Hypochlorous Acid-Induced Base Modifications in Isolated Calf

Large concentration-dependent increases in pyrimidine oxidation products. [thymine glycol (cis/trans), 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxyhy...
0 downloads 0 Views 187KB Size
Download PDF
1240

Chem. Res. Toxicol. 1997, 10, 1240-1246

Hypochlorous Acid-Induced Base Modifications in Isolated Calf Thymus DNA Matthew Whiteman,* Andrew Jenner, and Barry Halliwell International Antioxidant Research Centre, Pharmacology Group, University of London, Kings College, Manresa Road, London SW3 6LX, England Received May 20, 1997X

Exposure of calf thymus DNA to hypochlorous acid/hypochlorite leads to extensive DNA base modification. Large concentration-dependent increases in pyrimidine oxidation products [thymine glycol (cis/trans), 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxyhydantoin] but not purine oxidation products (8-hydroxyguanine, 2- and 8-hydroxyadenine, FAPy guanine, FAPy adenine) were observed at pH 7.4. In addition, large increases in 5-chlorouracil (probably formed from 5-chlorocytosine during sample preparation), a novel chlorinated base, were observed. Addition of HOCl to DNA already damaged by •OH generated by a mixture of ascorbate, copper(II) chloride, and hydrogen peroxide showed that hypochlorous acid led to a loss of 8-hydroxyguanine, 2- and 8-hydroxyadenine, FAPy guanine, FAPy adenine, and 5-hydroxycytosine in a concentration- and pH-dependent manner. Nevertheless, time course studies suggested that the formation of purine oxidation products in isolated DNA by hypochlorous acid was not a major oxidation pathway. If this pattern of damage, especially the production of 5-chlorocytosine, is unique to hypochlorous acid, it might act as a “fingerprint” of damage to DNA by HOCl.

Introduction The enzyme myeloperoxidase (MPO) is released by phagocytic cells at sites of inflammation and catalyzes the formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride ions (Cl-) (eq 1). It has been claimed that a minimum of 25-40% of the H2O2 generated by activated neutrophils is used to form HOCl (1, 2). MPO

H2O2 + Cl- 98 HOCl + OH-

(1)

The pKa of HOCl is 7.46, and so it is approximately 50% ionized to OCl- at pH 7.4 (3). In this paper we use “hypochlorous acid” to refer to the mixture of HOCl and OCl- species. Thermodynamic calculations have shown HOCl to be both a one- and a two-electron oxidant (4). It is strongly microbicidal, and exposure of many bacteria to HOCl leads to cell death within seconds (5) via mechanisms involving ATP depletion and inhibition of membrane transporters and respiratory enzymes (6). In mammalian systems, HOCl is capable of oxidizing many important biological molecules such as sulfhydryl and thioether moieties (7, 8), plasma membrane ATPases, collagen, ascorbate, proteins including R1-antiproteinase, nucleotides, and DNA repair enzymes (2, 7-14), and it is also capable of chlorinating fatty acid residues and cholesterol in cell membranes (15). HOCl has also been shown to react, apparently independently of metal ions, with superoxide (O2•-) to generate hydroxyl radical (16) by processes analogous to and apparently some orders of magnitude faster than the Haber-Weiss reaction (17) (eq 2). HOCl may also react with copper(I) and iron(II) salts to form •OH (11, 17). Reaction (2) below has been shown to occur in neutrophils (16). * Address correspondence to this author. Tel: 0171-333-4713. Fax: 0171-333-4949. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, November 1, 1997.

S0893-228x(97)00086-6 CCC: $14.00

HOCl + O2•- f O2 + Cl - + •OH

(2)

HOCl + Fe2+ f Cl- + •OH + Fe3+

(3)

While some reports of HOCl-mediated chlorination of individual DNA bases have been published (18-24), the products of reaction of intact DNA with HOCl have yet to be identified. In this study, we show that HOCl causes significant base modification to isolated DNA. We also show that the pattern of damage caused by HOCl is unlike that produced by other reactive species so far investigated.

Materials and Methods Materials. Chemicals were of the highest quality available. Calf thymus DNA (Sigma type I), ascorbic acid, methionine, hydrogen peroxide, copper(I and II) chloride, iron(II and III) chloride, 6-azathymine, 2,3-diaminopurine, 8-bromoadenine, 5-hydroxyuracil (isobarbituric acid), 4,6-diamino-5-formamidopyrimidine (FAPy adenine),1 2,5,6-triamino-4-hydroxypyrimidine, 5-(hydroxymethyl)uracil, 5-chlorocytosine arabinoside, and 5-chlorouracil were purchased from Sigma Chemical Co. (Poole, Dorset, U.K.). 8-Hydroxyguanine was purchased from Aldrich. 8-Hydroxyadenine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) were synthesized (courtesy of Dr. H. Kaur, Kings College, London) by, respectively, treatment of 8-bromoadenine with concentrated formic acid (95%) at 150 °C for 45 min with purification by crystallization with water, as described in ref 25, and treatment of 2,5,6-triamino4-hydroxypyrimidine with concentrated formic acid with purification by crystallization from water, as described in ref 26. Thymine glycol was synthesized by reaction of 5-methyl1 Abbreviations: GC-MS, gas chromatography-mass spectrometry; FAPy adenine, 4,6-diamino-5-formamidopyrimidine; FAPy guanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 8-OH guanine, 8-hydroxyguanine; 2-OH adenine, 2-hydroxyadenine; 8-OH adenine, 8-hydroxyadenine; 5-(OH,Me) uracil, 5-(hydroxymethyl)uracil; 5-(OH,Me)hydantoin, 5-(hydroxymethyl)hydantoin; 5-OH hydantoin, 5-hydroxyhydantoin; 5-Cl uracil, 5-chlorouracil; 5-OH uracil, 5-hydroxyuracil; 5-OH cytosine, 5-hydroxycytosine; TCMS, trimethylchorosilane; TMS, trimethylsilyl.

