Examination of Hypochlorous Acid-Induced Damage to Cytosine

Department of Basic Science, Loma Linda University School of Medicine, Loma Linda, California. Chem. Res. Toxicol. , 2008, 21 (6), pp 1211–1218. DOI...
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Chem. Res. Toxicol. 2008, 21, 1211–1218

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Examination of Hypochlorous Acid-Induced Damage to Cytosine Residues in a CpG Dinucleotide in DNA Joseph I. Kang, Jr. and Lawrence C. Sowers* Department of Basic Science, Loma Linda UniVersity School of Medicine, Loma Linda, California ReceiVed January 25, 2008

Inflammation-mediated, neutrophil-derived hypochlorous acid can damage DNA and result in the chlorination damage products 5-chlorocytosine and 5-chlorouracil as well as the oxidation damage products 5-hydroxycytosine and 5-hydroxyuracil. While 5-chlorocytosine could potentially perturb epigenetic signals if formed at a CpG dinucleotide, the remaining products are miscoding and could result in transition mutations. In this article, we have investigated the reaction of hypochlorous acid with an oligonucleotide site-specifically enriched with 15N to probe the reactivity of cytosine at CpG. These experiments demonstrate directly the formation of 5-chlorocytosine at a CpG dinucleotide in duplex DNA. We observe that chlorination relative to oxidation damage is greater at CpG by a factor of approximately two, whereas similar amounts of 5-chlorocytosine and 5-hydroxycytosine are formed at two non-CpG sites examined. The relative amounts of deamination of the cytosine to uracil derivatives are similar at CpG and nonCpG sites. Overall, we observe that the reactivity of cytosine at CpG and non-CpG sites toward hypochlorous acid induced damage is similar (5-chlorocytosine > 5-hydroxycytosine > 5-hydroxyuracil > 5-chlorouracil), with a greater proportion of chlorination damage at CpG sites. These results are in accord with the potential of inflammation-mediated DNA damage to both induce transition mutations and to perturb epigenetic signals. Introduction Purine and pyrimidine bases in the DNA of all organisms are continuously damaged by hydrolysis, oxidation, and alkylation under normal physiological conditions (1, 2). The level of damage can increase substantially because of chemical and biological stress. Reactive molecules associated with inflammation, including the hypohalous acids, are recognized increasingly as additional and potentially important contributors to DNA damage (3–19). Hypohalous acids, HOCl1 and HOBr, derived from activated neutrophils and eosinophils, respectively, are known to react with DNA resulting in an array of damage. Among the potential damage products are 5-hydroxycytosine (HOC) and 5-chlorocytosine (ClC) as well as the corresponding deamination products 5-hydroxyuracil (HOU) and 5-chlorouracil (ClU). Among these, the formation of HOC, HOU, and ClU could lead to miscoding, base misincorporation, and mutation if unrepaired prior to DNA replication (17–28). In contrast, ClC would not be expected to be miscoding; however, the formation of ClC in the CpG dinucleotide could perturb epigenetic patterns in DNA by causing inappropriate methyltransferase-mediated cytosine methylation and by increasing the binding affinity of methylbinding proteins involved in subsequent histone modification * To whom correspondence should be addressed. Department of Basic Science, Loma Linda University School of Medicine, Alumni Hall for Basic Science, Room 101, 11021 Campus Street, Loma Linda, CA 92350. Tel: 909-558-4480. Fax: 909-558-4035. E-mail: [email protected]. 1 Abbreviations: MPO, myeloperoxidase; HOCl, hypochlorous acid; HOBr, hypobromous acid; dC, 2′-deoxycytidine; CldC, 5-chloro-2′-deoxycytidine; CldU, 5-chloro-2′-deoxyuridine; HOdC, 5-hydroxy-2′-deoxycytidine; HOdU, 5-hydroxy-2′-deoxyuridine; ClC, 5-chlorocytosine; ClU, 5-chlorouracil; HOC, 5-hydroxycytosine; HOU, 5-hydroxyuracil; BrU, 5-bromouracil; GC/MS, gas-chromatography/mass spectrometry; 5mC, 5-methylcytosine; TBDMS, tert-butyldimethylsilyl; MTBSTFA, N-methylN-(tert-butyldimethylsilyl)trifluoroacetamide.

