Hypochlorite-Induced Damage to Nucleosides: Formation of

Hypochlorite-Induced Damage to Nucleosides: Formation of Chloramines and Nitrogen-Centered. Radicals. Clare L. Hawkins and Michael J. Davies*. The EPR...
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Chem. Res. Toxicol. 2001, 14, 1071-1081

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Hypochlorite-Induced Damage to Nucleosides: Formation of Chloramines and Nitrogen-Centered Radicals Clare L. Hawkins and Michael J. Davies* The EPR Group, Heart Research Institute, Camperdown, Sydney, New South Wales 2050, Australia Received April 9, 2001

Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. HOCl is a key bactericidal agent, but can also damage host tissue. As there is a strong link between chronic inflammation and some cancers, we have investigated HOCl damage to DNA bases. We show that reaction of HOCl with the exocyclic -NH2 groups of cytidine, adenosine, and guanosine, and the ring NH groups of all bases, yields chloramines (RNHCl/RR′NCl). These are the major initial products. Chloramine decay can be accelerated by UV light and metal ions, and these reactions, together with thermal decomposition, give rise to nucleoside-derived nitrogen-centered radicals. Evidence is presented for the rapid addition of pyrimidine-derived nitrogen-centered radicals to another parent molecule to give dimers. Experiments with nucleoside mixtures show that the propensity for radical formation is cytidine > adenosine ) guanosine > uridine ) thymidine. These data are inconsistent with the selectivity of HOCl attack and the stability of the resulting chloramines, but can be rationalized if chlorine transfer between bases is rapid and yields the most stable chloramine, with such transfer preceding radical formation. Thus, though thymidine is the major initial site of chloramine formation, rapid chlorine atom transfer generates cytidine and adenosine chloramines. These reactions rationalize the preferential formation of chlorinated cytidine and adenosine in DNA. The respiratory burst of activated phagocyte cells both in vivo and in vitro is known to result in the generation of H2O2 and O2•- and the release of the heme enzyme myeloperoxidase (MPO) (1, 2). This enzyme catalyzes the reaction of H2O2 with physiological concentrations of Clions to give the potent oxidant HOCl. The pKa of HOCl is 7.59 (3), thus at physiological pH, a mixture of both HOCl and -OCl is present; HOCl is used throughout to designate this mixture. HOCl1 plays an important role in bacterial cell killing (4), but excessive or misplaced generation of HOCl is known to cause damage to tissues (5). This is believed to be important in a number of diseases, and considerable evidence has accumulated for a link between chronic inflammation and some cancers (6, 7). HOCl is known to react with a number of biological targets including DNA, proteins, lipids, and cholesterol (5, 8-16). Reaction of HOCl with DNA can result in both structural changes, and chemical modification, with the heterocyclic (ring) NH groups of guanosine and thymidine derivatives more reactive than the exocyclic NH2 groups of guanosine, adenosine, and cytidine derivatives (1720). Reaction of HOCl with these groups results in the formation of semi-stable chloramines (RNHCl and RR′NCl species) (21-24), which can lead to the dissociation of double-stranded DNA due to the disruption of hydrogen * To whom correspondence should be addressed. Phone: +61 2 9550 3560. Fax: +61 2 9550 3302. E-mail: [email protected]. 1 Abbreviations: DMPO, 5,5-dimethyl-1-pyrroline N-oxide; HOCl, the physiological mixture of hypochlorous acid and its anion -OCl; MA, modulation amplitude; MNP, 2-methyl-2-nitrosopropane; NBS, Nbromosuccinimide; TNB, 5-thio-2-nitrobenzoic acid.

