Photochemical Deamination and Demethylation of 5-Methylcytosine

Cytosine methylation is believed to play a pivotal role in eucaryotic cellular development as well as in viral latency. We have been investigating che...
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Chem. Res. Toxicol. 1996, 9, 745-750

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Photochemical Deamination and Demethylation of 5-Methylcytosine Eric Privat and Lawrence C. Sowers* Division of Pediatrics, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, California 91010 Received November 1, 1995X

Cytosine methylation is believed to play a pivotal role in eucaryotic cellular development as well as in viral latency. We have been investigating chemical mechanisms for the perturbation of methylation patterns, including the effects of ultraviolet radiation. We observed that, upon exposure to UV light, 5-methylcytosine (5mC) was converted to thymine, cytosine, and a series of 5-substituted cytosine derivatives as analyzed by gas chromatography/mass spectrometry. Deamination of 5mC to thymine proceeds via formation of the intermediate photohydrate. Formation of 5-substituted cytosine derivatives results from oxidation of the 5-methyl group with initial formation of 5-(hydroxymethyl)cytosine (hmC). Upon exposure to UV light, hmC is converted to cytosine. The conversion of hmC to cytosine likely results from photohydration and elimination of formaldehyde. It is proposed that endogenous oxidation and hydrolysis could result in demethylation of 5mC residues in DNA. Whereas hydrolytic deamination of 5mC to thymine has been widely discussed, demethylation of 5mC has not as yet been described.

Introduction 5-Methylcytosine (5mC)1 is a modified base found in most higher organisms. Cytosine residues in DNA are methylated enzymatically, usually in the CpG dinucleotide. In humans, approximately one in twenty cytosine residues are methylated (1-3). Several roles have been ascribed to cytosine methylation, including regulation of transcription during cellular differentiation (4, 5) and silencing of invading viral genomes (6, 7). Recently, however, significant attention has been paid to the potential role of hyper- and hypomethylation in the initiation and progression of human tumors (8-10). Despite the potentially pivotal role of cytosine methylation in gene control, significantly less attention has been focused on the chemical reactivity of 5mC than on the predominant DNA bases. This likely is because 5mC is a chemically minor component of DNA. The rationale for the study reported here is based upon our presumption that, although minor in quantity, the specific placement of cytosine methyl groups along the genome is likely to be very important. Perturbation of methylation patterns by chemical reactions of 5mC may therefore be of profound biological significance. Cytosine is known to undergo photohydration when exposed to UV radiation. Cytosine photohydrates can dehydrate re-forming cytosine or deaminate, forming the corresponding uracil photohydrates. Uracil photohyrates can then dehydrate, forming uracil (11, 12). If unrepaired in DNA, this sequence of reactions would generate a transition mutation. In vivo, both the photohydrates (13-15) and uracil (16) are removed from DNA by specific glycosylases. In contrast, thymine and 5mC form photohydrates less efficiently than the unsubstituted pyrimidines (17-22). Recently, two groups have investigated the potential formation of 5mC photohydrates in oligonucleotides using Abstract published in Advance ACS Abstracts, May 1, 1996. Abbreviations: 5mC, 5-methylcytosine; hmC, 5-(hydroxymethyl)cytosine. X 1

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Escherichia coli endonuclease III, which excises ringsaturated pyrimidines; however, these groups have reported conflicting results (23, 24). Although thymine is relatively resistant to photohydration, the thymine methyl group is known to undergo a series of oxidation reactions, generating sequentially the 5-(hydroxymethyl), formyl, and carboxyl derivatives (25). The 5-carboxyuracil derivative may be photochemically decarboxylated to uracil (25). The purpose of this study was to examine the photochemical reactivity of 5mC in aqueous solution, with a focus on deamination of 5mC to thymine and oxidation of the methyl group forming cytosine.

