +81-93-601-2199. E-mail: h-kasai@med.uoeh-u.ac.jp. an active site for free radical reactions, in addition to the C-8 of purines and the C-6 position of pyrimidines, on the basis of quantum mechanical calculations (9). In this study, environ mental tumor promoters, cumene hydroperoxide (CuOOH) and t-butyl hydroperoxide (BuOOH), which are known to generate methyl radicals (10), were tested for the formation of m5dC from dC or in DNA. Experimental Procedures Materials. Deoxycytidine (dC), calf thymus DNA, poly(dGdC)*poly(dG-dC), poly(dG)*poly(dC), and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were purchased from SigmaAldrich (St. Louis, MO). dC was purified by repeated rounds of HPLC (Capcell Pak C18, 5 pm, 10 mm x 250 mm, Shiseido Fine Chemicals, Japan; elution, 5% methanol in water) to remove the small amount of contaminating 5-methyl-dC. a-(4Pyridyl-1-oxide)-A-tert-butylnitrone (POBN) was a product of Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). CuOOH (80% solution) was a product of Lancaster (Morecambe, England). BuOOH (70% solution) and ferrous sulfate (FeSO4*7H2O) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The FeSO4 solution (100 mM) was prepared just before use for reactions. The anti-5-methyldC monoclonal antibody was a gift from Dr. Kazuaki Watanabe, Toray Research Center (Kamakura, Japan) (11). Reaction of dC with CuOOH/Fe2+ and Analysis of the Product by HPLC. Detailed reaction conditions are described in the legends to Figures 1-3. After the reaction, the solution was centrifuged, and an aliquot of the supernatant was injected into the HPLC column (YMC-Pak ODS-AM, 4.6 mm x 250 mm, particle size, 5 pm; elution, 5% methanol, 0.9 mL/min) connected with a photodiode array UV detector (HewlettPackard 1100 HPLC Detection System). Reaction of DNA Polymer with CuOOH/Fe2+. Detailed reaction conditions are described in the legend to Figure 4. After 10.1021/tx900099s CCC: $40.75 2009 American Chemical Society Published on Web 05/18/2009 Communications Chem. Res. Toxicol., Vol. 22, No. 6, 2009 985 Figure 1. Detection of m5dC in the reaction mixture of dC, Fe2+, and CuOOH by HPLC. The reaction mixture (final volume, 0.32 mL), containing dC (final concentration, 5.46 mM), FeSO4 (6 mM), and CuOOH (63 mM) in 20 mM phosphate buffer (pH 7.4), was reacted in a sealed plastic tube (tube volume, 2 mL) by vigorous shaking at 20 C. After a 1 h of reaction, the solution was centrifuged, and an aliquot of the supernatant was injected into the HPLC apparatus. (A) Chromatogram of the m5dC standard and its UV spectrum (inset), (B) chromatogram of the reaction mixture and UV spectrum of the peak at 18.5 min (inset), and (C) chromatogram of the control reaction mixture without CuOOH. Figure 2. Dose and time dependency of m5dC formation in the dC/ CuOOH/Fe2+ reaction. (A) Dose dependency: The reaction conditions were the same as those in Figure 1, except that three different concentrations of CuOOH (7, 21, or 63 mM) were used. Mean values of duplicate experiments are plotted. (B) Time dependency: The reaction conditions were the same as those in Figure 1, except that the reaction was stopped at 5, 20, and 60 min. Mean values of duplicate experiments are plotted. a 5 min reaction, the reaction mixture was centrifuged, and the supernatant (290 uL) was mixed with 87 uL of 5 M NaCl and 754 uL of cold ethanol and kept at 5 C to precipitate the DNA. The DNA was recovered, washed with cold ethanol, dried under reduced pressure, and then dissolved in 260 juL of 1 mM EDTA (pH 8.0). For the LC/MS/MS analysis, a 170 uL aliquot of the sample was digested with 14 units of nuclease P1 and 4 units of alkaline phosphatase. For immunodot blot analysis, the DNA solution was centrifuged, and the supernatant was passed through a centrifugal filter device (Amicon Microcon YM-100) to recover the high molecular weight DNA (MW > 100000). The DNA trapped by the filter was dissolved in 150 uL of 1 mM EDTA (pH 8.0). Detection of m5dC in DNA by Immunodot Blot Analysis. The immunodot blot analysis was performed by basically the same method as previously reported (12). A calf thymus DNA sample (1 mg/mL PBS) was sonicated to obtain fragments of DNA. The DNA solution was then heat-denatured and diluted with 2 M ammonium acetate to an appropriate concentration (1-100 ng/mL). When the oligonucleotide was used, the sonication step was