and chromosome condensation (17–19). Disruptions of epigenetic patterns are reported in numerous human tumors and are considered critical for understanding cancer etiology (29, 30). The biological consequences of cytosine damage depend upon the product formed and the base sequence in which the damage occurs. Sensitive and specific methods have been developed to identify this series of cytosine damage products. However, these methods require enzymatic or acid hydrolysis of DNA to the corresponding deoxynucleosides or free bases, resulting in the loss of information on the sequence dependence of cytosine damage. Central to understanding the impact of HOClmediated damage on epigenetic patterns is information on the reactivity of cytosine in a CpG dinucleotide in duplex DNA. In order to probe the reactivity of a specific cytosine residue, we have adopted an approach referred to by our group and others as mass tagging (31–35). In this approach, individual residues can be distinguished by increasing their mass with stable isotopes. As the corresponding reaction products would also be isotopically enriched, the sequence dependence of reactivity can be assessed using standard mass spectrometry methods. In this article, we have constructed an oligonucleotide duplex containing a target CpG dinucleotide in which the nontarget cytosine residues have been enriched with 15N. This oligonucleotide was reacted with HOCl, hydrolyzed, and analyzed by gaschromatography/mass spectrometry (GC/MS). Results confirm the formation of the expected products HOC, HOU, ClC, and ClU; however, the relative formation of the chlorination and oxidation damage products is different at a CpG dinucleotide compared with other cytosine residues in the molecule. This study provides direct evidence for the formation of ClC at a CpG dinucleotide, consistent with our proposal that inflammation-mediated reactive molecules could result in epigenetic changes frequently seen in human tumors.

10.1021/tx800037h CCC: $40.75  2008 American Chemical Society Published on Web 06/16/2008

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Material and Methods The phosphoramidites of the normal DNA bases and Poly-Pak II cartridges were obtained from Glen Research. N-methyl-N-(tertbutyldimethylsilyl)trifluoroacetamide supplemented with 1% tertbutyldimethylchlorosilane (MTBSTFA with 1%TBDMCS) was purchased from Pierce. Urea-15N (98% + 15N-enriched) was obtained from Cambridge Isotope Laboratories. Chelex-100 resin was purchased from Bio-Rad. Oligonucleotide Synthesis and Characterization. Standard phosphoramidites and oligonucleotide synthesis reagents (36) were obtained from Glen Research. Synthesis was preformed with a Pharmacia Gen Assembler. Enriched (15N3) 2′-deoxycytidine (dC) was prepared as previously described (37). Enriched dC was converted to the 5′-dimethoxytrityl-3′-cyanoethylphosphoramidite as previously described (38). Standard synthetic conditions were utilized for the incorporation of the enriched dC phosphoramidite into the oligonucleotide. Following synthesis, oligonucleotides were deprotected and released from the resin by treatment with concentrated aqueous ammonia at 60 °C overnight. Purification utilized Poly-Pak II cartridges. Base composition was confirmed by acid hydrolysis and analysis of the corresponding tert-butyldimethylsilyl ethers by GC/MS (39). Reaction of HOCl with an Oligonucleotide Duplex. Standard and isotopically enriched oligonucleotides were reacted with HOCl in buffered solution. All solutions were treated with Chelex-100 resin before use. HOCl concentration was quantified spectrophotometrically at 290 nm (pH 12, e ) 350 M-1 cm-1) immediately prior to reaction (40). In a standard reaction, 55 nmol of oligonucleotide in 400 uL (137.5 µM) of 50 mM sodium phosphate buffer (pH 7.4) was incubated with 0-200 nmol of HOCl (0-500 µM) for specified periods of time at 37 °C. The reaction was quenched with the addition of excess ice cold L-Methionine. The samples were then dialyzed against water for over 18 h. Following dialysis, solvent was removed under reduced pressure. Oligonucleotides were hydrolyzed with 200 µL of 88% formic acid in sealed vials at 140 °C for 30 min. Formic acid was evaporated under reduced pressure, and the samples were derivatized with 200 µL of MTBSTFA (1%TBDMCS)/acetonitrile (1:1, v/v) at 140 °C for 30 min. GC/MS Analysis of Damaged Bases. GC/MS analysis was conducted in electron impact (EI) mode with an Agilent 6890 GC containing an HP-5 ms column (dimensions 30 m × 0.25 mm × 0.25 µm) interfaced directly to a 5973 inert mass-selective detector fitted with a series 7683B autosampler/autoinjector. Injector and interface temperatures were 250 and 280 °C, respectively. The initial GC temperature was 100 °C for 2 min with a 10 °C min-1 increase up to 230 °C and a final 30 °C min-1 rise to a final temperature of 260 °C. EI source temperature was 230 °C, and quadrupole temperature was 150 °C, with electron energy at 70 eV. Helium gas, obtained from Gilmore (El Monte, CA), was of research grade. A volume of 1 µL was injected in split mode (20:1). Data were collected in scan or selected ion mode, depending upon the specific experiment. Standards of HOdC, HOdU, CldC, and CldU were prepared and characterized as previously described (41). The internal standard 5-bromouracil was obtained from Sigma and used to prepare calibration curves. Deamination of cytosine and cytosine damage products can occur during oligonucleotide hydrolysis and derivatization. While silylation with BSTFA containing 1% TMCS (resulting in the trimethylsilyl derivatives) results in significant deamination (42), we have observed that the silylation reported here with MTBSTFA containing 1% TBDMCS results in substantially less deamination, perhaps because the steric bulk surrounding the derivatized 4-amino group inhibits subsequent deamination. In control experiments using the latter, the conversion of CldC to ClU from acid hydrolysis and derivatization never exceeded 2%, and the conversion of HOdC to HOU was often undetectable and never exceeded 1%. Quantitation of Damaged Bases with Overlapping Mass Spectra. In experiments with isotopically enriched pyrimidines, the enrichment with 2 or 3 15N atoms is not sufficient to achieve