Materials and Methods The free bases 5mC, hmC, 5-carboxycytosine, cytosine, thymine, 5-(hydroxymethyl)uracil, 5-formyluracil, 5-carboxyuracil, 5-hydroxyuracil, and uracil were obtained from Sigma Chemical Co. (St. Louis, MO). 5-Hydroxycytosine was prepared according to Moschel and Behrman (26). We attempted to prepare 5-formylcytosine by cerium oxidation of hmC (27); however, in our hands the expected conversion was not observed. Alternatively, hmC was oxidized with MnO2 in DMSO to the 5-formyl derivative according to a published method (28). Thymine hydrate (cis-6-hydroxy-5,6-dihydrothymine) was prepared by the method of Cadet and Teoule (29) and purified by HPLC (30). Thymine hydrate thus obtained reverted completely to thymine upon heating in acid solution. The identities of all free base standards were verified by UV and NMR spectroscopy and mass spectrometry as described below. All proton NMR spectra were obtained in D2O using a Varian Unity 300 MHz NMR spectrometer. Gas chromatography/mass spectrometry was conducted with a Hewlett Packard Series 5890 gas chromatograph interfaced to a Series 5870 mass spectral detector. To obtain reference mass spectra of potential 5mC photochemical reaction products, aqueous samples of standard compounds were prepared at a concentration of approximately 0.1 mM. A quantity (500 µL) of each standard solution was placed in a 1 mL reactivial (Pierce), and solvent was removed under reduced pressure. The dried samples were reconstituted in 100 µL of acetonitrile and 100 µL of bis(trimethysilyl)trifluoroacetamide which were sealed and heated at 180 °C for 30 min. Each silylated sample (1 µL) was injected onto the gas chro-

© 1996 American Chemical Society

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Privat and Sowers

Table 1. Chromatographic and Mass Spectral Data for 5mC Reaction Products retention M•+ time (min) (m/z)

no.

pyrimidine

1 2 3 4 5 6 7 8 9

5-methylcytosine 5-(hydroxymethyl)cytosine 5-formylcytosine 5-carboxycytosine cytosine 5-hydroxycytosine thymine 5-(hydroxymethyl)uracil 5-formyluracil

10 5-carboxyuracil 11 uracil 12 cis-6-hydroxy-5,6dihydrothymine

11.9 14.3 13.0 15.2 11.6 13.7 9.9 13.4 12.2 14.6 9.1 12.5

characteristic fragments (m/z)

269 357 283 371 255 343 270 358 284

254, 269 357, 342, 254 254, 268, 240, 283 356, 254, 371 254, 240 328, 343, 240, 254 255, 270 358, 343, 255 269, 256, 255, 241, 284 372 255, 357, 372 256 241, 255 360 345, 360, 255, 271

matograph using run parameters described by Dizdaroglu et al. (31). UV irradiation was performed with a Hanovia 450 W photochemical apparatus. The UV source was mounted within a Vycor filter, which blocks irradiation below 220 nm, and housed within a water-cooled quartz jacket. The UV light assembly was immersed within a glass well containing a solution of the free base which was stirred and open to the air. Photochemical reactions were also conducted at low oxygen concentrations generated by purging the reaction solution with nitrogen for 30 min prior to and during irradiation. CAUTION: Do not expose eyes or skin to ultraviolet light. Wear appropriate eye protection. The output energy of the UV lamp was measured with an Oriel Merlin radiometer Model 70100 with a pyroelectric detector head. The detector head was placed 13 cm from the quartz jacket which held the UV lamp inside the Vycor filter. Output energy, measured at three different wavelengths using 5 nm bandpass interference filters at 242, 260, and 282 nm, was 25.7, 45.8, and 17.2 W/cm2, respectively. A solution of each free base studied (0.1 mM) was prepared in distilled, deionized water. The pH was adjusted to neutrality prior to irradiation; however, no buffer was added. Samples (1 mL) were removed at selected time intervals, concentrated, dried, and analyzed by GC/MS.

Results On the basis of previously published studies with both cytosine and thymine, we anticipated that 5mC might undergo both photohydration and deamination as well as oxidation of the methyl group. A series of standard compounds expected from these potential reaction pathways were analyzed by GC/MS. The results are presented in Table 1 in which the retention time and characteristic ion fragments are given for each compound. Essentially all of the cytosine derivatives have a common fragment at m/z 254 due to cleavage of the 5-substituent. Similarly, uracil derivatives have a characteristic fragment at m/z 255. Monitoring the photochemical reaction at m/z 254 and 255 could thus reveal products derived from oxidation of the methyl group (m/z 254) as well as deamination following photohydration (m/z 255). Among this series of compounds, chemical characterization of 5-formylcytosine has not appeared previously in the literature. 5-Formylcytosine was prepared by permanganate oxidation of hmC in DMSO and purified by silica gel chromatography. The NMR spectrum showed two low-field singlet resonances at 9.5 and 8.4 ppm, corresponding to the formyl and H6 proton resonances, respectively. The mass spectrum of silylated 5-formyl-

Figure 1. Mass spectrum of silylated 5-formylcytosine. The molecular ion is m/z 283. Predominant fragments include loss of a methyl group (m/z 268) and loss of the formyl group (m/z 254).