omitted. Single-stranded DNA and oligo nucleotides (containing 0.1-10 ng DNA or 50 ug oligonucle- 986 Chem. Res. Toxicol., Vol. 22, No. 6, 2009 Communications Figure 3. Inhibition of m5dC formation from dC by TEMPO (A) or POBN (B). The reaction conditions were the same as those in Figure 1, except that the reaction time was 20 min. The reaction was conducted in the presence or absence of TEMPO and POBN. The ratio to the m5dC yield without TEMPO or POBN (1.0) is shown. Figure 4. Detection of m5dC in DNA polymers by an immunodot blot analysis. The reaction mixture (final volume, 0.32 mL) contained poly(dGdC)poly(dG-dC) or poly(dG)poly(dC) (final concentration, 10 A260 OD units/mL), FeSO4 (6 mM), and CuOOH (63 mM) in 20 mM phosphate buffer (pH 7.4) and was reacted in a sealed plastic tube (tube volume, 2 mL) by vigorous shaking at 20 C. After 5 min, the polymers were recovered from the reaction mixture, as described in the Experimental Procedures, and were used for the analysis. As positive controls, m5dC in various amounts of calf thymus DNA was visualized. As negative controls, DNA polymers without treatment were analyzed. otide /100 /iL sample) were immobilized on a nitrocellulose membrane (0.45 im, Bio-Rad Laboratories, CA) using a Bio Dot Microfiltration Apparatus (Bio-Rad Laboratories). The wells were rinsed with 200 fiL of 2 M ammonium acetate. The filter was subsequently removed from the support, and the DNA was cross-linked to the nitrocellulose using a Spectrolinker XL 1000 UV (Spectronics Co., NY). The membrane was washed twice for 5 min with PBS-Tween 20 (0.05%) (PBS-T) containing 2% ECL Advance Blocking Agent (GE Healthcare, Buckingham shire, United Kingdom) (blocking solution). The membrane was then incubated overnight at 4 C with the blocking solution containing an anti-m5dC monoclonal antibody (1.3 ig/mL) (11). The membrane was washed three times with PBS-T and was then incubated with the secondary antibody diluted 1:75000 in the blocking solution (ECL Anti-Mouse IgG Horseradish Peroxidase-Linked Species-Specific Whole Antibody, from sheep, GE Healthcare) for 2 h at room temperature. The membrane was washed four times with PBS-T. The enzymatic activity was visualized with an Amersham ECL advance Western blotting detection kit (GE Healthcare). The chemilu minescence output from the membrane was imaged using a CCD imager (Light Capture AE-6972, ATTO, Tokyo, Japan). LC/MS/MS Analysis. The LC/MS/MS data were acquired on a Waters Micromass Quattro Ultima Pt triple quadrupole mass spectrometer with an ESI source (Waters Corp., Milford, MA). It was operated in the positive ion mode with a potential of 35 V. The desolvation temperature was 350 C, and the ion source temperature was 120 C. The collision energy was 11 eV. HPLC was performed using a Waters Alliance 2695 system (Waters Corp.), with a Capcell Pak C18 MG column, 5 im, 2.0 mm x 250 mm (Shiseido Fine Chemicals, Japan); column temperature, 40 C; elution, 8% aqueous methanol containing 10 mM ammonium formate; and elution speed, 0.2 mL/min. Results Reaction of dC with CuOOH/Fe2+. When dC was reacted with CuOOH in the presence of Fe2+ at pH 7.4, the formation of m5dC was clearly identified by HPLC equipped with a photodiodo array UV detector (Figure 1). The retention time and the UV spectrum of the reaction product were exactly the same as those of the authentic m5dC. Its formation was dependent on the concentration of CuOOH (Figure 2A), and the reaction was rather rapid, due to its radical character. The reaction was approximately 70% complete within 5 min (Figure 2B). The methyl radical is produced by the reduction of CuOOH by Fe2+. It is reasonable to speculate that the methyl radical formation rate and the m5dC formation rate are dependent upon Communications (17) Augusto, O., Du Plessis, L. R., and Weingrill, C. L. V. (1985) Spin trapping of methyl radical in the oxidative metabolism of 1,2dimethylhydrazine. Biochem. Biophys. Res. Commun. 110, 625-631. (18) Goria-Gatti, L., Iannone, A., Tomasi, A., Poli, G., and Albano, E. (1992) In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin-trapping study. Carcinogenesis 13, 799 805. (19) Salaspuro, V., and Salaspuro, M. (2004) Synergistic effect of alcohol drinking and smoking on in vivo acetaldehyde concentration in saliva. Int. J. Cancer 111, 480-483. 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