Kang and Sowers complete separation of the mass spectra of the unenriched and enriched species. To correct for overlap, the relative intensities of the isotope lines in the mass spectra of the unlabeled (lighter) derivatives were calculated on the basis of the molecular formula of a specific ion. The theoretical peak intensities in the isotope cluster of the M-57 peak for derivatized cytosine (C12H24N3OSi2) are 73.1% (282 Da), 18.4% (283 Da), 7.1% (284 Da), 1.2% (285 Da), and 0.2% (286 Da). The theoretical and experimental isotope clusters agree routinely within 2%. Enrichment of cytosine with 15N in all three nitrogen positions increases the mass by 3 Da and shifts the corresponding isotope cluster 3 Da to heavier mass. When examining a mixture of the unenriched cytosine derivative (282 Da) and 15N3-enriched cytosine (285 Da), the mass spectra of the two isotopomers will partially overlap. The peak at 282 Da arises from the unenriched isotopomer exclusively. Characterization of 15N3dC used for phosphoramidite synthesis showed the percentage of the unenriched isotopomer (282 Da) to the enriched isotopomer (285 Da) to be 0.013%. Therefore, the enriched analogue contributes negligibly to the 282 Da peak. However, the peak at 285 Da contains contributions predominantly from the 15N3-cytosine enriched isotopomer and heavier isotopes of C, H, N, O, and Si at natural abundance from the unenriched cytosine. The contribution from unenriched cytosine to the 285 Da peak from heavier isotopes at natural abundance is 1.6% of the 282 Da peak. In a similar manner, overlapping contributions can be determined for the cytosine damage products. The contribution of the unenriched isotopomer of HOC (412 Da) to the 415 peak is 4.1%. The contribution of the unenriched isotopomer of ClC (316 Da) to the 319 peak is 9.7%. The greater overlap in the chlorinated species results from the presence of chlorine isotopes at 35 Da (75.53%) and 37 Da (24.47%). The overlap for the deaminated derivatives, HOU and ClU, is greater than the corresponding cytosine derivatives because the 15N at the 4-position is lost during deamination. The contribution of the unenriched isotopomer of HOU (413 Da) to the 415 peak is 17%, and the contribution of the unenriched isotopomer of ClU (317 Da) to the 319 peak is 42.3%. In the design of the experiments reported here, the target CpG could have been enriched, as opposed to enrichment of the nonCpG sites. However, the primary objective was to provide direct evidence for damage at the CpG site. By labeling the non-CpG sites, the masses of the corresponding damage products would be shifted to higher mass, allowing direct examination of the products generated at the unenriched CpG site without requiring correction for overlapping isotopes. While the spectra of the unenriched derivatives reported here overlap partially with the enriched derivatives, requiring correction in order to determine the amounts of the enriched deriVatiVes, the experiments reported here utilize a common synthetic oligonucleotide in which the ratio of enriched to unenriched parent cytosine is constant. Therefore, comparison of the relative amounts of enriched and unenriched cytosine damage products provides a reliable indicator of the relative reactivity of cytosine at the CpG position relative to the non-CpG sites, independent of quantitation based upon use of the 5-bromouracil internal standard.