Figure 2. Total ion chromatogram of the products obtained from the UV irradiation of 5mC at 2 h. The mass spectrometer was operated in the selected ion mode at m/z 254 and 255. The identity of several of the peaks is indicated by the number above the peak which corresponds to the derivatives listed in Table 1.

cytosine is shown in Figure 1. The molecular ion (M•+) is observed at m/z 283, and other prominent fragments include m/z 268 (M - CH3)+ and m/z 254 (M - formyl)+. To study the generation of products formed by photochemical reaction of 5mC in aqueous solution, UV irradiation was allowed to proceed for 2 h, and 1 mL of the irradiated solution was concentrated, derivatized, and analyzed by GC/MS operating in the selected ion mode at m/z 254 and 255. A series of peaks were obtained, as shown in Figure 2. In order to unambiguously identify these peaks, 10 mL of the irradiated solution was concentrated and analyzed with the mass spectrometer in the scan mode to obtain the mass spectrum of each of the resulting peaks. Peaks were identified by their characteristic retention times and mass spectra, and the identification of peaks is indicated in Figure 2. The numbers above the peaks correspond to the compounds shown in Table 1. Derivatives identified as photochemical reaction products of 5mC include thymine, cis-6hydroxy-5,6-dihydrothymine, hmC, 5-carboxycytosine, 5-formylcytosine, cytosine, uracil, and 5-hydroxycytosine. The appearance of these products demonstrated that both deamination and oxidation of the methyl group of 5mC can occur. Surprisingly, products derived from methyl group oxidation appeared to be as predominant as the deamination product, thymine, with cytosine being one of the major products. On the basis of the previous study by Alca´ntara and Wang (25) on the photochemical oxidation of thymine, we anticipated that the 5-substituted cytosine derivatives and cytosine might result from

Photochemical Reactions of 5-Methylcytosine

Figure 3. Reaction pathways for the photochemical decomposition of 5mC. The upper pathway is conversion of 5mC to cytosine via sequential oxidation of the methyl group. The lower pathway is the conversion of 5mC to thymine by photohydration, deamination, and dehydration.

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Figure 5. Selected ion chromatogram of the reaction products derived from UV irradiation of hmC. The ion current profile is the sum of the ion chromatograms of 254 and 255 amu. The numbers in the figure near each peak correspond to the derivatives listed in Table 1. The expected location of 5-(hydroxymethyl)uracil, which was barely detectable, is indicated by the arrow beneath the number 8.

from the photolysis of 5mC, may have induced a secondary set of products including 5-hydroxycytosine.

Discussion

Figure 4. Selected ion chromatogram of the reaction products derived from UV irradiation of 5-carboxycytosine. The ion current profile is the sum of the ion chromatograms of 254 and 255 amu. The numbers in the figure near each peak correspond to the derivatives listed in Table 1.

sequential oxidation of 5mC to the 5-(hydroxymethyl), 5-formyl, and 5-carboxy derivatives. Cytosine could then result from the decarboxylation of 5-carboxycytosine (Figure 3). When photolysis was repeated with a deoxygenated solution, products derived from oxidation of the methyl group were not observed. To determine if such a sequential oxidation scheme would convert 5mC to cytosine, 5-carboxycytosine was exposed to UV light and the products were analyzed by GC/MS. After 2 h of irradiation, we observed that only a small portion of the 5-carboxycytosine was converted to cytosine, whereas the predominant reaction product was 5-carboxyuracil (Figure 4). These data suggested that the observed 5mC to cytosine conversion might not occur via the four step sequential oxidation scheme (Figure 3), analogous to that proposed for thymine (25). We therefore investigated an earlier step, photochemical oxidation of hmC, by exposing hmC to UV light and analyzing the resulting products. We observed substantial generation of cytosine, with formation of 5-formylcytosine and 5-(hydroxymethyl)uracil occurring to a lesser extent. The formation of products from the photochemical oxidation of hmC is shown in Figure 5. An additional product of interest was observed to result from photochemical oxidation of 5mC. This product, 5-hydroxycytosine, has been recently shown to be a highly mutagenic DNA damage product derived from cytosine (32, 33). Irradiation of cytosine under similar conditions failed to generate 5-hydroxycytosine. It is likely therefore that reactive oxygen species or organic radicals, derived