bonding (18, 25). The heterocyclic chloramines formed with guanosine and thymidine react more rapidly with thiols and other primary amines (via chlorine atom transfer) than the exocyclic NH2-derived chloramines formed on guanosine, adenosine, and cytidine (17-19). A number of stable chlorinated products have been detected from reaction of HOCl with free bases, nucleosides, RNA, and DNA (8, 26, 27). These include 5-chlorocytosine (22, 23, 27), 5-chlorouracil (24, 28) and 8-chloroadenine (29). Early studies suggested that the formation of 5-chlorocytosine occurs via an acid-catalyzed rearrangement reaction of an initial cytosine chloramine (22, 23), though a more recent study has suggested that this material arises via direct chlorination of the ring by Cl2 (27). The mechanism of formation of 8-chloroadenine has not been fully established (29). HOBr has been shown to brominate uracil and derivatives to 5-bromouracil; the detection of this same product with preformed bromamines (RNHBr) suggests that these materials are likely intermediates in the formation of this product (30, 31). Some of these chlorinated and brominated species exert potent biological effects, with 8-chloroadenine reported to induce apoptosis, while 5-bromouracil is mutagenic (31). Previous studies have provided evidence for the involvement of radical species in the HOCl-mediated oxidation of adenosine and related compounds (32-34). EPR spin trapping studies carried out with HOCl-treated adenosine 5′-monophosphate (AMP) and the spin trap DMPO resulted in the detection of a mixture of different nitrogen-centered radical adducts. These adducts were assigned to nitrogen-centered radicals formed at the N1,

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N3, and N6 positions of the adenosine ring, generated via the formation and subsequent decomposition of the corresponding chloramines (32-34). In light of these previous reports on radical formation from HOCl-treated AMP and our recent studies on the generation of nitrogen-centered radicals from amino acid-, peptide-, and protein-chloramines (10, 11, 35), we hypothesized that chloramines formed on other DNA and RNA bases might also yield nitrogen-centered radicals. In this study we have therefore examined the formation and decomposition of nucleoside chloramines and the generation of nitrogen-centered radicals from these materials.

Materials and Methods Materials. The water used was filtered through a four-stage Milli-Q system (Millipore-Waters, Lane Cove, NSW, Australia). pH Control was achieved by the use of 0.1 M, pH 7.4, phosphate buffer pretreated with Chelex resin (BioRad, Hercules, CA) to remove any contaminating trace metal ions. The nucleosides and various derivatives were obtained from either Sigma Chemical Co. (St Louis, MO) or ICN (Seven Hills, NSW, Australia). 5,5-Dimethyl-1-pyrroline N-oxide (DMPO; ICN, Seven Hills, NSW, Australia) was purified before use by treatment with activated charcoal. Stock solutions of 2-methyl-2-nitrosopropane (0.1 M) (MNP; Aldrich, Castle Hill, NSW, Australia) were generated by dissolution of the solid dimer in acetonitrile overnight at -20 °C; these solutions were diluted into the incubations such that the final acetonitrile concentration was e5% v/v. HOCl solutions were prepared immediately before use by dilution of a concentrated stock solution [ca. 0.5 M in 0.1 M NaOH (BDH, Poole, Dorset, U.K.)] into 0.1 M phosphate buffer, pH 7.4. HOCl concentrations were determined from the absorbance of -OCl at 292 nm at pH 12 using a molar extinction coefficient of 350 M-1 cm-1 (3). All other chemicals were of analytical reagent grade. Chloramine Determination. Chloramine concentrations were determined by the reaction with 5-thio-2-nitrobenzoic acid (TNB) as described previously (11, 36). TNB (typically 35-40 µM) was prepared from the disulfide 5,5′-dithio-2-nitrobenzoic acid (DTNB; 1 mM) by exposure to NaOH (50 mM) for 5 min before dilution into 0.1 M, pH 7.4, phosphate buffer. The concentration of TNB consumed after reaction with the various chloramines for 15 min was determined at 412 nm by using a molar extinction coefficient of 13 600 M-1 cm-1 (36). UV Photolysis. Illumination of chloramine solutions with UV light was carried out using an unfiltered 125 W mercury lamp (Osram HQL). Samples were photolyzed at a distance of 30 cm and held in open sample tubes or in a standard EPR suprasil quartz flattened aqueous sample cell. Electron Paramagnetic Resonance (EPR) Spectroscopy. EPR spectra were recorded at room temperature using a Bruker EMX X-band spectrometer with 100 kHz modulation and a cylindrical ER4103TM cavity. Samples were contained in a flattened, aqueous-sample cell (WG-813-SQ; Wilmad, Buena, NJ) and recording of the spectra was initiated within 2 min of the addition of the spin trap to the samples unless stated otherwise. The spin trap was added into the reaction mixture 2-60 min after the HOCl to avoid direct reaction of HOCl with the spin trap. Experiments involving the addition of Fe(II) or Cu(I) ions were carried out in the absence of O2 (by gassing with O2-free nitrogen) to prevent autoxidation of the metal ions and subsequent formation of H2O2. Hyperfine couplings were measured directly from the field scan and confirmed by simulation with the program WINSIM (37). This software is freely available at the NIEHS website (http://EPR.niehs.nih.gov). Correlation coefficients between simulated and experimental spectra were >0.95. Typical EPR spectrometer settings were gain, 1 × 105106; modulation amplitude, 0.01-0.05 mT; time constant, 0.16 s; scan time, 84 s; resolution, 1024 points; center field, 348 mT;