Results A 2′-deoxycytidine analogue enriched with 15N in the N(1) and N(3) ring positions was prepared and site-specifically incorporated into a self-complementary 12-mer oligonucleotide as shown in Figure 1. Oligonucleotide deprotection in 15Nenriched ammonium hydroxide results in additional 15Nenrichment at the N(4) amino position. The self-complementary 12-mer oligonucleotide examined here showed typical UVdependent changes in the UV spectrum as shown in Figure 2. Unlabeled oligonucleotide (13.75 nmol) was dissolved in 100 µL of 50 mM sodium phosphate buffer (pH 7.4) to a concentration of 137.5 µM. The observed melting temperature of the oligonucleotide, 50 °C, is close to the value of 51.3 °C predicted

HOCl Damage to CpG

Figure 1. Oligonucleotide sequence and structure of 15N3 2′-deoxycytidine used in this study. The cytosine residues at positions 1 and 5 (bold italic) have been isotopically enriched. The unenriched cytosine residue at the 6 position (CpG) gives rise to damage products ClC, ClU, HOC, and HOU, whereas the enriched cytosine residues at positions 1 and 5 give rise to isotopically enriched products of higher mass. The mass of the predominant ion used for measurement of the damage product is shown in the figure.

Figure 2. Melting temperature study for the oligonucleotide shown in Figure 1. Unlabeled oligonucleotide (13.75 nmol) was dissolved in 100 µL of 50 mM sodium phosphate buffer (pH ) 7.4) to a concentration of 137.5 µM. Data were collected at 0.5 °C intervals. The Tm of the duplex was determined using the hyperchromicity method of Cary Win UV Thermal software (Varian) with the average of 3 independent Tm measurements.

for this sequence using the online prediction tool, HyTher server (Department of Chemistry, Wayne State University). This selfcomplementary oligonucleotide would be predominantly in a duplex at and below physiological temperature (37 °C). Preliminary experiments were conducted with an unenriched oligonucleotide containing only standard, unenriched bases. The unenriched oligonucleotide was incubated with increasing concentrations of HOCl for 60 min. Base composition analysis by GC/MS following formic acid hydrolysis and silylation with MTBSTFA/TBDMCS revealed the presence of several peaks not observed in the untreated oligonucleotide (Figure 3). The damaged bases giving rise to the observed additional peaks were identified as ClC, HOC, HOU, and ClU on the basis of characteristic retention times and mass spectra. The predominant ion in the mass spectra for each of these compounds is the M-57 peak resulting from the loss of a tert-butyl group as shown in Figure 4. The isotope cluster of the silylated HOC and HOU (Figure 4C and D) show a typical pattern for molecules of this size in that the monoisotopic line is the highest intensity, and the corresponding lines at higher mass resulting from the enrichment of heavier isotopes of C, H, N, O, and Si at natural abundance progressively decrease in size. In contrast, the isotope cluster for ClC and ClU show a typical pattern for a chlorinecontaining molecule of this size in that the M + 2 peak is larger than the M + 1 peak (Figure 4A and B). Calibration curves were constructed relating peak area to base concentration using 5-bromouracil as the internal standard. Peak