The solution photochemistry of 5mC is complex, with a wide array of potential reaction products. It has been demonstrated that, in both aqueous solution and organic solvents, 5mC can undergo UV-induced ring opening and contraction (34, 35). In DNA, 5mC and the predominant pyrimidines, thymine and cytosine, might also undergo pyrimidine dimer formation in the appropriate sequence (21). In addition to these reactions, 5mC may undergo reactions with water and oxygen generating monomeric adducts. This latter group of reactions, predicted based upon the photochemistry of thymine and cytosine, include photohydration followed by hydrolytic deamination and oxidation of the methyl group. The studies reported here were designed primarily to investigate the potential formation of monomeric pyrimidine adducts derived from 5mC in aqueous solution. UV irradiation of 5-methylcytosine in aerated aqueous solution generated a complex group of products as shown in the chromatogram of Figure 2. Peaks were identified by examination of mass spectra and characteristic retention times (Table 1). The most prominent of the products were thymine and, surprisingly, cytosine. From this collection of products, we can surmise the reaction pathways as described below. Thymine would be expected to result sequentially from photohydration of 5mC, deamination, and dehydration (Figure 3, lower). Peak 12 in Figure 2, cis-6-hydroxy5,6-dihydrothymine, was observed, supporting the proposed mechanism for conversion of 5mC to T. None of the peaks in the chromatogram of Figure 2 had a mass spectrum consistent with the photohydrate of 5mC. Once formed, the photohydrate of 5mC must rapidly deaminate, forming thymine hydrate, or dehydrate, re-forming 5mC. Previously, Vairapandi and Duker (23) inferred the presence of 5-methylpyrimidine photohydrates in oligonucleotides based upon susceptibility to enzymatic removal. It is important to note, however, that Teebor and co-workers (24) have recently presented data which indicated that 5mC photohydration may be much less efficient in DNA. The data reported here clearly demonstrate that 5mC deamination to thymine via photohydration in solution is chemically feasible. Determination

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of the extent of such a reaction in DNA will require further study. Whereas pyrimidine methylation reduces the efficiency of photohydration, formation of 5mC does introduce the potentially reactive methyl group. The most surprising result of this study was the abundance of products resulting from oxidation of the 5mC methyl group. As can be seen by inspection of Figure 2, methyl oxidation products (peaks 11, 5, 3, 2, and 4, Figure 2) were as abundant as the deamination product, thymine (peak 7). Generation of the methyl group oxidation products is consistent with a previous study on the oxidation of the thymine methyl group (25). Although it is known that 5mC may be oxidized to hmC by reactive oxygen species (36), the mechanism for the photochemical conversion of 5mC to hmC in oxygenated solution has not yet been described. A mechanism may be postulated, however, based upon the observed oxygen dependence of the reaction and results of previous studies. It has been reported that, in a frozen matrix, UV irradiation of 5mC gives rise to a long-lived excited triplet state which can be observed by electron spin resonance spectroscopy (37). In solution, such electronically excited pyrimidines may transfer an electron to ground state oxygen, generating a radical cation (38, 39) as demonstrated for both thymine and uracil. The radical cation of 5mC may deprotonate, forming a methylene radical as has been demonstrated for the radical cation of 5mC generated by irradiation (40). The methylene radical can then combine with oxygen, forming a hydroperoxide, which would then decompose, forming either hmC or 5-formylcytosine (41, 42). While both hmC and 5-formylcytosine are observed upon UV irradiation of 5mC in aqueous solution, we also observed cytosine at all irradiation times in greater amounts than any of the 5-substituted cytosine derivatives. This result suggested that either all of the subsequent oxidation reactions are relatively fast, or the production of cytosine does not exclusively result from the sequential oxidation scheme. To probe the mechanism for the photochemical conversion of hmC to cytosine, we investigated the sequential oxidation scheme starting with the last step, decarboxylation of 5-carboxycytosine. We observed that photodecarboxylation of 5-carboxycytosine to cytosine does occur; however, the efficiency of the reaction is extremely low (Figure 4). The predominant photochemical reaction of 5-carboxycytosine is deamination to 5-carboxyuracil rather than decarboxylation. In the reaction of 5mC, significant cytosine was observed, with no observable 5-carboxyuracil. If cytosine was generated from 5mC predominantly by formation and decarboxylation of 5-carboxycytosine, we would also expect to have observed 5-carboxyuracil. While our results do demonstrate the plausibility of the sequential oxidation scheme, which invokes decarboxylation of 5-carboxycyctosine to cytosine, the absence of 5-carboxyuracil among the 5mC reaction products argues for the existence of an additional mechanism for the conversion of 5mC to cytosine. The first step in the methyl oxidation scheme is formation of hmC as described above. Oxidation of hmC by the sequential pathway (Figure 3, upper) would yield the 5-formyl derivative, followed by 5-carboxycytosine. When hmC was exposed to UV radiation, however, we observed that cytosine was generated very efficiently. Indeed, cytosine was formed to a significantly greater extent than 5-formylcytosine (Figure 5). These data