Hawkins and Davies field scan, 6 mT (MNP experiments) or 8 mT (DMPO experiments); power, 25 mW; frequency, 9.76 GHz; with four scans averaged. For further details see figure legends.

Results Formation and Stability of Chloramines. Direct analysis of the formation and decomposition of basederived chloramines by UV spectroscopy proved inaccurate due to the intense UV-absorptions of the parent nucleosides (data not shown). Consequently, the concentration and decay of nucleoside-derived chloramines was examined using the TNB assay, with the initial yield of these materials assessed 5 min after addition of HOCl (0.25 mM) to the substrate (1.25 mM) at 4 °C. Previous kinetic studies have shown that complete consumption of HOCl occurs over such a time interval at these concentrations [cf. rate constants of 3 × 104 M-1 s-1 for TMP and UMP, 100 M-1 s-1 for CMP, 6.4 M-1 s-1 and 2.4 M-1 s-1 for the exocyclic NH2 groups of AMP and GMP respectively, and 2.1 M-1 s-1 for the heterocyclic NH of GMP (17-19)]. The initial yield of chloramines detected with the pyrimidine bases accounted for 95-100% of the initial HOCl, whereas ca. 85 and 60% conversion was observed for adenosine and guanosine, respectively. The stability of the resulting chloramines was investigated by incubation of the HOCl-treated samples for varying periods at 4, 20, and 37 °C before assay using TNB. With cytidine, only a slow loss of chloramines was detected at 37 °C, or lower temperatures, over 24 h (Figure 1a). In contrast, a rapid loss of chloramines was observed with uridine and thymidine at 37 °C (Figure 1a), though this was slower at lower temperatures (data not shown). With adenosine and guanosine, bi-phasic behavior was observed at all temperatures examined, with a rapid initial loss followed by a second slower phase (Figure 1b), consistent with the presence of multiple chloramine species which decay at different rates. With both these substrates significant chloramine yields were still detectable after 24 h at 37 °C. In contrast, chloramines formed on inosine, where the exocyclic NH2 group of adenosine is replaced by a keto function, decayed rapidly and in a mono-phasic manner over time (Figure 1b). Preincubation of each of these chloramines (250 µM) with methionine (50 mM) for 1 min before the addition of TNB resulted in the complete loss of TNB-reactive material, consistent with the rapid scavenging of the chloramines by methionine; this is in accord with previous data (11, 16). The difference in thermal stability of the chloramines formed on cytidine compared to uridine, thymidine and inosine is attributed to the formation of more stable exocyclic NH2-derived species with cytidine, compared to the short-lived, ring-derived species generated with the other bases. Similarly, the initial rapid chloramine loss detected with HOCl-treated adenosine and guanosine is ascribed to the decay of ring-derived chloramines, with the slower, secondary phase due to the decay of the more stable, exocyclic, NH2-derived chloramines. The rate of decay of the chloramines formed with uridine and a fixed concentration of HOCl (0.25 mM) was independent of the uridine concentration over the range 1.25-10 mM, at 37 °C (Figure 2a). In contrast, the rate of decay of the adenosine-derived chloramines was dependent on the initial substrate concentration (Figure 2b). Thus, the rapid initial phase of chloramine loss,