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Figure 3. Gas chromatograph of ClU, ClC, HOC, and HOU obtained from reacting unenriched oligonucleotide (137.5 µM) with 500 µM HOCl at 37 °C for 1 h. Data were collected with selected ion monitoring for ClU (ion 317), ClC (ion 316), HOC (ion 412), and HOU (ion 413) to obtain the chromatograph.

areas for a selected ion were then used to determine the amount of each of the modified bases as well as the precursor cytosine in each sample. The amount of each of the modified bases formed increased with the concentration of HOCl present in the incubation mixture (Figure 5). The predominant products formed are ClC and HOC. The corresponding deamination products HOU and ClU were minor products in all reactions examined. The relatiVe amounts of the four reaction products measured did not change significantly with incubation time or HOCl concentration. The molar amounts of the four damage products observed are presented in Table 1. In order to examine the sequence-dependent reactivity of HOCl toward cytosine, the above experiments were repeated with the oligonucleotide containing the enriched cytosine residues at positions 1 and 5 and unenriched cytosine at the target CpG (position 6, Figure 1). Base analysis by GC/MS following acid hydrolysis of the enriched oligonucleotide was consistent with the expected composition. Analysis of the cytosine peak, but no others, revealed the presence of enriched bases. The ratio of unenriched to enriched cytosine was 2:1 as shown in Figure 6A. The site-specifically isotopically enriched oligonucleotide was then reacted with HOCl as described above. Following oligonucleotide hydrolysis and GC/MS analysis, peaks with retention times corresponding to ClC, HOC, ClU, and HOU were observed in scan mode. In Figure 6B, the selected ion chromatogram at 316 and 319 Da, corresponding to formation of ClC, is shown. The peak at 316 arose from formation of ClC at the CpG target site (position 6, Figure 1). The peak at 319 Da arose predominantly from the formation of ClC at the two nonCpG sites at C1 and C5. The mass spectra of the four damage products derived from the enriched oligonucleotide (Figure 7) are more complicated than those arising from the unenriched oligonucleotide (Figure 4) because of the presence of both isotopically enriched and unenriched species. In each case, the observed mass spectrum results from the partial overlap of the corresponding mass spectra for the isotopically enriched and unenriched damaged bases. For the cytosine derivatives, the mass spectral lines arising from the enriched cytosine at positions 1 and 5 are all increased by three mass units. The lines in the spectra of the cytosine damage products arising from the unenriched cytosine at position 6 are not increased in mass and correspond to the lines observed in the spectra of the unenriched damage products. The lines arising from enriched cytosine for the deamination products ClU and HOU are shifted by 2 mass units to higher mass. The conversion of the cytosine to uracil analogues results from the loss of the 15 N-enriched N(4) amino group.

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Figure 4. Mass Spectra of ClC (A), ClU (B), HOC (C), and HOU (D) from unenriched oligonucleotide reacted with 500 µM HOCl at 37 °C for 1 h.

Figure 5. Amounts of chlorinated and oxidized cytosine products formed from the reaction of unenriched oligonucleotide with 0-500 µM HOCl for 1 h at 37 °C. Data are normalized to the amount of cytosine measured. Error bars represent (1 standard deviation.

Table 1. Amounts of Cytosine Reaction Products for Both Unenriched and Enriched Oligonucleotides Reacted with 500 µM HOCl at 37°C for 1 ha 15 15 unenriched N3-enriched N3-enriched oligonucleotide oligonucleotide oligonucleotide product (nmol/µmol C) CpG (nmol/µmol C) non-CpG (nmol/µmol C)

ClC HOC ClU HOU

54.7 (3.7) 36.7 (0.3) 1.40 (0.2) 10.7 (0.2)

17.3 (1.5) 6.40 (0.5) 0.80 (0.09) 2.20 (0.15)

21.7 (1.7) 21.8 (1.6) 1.00 (0.1) 6.00 (0.2)

a Data are given in nmol of product per µmol of cytosine. Parentheses indicate 1 standard deviation from triplicate data.