Privat and Sowers

Figure 6. Mechanisms for hmC synthesis and hydrolysis of hmC. (A) Synthesis with formaldehyde in basic solution, and (B) deformylation following photohydration.

suggest that hmC undergoes two independent UVinduced reactions, one involving further oxidation of the (hydroxymethyl) group, the other involving direct conversion to cytosine. The mechanism by which cytosine could be generated directly from hmC is not readily apparent based upon previously described photochemical reaction pathways; however, an examination of the mechanism of synthesis of hmC is informative. In basic solution, pyrimidines and their corresponding nucleosides and nucleotides react with formaldehyde to form the corresponding 5-(hydroxymethyl) derivatives (43, 44). The mechanism for this reaction is shown in Figure 6A. Photochemical hydration of hmC followed by elimination of formaldehyde (Figure 6B) is essentially the reverse of the synthetic reaction. Indeed, as noted by Flaks and Cohen (45), and by Alegria (44), 5-(hydroxymethyl)pyrimidine derivatives prepared with isotopically labeled formaldehyde lose label when stored in alkaline solution. Conversion of hmC to cytosine, via the photohydrate, is a plausible mechanism to explain our experimental results. Conversion of 5mC to cytosine would therefore involve initial photochemical oxidation of the methyl group followed by photohydration and elimination of formaldehyde. Photohydration and deamination of cytosine has long been considered a mechanism for UV-induced transition mutations in vivo (11-15). It is proposed that UVinduced demethylation of 5mC as described here could also occur. Within this context, it is reported that latency of several pathogenic human viruses, including herpes simplex, EBV, and HIV (46-51), is maintained by enzymatic cytosine methylation. It is also known that oxidizing conditions and UV light (50) efficiently induce virus reactivation. Although the mechanism for reactivation is as yet unknown, demethylation of 5mC, as reported here, may be a plausible explanation. Demethylation of 5mC to cytosine, as with deamination of 5mC to thymine, is likely to be of profound biological significance. The mechanism described here for the demethylation of 5mC may well occur even in the absence of UV light. Endogenous oxidation of the thymidine methyl group with formation of 5-(hydroxymethyl)-2′deoxyuridine is considered to be an important endogenous DNA damage reaction (52, 53). In free radical competition reactions, the methyl group of 5mC has been shown to be slightly more reactive than that of thymine (54), leading to the prediction that 5mC oxidation may also be an important source of endogenous DNA damage. Using 32P-postlabeling, Steinberg et al. (55) have reported the presence of 5-(hydroxymethyl)-2′-deoxycytidine in calf

Photochemical Reactions of 5-Methylcytosine

thymus DNA, and Teebor and co-workers (56) have identified a mammalian glycosylase activity which removes hmC from DNA. The second of the two steps required for converting 5mC to cytosine is loss of formaldehyde. Photochemical conversion of hmC to cytosine is demonstrated here via formation of a proposed intermediate photohydrate (Figure 6B). As the elimination of formaldehyde is a hydrolysis reaction, it may also occur even in the absence of UV light. We noted previously that deformylation of 5-(hydroxymethyl)pyrimidine derivatives is known to occur in alkaline solution. Such a reaction would also be expected at neutral pH, although at a slower rate. Deamination reactions of cytosine and 5mC are known hydrolytic reactions both in solution and in DNA (57, 58). Although these deamination reactions are chemically slow under physiological conditions, they are considered to be very important sources for the generation of spontaneous mutations in vivo. Deamination of 5mC to thymine is considered to be one of the most important modifications in human tumors (9). Both oxidation of 5mC to hmC and hydrolysis of hmC to cytosine are chemical reactions which could be expected to proceed in vivo and thus represent a new pathway for the perturbation of methylation patterns.

Acknowledgment. This work was supported in part by the National Institutes of Health (GM 50351 and CA 33572).

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