Nucleoside Radicals Generated by HOCl

Figure 1. Time-dependent decay of nucleoside-derived chloramines generated on reaction of the bases (1.25 mM) with HOCl (250 µM) for 5 min at 4 °C prior to incubation at 37 °C. (a) Decay of chloramines formed on thymidine (9), uridine (b), cytidine (2) at 37 °C. Initial chloramine yields were 220 µM (thymidine), 215 µM (uridine), and 245 µM (cytidine). (b) Decay of chloramines formed on adenosine (9), guanosine (b), inosine (2) at 37 °C. Initial chloramine yields were 200 µM (adenosine), 140 µM (guanosine) and 225 µM (inosine). Results are means ( SD for triplicate experiments.

attributed to the ring-derived species, observed with 1.25 mM adenosine was not detected with higher concentrations of adenosine (2.5-10 mM) and identical concentrations of HOCl (0.25 mM). This is consistent with a change in the population of chloramines present as the substrate: HOCl ratio is increased. This interpretation is supported by the observation that addition of adenosine (10 mM) to preformed uridine chloramines (1.25 mM uridine, 250 µM chloramine), resulted in the loss of the unstable uridine-derived species and the formation of a long-lived chloramine (Figure 2c). This effect, together with the observation that only long-lived chloramines were detected with adenosine when high substrate:HOCl ratios (>10:1) were employed, is consistent with rapid chlorine transfer from ring NH positions to exocyclic NH2 positions, resulting in the formation of the more stable exocyclic chloramines. This interpretation is in accord with previous studies that have shown that heterocyclic chloramines can react rapidly with free amine functions by chlorine atom transfer (18, 19). UV-photolysis of nucleoside-derived chloramines (formed as outlined above) at 20 °C resulted in an enhanced loss of chloramines compared to the nonirradiated controls. The rate of UV-induced decomposition of cytidine, thymidine, and uridine chloramines was similar with an 80% loss in chloramine concentration observed on photolysis,

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Figure 2. Time-dependent decay of chloramines formed on treatment of (a) uridine and (b) adenosine with HOCl (250 µM) for 5 min at 4 °C before incubation at 37 °C. Concentration of nucleoside in each case 1.25 mM (b), 2.5 mM (9), 5 mM ([), and 10 mM (2). (c) Decay of preformed uridine chloramines (1.25 mM uridine treated with 250 µM HOCl for 5 min at 4 °C) in the presence (O) and absence (b) of added adenosine (5 mM). Results are means ( SD for triplicate experiments.

compared to a 10% loss in the nonirradiated controls, after 15 min (Figure 3a). A similar enhanced rate of chloramine loss was observed with adenosine and guanosine (Figure 3b). Experiments with added Fe(II) or Cu(I) did not give meaningful results, due to direct reaction of these metal ions with TNB. Formation of Radicals on Reaction of HOCl with Nucleosides and Related Compounds: Pyrimidine Nucleosides. Treatment of cytidine (25 mM) with HOCl (6.25 mM) for 5 min at 20 °C and pH 7.4, before the addition of DMPO (125 mM), resulted in the detection of weak EPR signals. This is consistent with the slow decay of cytidine chloramines at this temperature. In contrast incubations carried out at 37 °C for 15 min in the presence of DMPO, gave an intense EPR signal (Figure 4a). This signal (aN 1.55 mT, aH 2.05 mT, aN 0.27 mT) is assigned to a nitrogen-centered radical adduct on the basis of the second small 1:1:1 nitrogen coupling. Omission of any component of the reaction mixture resulted in the loss of this signal. In the absence of the spin trap no signals were detected, whereas on omission of the

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Figure 3. Stability of nucleoside chloramines produced on treatment of the nucleosides (1.25 mM) with HOCl (250 µM) for 5 min at 4 °C in the presence (open symbols) and absence (closed symbols) of UV light. (a) Pyrimidine bases: cytidine (b, O); uridine (9, 0); thymidine in dark ([, ]); (b) purine bases: adenosine in dark (b, O); guanosine in dark ([, ]). Results are means ( SD for triplicate experiments.