Because the mass spectra of the enriched and unenriched derivatives are partially overlapping, the amount of each heavy derivative cannot be obtained directly from measuring relative peak areas. For example, the 316 line of ClC arises exclusively from the M-57 ion of unenriched cytosine. However, the 319 line arises predominantly from the M-57 ion of an enriched (+3) cytosine residue, but has a significant contribution from the 319

Figure 6. Extracted ion chromatographs of (A) Ions 282 and 285 for cytosine from isotopically enriched oligonucleotide incubated in buffer for 1 h at 37 °C (negative control) and (B) Ions 316 and 319 for ClC from isotopically enriched oligonucleotide reacted with 500 µM HOCl for 1 h at 37 °C. Note while the area of Ion 282 appears to be approximately half the area of Ion 285 indicating a 1 to 2 ratio of species, the area for Ion 316 is a much higher percentage of the area for Ion 319 indicating a larger ratio of the unenriched species.

line of the unenriched cytosine residue due to the natural abundance of heavier isotopes. The relative contribution of

HOCl Damage to CpG

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Figure 7. Mass Spectra of ClC (A), ClU (B), HOC (C), and HOU (D) from isotopically enriched oligonucleotide reacted with 500 µM HOCl for 1 h at 37 °C.

isotopes at natural abundance to the peaks within the isotope cluster can be determined as described in Materials and Methods and subtracted from the peaks arising from the isotopically enriched species. Using this approach, the amounts of each of the four damage products investigated here, derived from the unenriched target CpG at position 6, could be measured independently of the enriched cytosines at positions 1 and 5. The results of this experiment are presented in Table 1.

Discussion DNA damage and tissue inflammation have both been associated with cancer development in man (43, 44). The results of emerging biochemical studies, including those reported here, may provide a mechanistic link between the two. It is well known that activated neutrophils generate reactive HOCl, a highly effective antimicrobial agent that can simultaneously cause substantial collateral damage to normal tissues (4–16). HOCl is known to react with thiols, nucleotides, proteins, and other biological substrates (4). Several previous studies have demonstrated that HOCl can cause a complex array of damaged bases, and in particular, pyrimidine damage products (4–16). Thymine yields primarily cis and trans glycols, whereas cytosine generates both chlorination and oxidation damage products (6). The sum of the thymine products has been found to be similar in magnitude to the cytosine-generated products. In contrast to hydrogen peroxide, HOCl generates only small amounts of oxidized purines, including 8-oxoguanine (6). Among the HOCl-generated cytosine damage products, HOC, HOU, and ClU have been reported in several previous studies (4–16). The chlorination damage product ClC has been reported in substantially fewer studies (7, 9, 14, 15), perhaps because of

its apparent lability under DNA hydrolysis conditions, primarily its deamination to ClU. Recently, we developed methods for the chemical synthesis of oligonucleotides containing ClC (42), and using these oligonucleotides, we have optimized methods for the recovery of ClC from DNA as described here. Using these methods, we identify ClC as a significant HOCl-mediated DNA damage product. A reaction scheme is proposed, on the basis of the observed products, showing initial formation of a dihydrocytosine intermediate leading to each of the four products examined (Figure 8). It is well established that ring-saturated dihydrocytosine intermediates are extremely susceptible to hydrolytic deamination (45, 46). A cytosine residue in DNA (1) could react with HOCl to yield the 5-chloro (2) or 6-chloro (3) intermediates. The 5-chloro intermediate (2) could dehydrate to ClC (6) or deaminate to the dihydrouracil intermediate (4) and then dehydrate to ClU (7). The 6-chloro intermediate (3) could eliminate HCl, generating HOC (8) or deaminate to the corresponding dihydrouracil analogue (5) and then dehydrate to HOU (9). In a previous study, the amount of HOC generated in DNA by HOCl exceeded the amounts of HOU by a factor of approximately two (6). In our study, we observe that the HOClgenerated levels of HOC are approximately three times higher than the amounts of HOU, consistent with overall lower levels of product deamination with the methods reported here. In the previous study, ClU and HOU levels were similar (6); however, here we observe substantially lower levels of ClU relative to HOU, again reflecting lower levels of ClC deamination using the methods reported here. With the inclusion of ClC reported here, the array of damaged bases generated by HOCl constitutes a unique chemical damage spectrum.