cytidine, strong artifactual signals arising from direct reaction of HOCl with DMPO were detected; the Nchloroimine derivative which gives rise to this signal has been characterized previously (38, 39). Identical, but more intense, signals were observed on addition of Cu(I) ions [generated by the sequential addition of Cu(II) (625 µM) and Ti(III) (500 µM) (40)] or Fe(II) ions (625 µM), to the preformed chloramines in the presence of DMPO (Figure 4b). Similar intense spectra were detected on UV-photolysis (60 s) of the preformed chloramines in the presence of DMPO (data not shown). Identical signals were observed with both cytosine and 2′-deoxycytidine (data not shown). No change in the nature of the cytidine-derived EPR signals was observed on varying either the concentration of HOCl or the time between treating the nucleoside with HOCl and adding the DMPO over the time periods examined (0-60 min). A significant reduction (approximately 80%) in the EPR signal intensity was observed on pretreatment of the HOCl-treated cytidine with methionine (50 mM) prior to the addition of the DMPO (data not shown). HOCl-treated N-3-methylated cytidine gave weak EPR signals in the presence of DMPO, consistent with the presence of both a nitrogen-centered radical adduct (aN 1.58 mT, aH 2.11 mT, aN 0.23 mT) and a more intense carbon-centered radical adduct (aN 1.56 mT, aH 2.25 mT) (Figure 4c). Addition of Cu(I) ions to the reaction mixture resulted in the loss of the nitrogen-centered radical, and an increase in the intensity of the carbon-centered radical. No EPR signals were detected with 2′-deoxycytidine where the N-4 (exocyclic NH2) group was dimethylated (data not shown). These data are consistent with the nitrogen-centered radical being located on the N-4 exocyclic NH2 group.

Hawkins and Davies

Figure 4. (a) EPR spectrum observed on reaction of cytidine (10 mM) with HOCl (2 mM) for 5 min at 20 °C before addition of DMPO (125 mM) and incubation for 15 min at 37 °C run with modulation amplitude (MA) 0.05 mT, gain 1 × 106. (b) As in panel a, except CuSO4 (625 µM) and TiCl3 (500 µM) were added and EPR spectrum acquired immediately after mixing; MA 0.01 mT, gain 1 × 105. (c) 3-Methylcytidine (10 mM) treated with HOCl (2 mM) for 5 min at 20 °C before addition of DMPO (125 mM); MA 0.05 mT, gain 1 × 106. Signals marked (b) are assigned to a nitrogen-centered radical adduct formed on the exocyclic NH2 group. Signals marked (O) are assigned to a carbon-centered radical adduct, and signs marked (×) to DMPOOH (aN ) aH1.49 mT).

No substrate-derived signals were observed with thymidine and uridine when DMPO was employed as the spin trap, with the latter added after 5 min, or greater time periods (up to 60 min). In all cases, only the N-chloroimine species derived from oxidation of DMPO was detected (spectrum not shown). As reaction of thymidine and uridine with HOCl is rapid [k ) ca. 3 × 104 M-1 s-1 (18)], HOCl consumption by the substrate will be complete in 30 min at 20 °C. At longer incubation times (>30 min at 20 °C) before addition of DMPO, a

Nucleoside Radicals Generated by HOCl

Figure 7. (a) EPR spectrum observed on treatment of adenosine (10 mM) with HOCl (2 mM) for 5 min at 20 °C before the addition of DMPO (125 mM) at pH 7.4. (b) As in panel a, except in the presence of CuSO4 (625 µM) and TiCl3 (500 µM). Signals are assigned to adenosine-derived nitrogen-centered radical adducts formed at (a) N1 (radical 1) and (b) the exocyclic NH2 (radical 2).

second nitrogen-centered radical was detected (radical 2: aN 1.55 mT, aH 2.02 mT, aN 0.27 mT; Figure 7b). This radical was also observed, at higher concentrations, when the HOCl-treated adenosine was incubated at 37 °C for short periods of time (e.g., 15 min) or on addition of Cu(I) ions (generated as above). Both signals were only detected in the presence of HOCl, adenosine and DMPO. Similar signals, and behavior, were detected with adenine (data not shown). No substrate-derived signals were detected with inosine, N6-dimethyladenosine, or 6-methyladenosine either in the presence or absence of Cu(I) ions. Experiments with 3-deaza- and 7-deaza-adenosine (i.e., with the N3 or N7 NH groups respectively replaced with a carbon atom) gave identical signals to those observed with adenosine. These data is consistent with the exocyclic N6 NH2 group being the source of one of these radicals, and N1 the other, but it does not allow an unequivocable assignment. However, the time frame over which these species are detected is consistent with radical 1 being the N1 species and radical 2 the exocyclic N6-derived species. Analogous incubations with HOCl-treated guanosine (2 mM and ca. 4 mM, respectively) and DMPO (125 mM) at pH 7.4 and 20 °C yielded complex spectra assigned to three nitrogen-centered radical adducts (radical 3, aN 1.53 mT, aH 2.00 mT, aN 0.30 mT; radical 4, aN 1.51 mT, aH 1.68 mT, aN 0.33 mT; radical 5, aN 1.50 mT, aH 1.50 mT, aN 0.37 mT; Figure 8a). Addition of Cu(I) to the reaction mixture gave identical, but more intense, signals. These signals were only observed in the presence of all of the

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Figure 8. (a) EPR spectrum observed on reaction of guanosine (saturated solution) with HOCl (2 mM) for 5 min at 20 °C before the addition of DMPO (125 mM), CuSO4 (625 µM) and TiCl3 (500 µM) at pH 7.4. (b) Computer simulation of panel a, using the parameters given in Table 1. (c) As in panel a, but with 60 min incubation at 20 °C before the addition of DMPO. (d) Computer simulation of panel c, using the parameters given in Table 1. Signals are assigned to three different guanosine nitrogen-centered radical adducts formed at N3 (9) (radical 3), the exocyclic NH2 (O) (radical 4), and N1 or N7 (1) (radical 5).

components of the reaction mixture. The ratio of these signals was dependent on the incubation time of the guanosine with HOCl before the addition of the spin trap. At short incubation times (80 and >60% of the added HOCl, respectively. The lower initial yield of purine chloramines compared to the pyrimidine species is attributed to the rapid decomposition of the purine ring-derived chloramines. These data are consistent with chloramine formation being the major, if not exclusive, mode of reaction of HOCl with pyrimidine and purine bases. This suggests that the previously detected carbon-chlorinated species [e.g., 5-chlorocytosine (46) and 8-chloroadenine (29)] are either minor initial products, or arise from the decomposition of chloramines. The latter explanation is consistent with previous data (23) on the kinetics of formation of these species. All the pyrimidine and purine chloramines decompose when incubated for extended periods at 20 and 37 °C; these species are significantly more stable at 4 °C. The rate of chloramine decomposition is dependent on the structure of the nucleoside and the chloramine

Nucleoside Radicals Generated by HOCl

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formed. Cytidine-derived chloramines are significantly more stable than the analogous uridine- and thymidinederived species. This has been attributed to the generation of exocyclic-NH2 derived chloramines with cytidine and heterocyclic-NH chloramines with uridine and thymidine. This is consistent with previous observations where the heterocyclic-NH chloramines of TMP, UMP, and GMP were shown to be more reactive than the exocyclic-NH2 chloramines formed with CMP, AMP, and GMP (17-20). Both short-lived and stable chloramines were observed in experiments with adenosine and guanosine. This suggests that a mixture of short-lived heterocyclic-NH chloramines, and more stable exocyclic-NH2 species, are formed with the purine bases. This observation agrees with a previous study where the N6 (exocyclic-NH2) chloramine from AMP was isolated and characterized by NMR (34). Furthermore, this interpretation is supported by the observation that the long-lived chloramines are not observed with inosine, which lacks an exocyclic-NH2 group. The ratio of chloramines formed at ring NH groups compared to the exocyclic NH2 position of purine bases depends on the substrate to HOCl molar ratio. Thus, an increase in the concentration of the more stable chloramines, attributed to the formation of the exocyclic species, is observed on increasing the relative concentration of adenosine compared to HOCl. This effect is believed to be due to chlorine transfer from the heterocyclic NH groups to the exocyclic NH2 group, and not to a difference in the initial site of HOCl attack, as this would not be expected to vary with the substrate concentration, provided the substrate remains in excess. Thus, a change from the kinetically preferred product (ring-derived chloramine) to the thermodynamically preferred product (exocyclic NH2 chloramine) is observed. This effect does not occur with uridine and thymidine, which only have heterocyclic NH groups, but chlorine transfer has been observed from preformed uridine chloramines to adenosine. Treatment of these species with UV-light (up to 15 min) prior to the reaction with TNB resulted in an enhanced rate of chloramine loss. This has been attributed to the UV-induced cleavage of the chloramine N-Cl bond (reaction 1), a process analogous to the formation of nitrogen-centered radicals on UV photolysis of N-haloamides (47, 48). The effects of Fe(II) and Cu(I) on chloramine stability could not be studied due to the direct reaction of the metal ions with the thiol group of TNB, but previous studies have shown that reaction 2 can be rapid (47, 49).