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Figure 8. Reaction Scheme for the conversion of a cytosine residue to the reaction products, ClC (6), ClU (7) HOC (8), and HOU (9).

The biological consequences of DNA damage have historically focused upon mutagenesis. Among the HOCl-generated DNA damage products, ClU, HOC, and HOU would be mutagenic, and collectively, they generate approximately half of the cytosine-derived HOCl-mediated damage products (Table 1). All of these damage products can be excised by glycosylases of the base-excision repair pathway (47–52). In contrast, ClC would not be expected to be mutagenic, and no repair activities have yet been reported for ClC (42). In contrast to mutagenesis, the similar size of the 5-chlorine substituent of ClC and the 5-methyl group of 5mC led us to suspect that ClC might mimic 5mC in critical DNA-protein interactions (17–19). In higher eucaryotes, cytosine methylation patterns, in concert with specific modified histone proteins, comprise an epigenetic code essential for tissue and cell-specific control of gene expression (53, 54). Disruption of the epigenetic code can result in inappropriate gene expression, and such defects are commonly observed in human tumors. Although aberrant cytosine methylation, leading to the silencing of tumor suppressor genes, is a characteristic of many human tumors (29, 30), mechanisms leading to inappropriate methylation have not yet been identified. The identification of ClC as an endogenous inflammationmediated damage product led to in Vitro studies that confirmed that ClC can mimic 5mC in enhancing the binding of methylation-sensitive DNA binding proteins and in directing the inappropriate methylation of previously unmethylated sites by the human cytosine methyltransferase, Dnmt1 (17–19). In eucaryotes, cytosine methylation occurs predominantly in the CpG dinucleotide. If adventitious chlorination of DNA, generating ClC, can result in perturbations of the epigenetic code, it must occur at the CpG dinucleotide. Current analytical methods for examining DNA damage (55–61), although highly sensitive and specific, could not be used to establish ClC formation at the CpG dinucleotide because the acid or enzymatic hydrolysis required prior to analysis results in the loss of sequence-specific information. Examination of a complex mixture of intact oligonucleotides by mass spectrometry methods (62–64) also would not reveal the sequence-specific information desired. We therefore adapted a mass-tagging approach as reported here. The resulting mass differences between ClC formed at a target CpG site versus ClC formed at non-CpG

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sites (Figure 7) provide direct evidence for the capacity of HOCl to form ClC at the CpG dinucleotide in duplex DNA. As shown in Table 1, the overall reactivity of cytosine residues at CpG and the two non-CpG sites, measured as the sum of the four products examined, is roughly equivalent. In both cases, the yield of the cytosine products, ClC and HOC, is approximately seven times the sum of the corresponding deamination products ClU and HOU. In contrast, differences in the yield of chlorination versus oxidation products are observed. At the non-CpG sites, the sum of the chlorination products (ClC and ClU) are similar to the sum of the oxidation products (HOC and HOU). However, the sum of the chlorination products at the CpG site is approximately twice that of the oxidation products. The mechanistic basis for this difference is as yet unknown; however, it might be ascribed to sequencespecific structural differences that modify the orientation of the initial attacking species leading to a greater relative amount of the 5-chloro intermediate (2 in Figure 8), as opposed to the 5-hydroxyl intermediate (3 in Figure 8) at the CpG site. In conclusion, the results reported here identify ClC as a significant HOCl-mediated DNA damage product and confirm that the collection of cytosine-derived damage products comprise a diagnostic signature of HOCl-mediated DNA damage. The use of the mass-tagging method allows direct demonstration of the formation of ClC at a CpG dinucleotide. These results strengthen the hypothesis that inflammation-mediated DNA damage can result in mutation as well as perturbations of the epigenetic code and might provide a mechanistic basis for the association between inflammation and cancer long suggested by epidemiologic studies (43, 44). Acknowledgment. This study was supported by funds from the National Cancer Institute.

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