RNHCl + UV light f Cl• + RNH•

(1)

Mn+ + RNHCl f M(n+1)+ + Cl- + RNH•

(2)

These decomposition pathways have been shown to give rise to nucleoside-derived nitrogen-centered radicals, though the nature of the nitrogen-centered radicals depends on both the observation time after initiation of the reaction, as a result of the different populations of chloramines present at different time points, and the substrate:HOCl molar ratio. This confirms and extends previous studies on AMP (34). The nitrogen-centered radicals observed with cytidine have been assigned to the exocyclic-NH2 radical on the basis that a similar signal is observed in experiments

Scheme 1. Proposed Mechanism for the Formation of C5-yl and C6-yl Radicals on Reaction of the Pyrimidine Bases (e.g., uridine) with HOCl

with the heterocyclic NH blocked material (i.e., 3-methylcytidine), and the loss of this species when the exocyclicNH2 group is blocked by methyl groups (i.e., with N,Ndimethyl-2′-deoxycytidine). The marginal difference in parameters of the nitrogen-centered radicals observed with cytidine and 3-methylcytidine is ascribed to alterations in the conformation of the ring, or the electron distribution, as a result of the presence of the methyl group. The lack of observable nitrogen-centered radicals with HOCl-treated uridine or thymidine in the presence of DMPO, and the detection of the N-chloroimine adduct from DMPO, is believed to be due to radical formation from the chloramine being uncompetitive compared to chlorination of DMPO. This difference in behavior between the cytidine chloramines, and those from uridine and thymidine, is consistent with previous kinetic studies where the heterocyclic chloramines of TMP and UMP were shown to chlorinate peptides [e.g., Gly-Gly-Gly, with k ) 5.8 × 103 and 1.4 × 104 M-1 s-1, respectively (1719)], but the exocyclic chloramine formed with CMP did not (17-19). The carbon-centered radicals detected with HOCltreated pyrimidine bases using both DMPO and MNP, are believed to arise as a result of the addition of an initial nitrogen-centered radical to the C5-C6 double bond of another base molecule to form a dimer (Scheme 1). Similar, but not identical, carbon-centered radicals have been observed on reaction of oxygen- and carboncentered radicals to the pyrimidine bases (HO•, t-BuO•, Ph•), and such addition reactions would be expected to be rapid (41-43, 50, 51). Though reaction of HOCl with metal ions, such as Fe(II), has been suggested to generate HO• (52, 53, though see also ref 54), an assignment of the observed species to the HO• adducts is inconsistent with the previously reported parameters for these adducts (41, 50, 51), and the observation of these adducts in the absence of added metal ions. Furthermore, experiments with the nitrogen-centered radical generated by UV-photolysis of NBS, confirms that such nitrogencentered radicals react rapidly with the pyrimidine bases, yielding MNP adducts with very similar parameters to those detected with the pyrimidine chloramines (Scheme 2). The initial radical observed with HOCl-treated adenosine has been assigned to the heterocyclic nitrogencentered radical formed on decomposition of a short-lived

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Scheme 2. Proposed Mechanism for the Formation of C5-yl and C6-yl Base-Derived Radicals on Reaction of the Pyrimidine Bases (e.g., uridine) with Succinimidyl Radicals

chloramine present at N1. The second nitrogen-centered radical detected at longer incubation times, and on addition of metal ions or UV-photolysis, is believed to be centered at N6, and arises from the decomposition of the exocyclic chloramine. This second radical has similar couplings to the AMP-derived N6 radical observed in previous studies, where the exocyclic N6 AMP-derived chloramine was isolated by HPLC and characterized by NMR spectroscopy (33). Similar nitrogen-centered radical adducts have been identified from the chloramines formed on HOCl-treated guanosine. However, two further nitrogen-centered radicals were also detected on decomposition of the N6 AMP chloramine in (unbuffered) solutions at pH ca. 6 in this previous study (32, 33). These species were tentatively assigned to the N1 and N3 nitrogen-centered radicals. The couplings of these N1 and N3 radicals proposed in (32, 33) are different to those assigned to the N1 species observed in the present investigation. The couplings of one of the radicals observed in these previous studies are however identical to the parameters of an artifactual species generated from 4-methyl-4-nitroso-pentanoic acid, which is an oxidation product of DMPO (55, 56). The couplings of the other radical observed previously are similar to this nitrogen-centered artifact species and, hence, may be a related product. These (artifactual) signals are not observed under the conditions employed in this study. HOCl treatment of equimolar mixtures of pairs of different nucleosides, or all four nucleosides together, using either DMPO or MNP as the spin trap, showed that propensity for radical formation is cytidine > adenosine ) guanosine > uridine and thymidine. Thus, the major radical adducts formed are the nitrogen-centered radicals formed from the exocyclic NH2-derived chloramines of cytidine or adenosine, and a ring-derived position on guanosine, when these bases are present. These results are inconsistent with previous studies which have concluded that the major initial site of chloramine formation, with the concentration of HOCl employed (i.e., where each base is individually present at a concentration in excess of the oxidant), is on thymidine [cf. rate constants of 3 × 104 M-1 s-1 for TMP, 1 × 102 M-1 s-1 for CMP 6.4 M-1 s-1 and 2.4 M-1 s-1 for the exocyclic NH2 groups of AMP and GMP respectively, and 2.1 M-1 s-1 for the heterocyclic NH of GMP (17-19)]. Furthermore, this propensity for radical formation is inconsistent with the

Hawkins and Davies

stability of the chloramines, when these are examined in isolation (cf. data above, which show that the chloramines formed on the exocyclic -NH2 groups of cytidine and adenosine which are the major sites of radical formation, as judged by EPR, are the most stable chloramines). These differences can be rationalized if chlorine transfer between the various nucleobases, when multiple species are present, is rapid compared to decomposition to give radicals, and that this process gives rise to the most thermodynamically stable chloramine. Such a process would give rise to the exocyclic chloramine precursors of the observed radicals. Such a suggestion is consistent with a report (18) that the heterocyclic chloramines formed with guanosine and thymidine react more rapidly with thiols and other primary amines (via chlorine transfer) than the exocyclic NH2-derived chloramines formed on guanosine, adenosine, and cytidine. Product studies with DNA suggest that reaction with HOCl results in the selective formation of 8-chloroadenine and 5-chlorocytosine (and 5-chlorouracil which arises from deamination of the altered cytidine) (29, 46). This is unexpected as the major site of initial chloramine formation predicted from reaction kinetics (17-19) would be at thymidine. These data can, however, be rationalized if rapid chlorine atom transfer occurs from initial thymidine-derived chloramines to the exocyclic NH2 groups of cytidine and adenosine, thereby giving rise to radicals and products from these bases.

Acknowledgment. The authors thank the Association for International Cancer Research (99152) and the Australian Research Council (A00001441 and F00001444) for financial support, Prof. R. T. Dean, Dr. R. P. Mason, and Dr. D. Pattison for helpful discussions, and Prof. B. C. Gilbert and Dr. W. Ho for collaborating on some preliminary experiments.

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