© 1997 American Chemical Society

Hypochlorous Acid Modifies DNA Bases

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1241

Figure 1. Mass spectrum of hydrolyzed and derivatized 5-Cl cytosine arabinoside. Abundant M+ and (M - 15)+ ions at m/z 290 and 275, respectively, correspond to the bis-Me3Si derivative. Highly characteristic “A+2” isotopic clusters indicate these ions contain a single Cl. Loss of Cl from M+ probably accounts for the ion at m/z 255. No peaks corresponding to 5-Cl cytosine were detected. uracil with OsO4 for 1 h at 60 °C, and excess OsO4 was removed by freeze-drying (27). Purity of standards (>99%) was assessed by mass spectrometry. 2-Hydroxyadenine, 5-hydroxycytosine, 5-hydroxyhydantoin, and 5-(hydroxymethyl)hydantoin were kind gifts from Dr. M. Dizdaroglu (National Institute of Standards and Technology, Gaithersberg, MD). Cellu.Sep dialysis membranes with a relative molecular mass cut off of 3500, silylation grade acetonitrile, and bis(trimethylsilyl)trifluoracetamide (BSTFA) (containing 1% trimethylchlorosilane, TMCS) were obtained from Pierce Chemical Co. (Rockford, IL.). Distilled water passed through a purification system (Elga, High Wycombe, Bucks) was used for all purposes. DNA Damage Induced by Hypochlorous Acid. All solutions were treated with Chelex-100 resin before use. Hypochlorous acid (HOCl) concentration was quantified immediately before use spectrophotometrically at 290 nm (pH 12,  ) 350 M-1 cm-1) (3). HOCl at various concentrations was added to a reaction mixture (final volume 2.0 mL) containing calf thymus DNA (0.5 mg/mL) in 50 mM phosphate buffer at the pH stated with or without CuCl2, CuCl, FeCl3, or FeCl2 (100 µM). All copper and iron salt solutions were prepared in water and added immediately prior to the experiments. Addition of HOCl did not significantly alter pH. After incubation at 37 °C for up to 1 h samples were dialyzed against water for 24 h. HOCl and DNA: Time Course Study. The reaction was quenched at various times with 10 mM ice-cold methionine, and the samples were stored on ice until dialysis against water for 24 h. Oxidation of DNA: Loss of Oxidized DNA on HOCl Addition. Oxidative DNA base damage was induced by incubating calf thymus DNA (2.0 mg/mL) with H2O2 (2.8 mM), CuCl2 (100 µM), and ascorbate (1 mM) in 50 mM phosphate buffer (pH 7.4) (28). After 1 h incubation the DNA was dialyzed against water (24 h). This damaged DNA solution was subsequently incubated with HOCl at various concentrations (0-500 µM) and pH as outlined above. Analysis of DNA Base Modification by Gas Chromatography-Mass Spectrometry. Preparation, hydrolysis, derivatization, and analysis of samples were essentially performed as described previously (28, 29). Briefly: DNA concentration was measured spectrophotometrically (E260 1.0 ) 50 µg/mL) after overnight dialysis. Aliquots of 100 µg of DNA were then freeze-dried under reduced pressure after addition of internal standards (0.5 nmol: 6-azathymine and 2,6-diaminopurine). Samples were subsequently hydrolyzed by addition of 0.5 mL of 60% formic acid and heating at 140 °C for 45 min in a vacuum. Samples were cooled, lyophilized, and finally derivatized under an N2 atmosphere in poly(tetrafluoroethylene)-capped hypovials (Pierce) by adding 100 µL of a BSTFA (+1% TMCS)/acetonitrile

(4:1, v/v) mixture. Samples were derivatized at 90 °C for 45 min and then analyzed by GC-MS (Hewlett-Packard 5890II gas chromatograph interfaced with a Hewlett-Packard 5917A mass selective detector). The injection port and the GC-MS interface were kept at 250 and 290 °C, respectively. Separations were carried out on a fused silica gel capillary column (12-m × 0.2mm i.d.) coated with cross-linked 5% phenylmethylsiloxane (film thickness 0.33 µM) (Hewlett-Packard). Helium was the carrier gas with a flow of 0.93 mL/min. Derivatized samples (1.0 µL) were injected into the GC port using a split ratio of 8:1. Column temperature was increased from 125 to 175 °C at 8 °C/min after 2 min at 125 °C, then increased from 175 to 220 °C at 30 °C/ min, held at 220 °C for 1 min, and finally increased from 220 to 290 °C at 40 °C/min and held at 290 °C for 2 min. Selected-ion monitoring was performed using the electron-ionization mode at 70 eV with the ion source maintained at 189 °C. Quantification of modified bases was achieved by relating the peak area of the compound with the internal standard peak area and applying the following formula:

concentration (nmol/mg of DNA) ) A/AIST × [IST] × (1/K) where K ) relative molar response factor for each damaged base, A ) peak area of product, AIST ) peak area of the internal standard, and [IST] ) concentration of internal standard (5 nmol/mg of DNA). K constants were calculated from the slopes of the calibration curves constructed using known concentrations of internal standards and authentic compounds. Values of K for pyrimidine and purine compounds ranged from 1.35 to 6.12 and from 0.61 to 4.89, respectively. Formation of 5-Chlorouracil during Hydrolysis of 5-Chlorocytosine Arabinoside. Confirmation of 5-Cl uracil formation during the acidic hydrolysis of 5-Cl cytosine nucleosides was achieved by hydrolyzing and derivatizing 1 mg of 5-chlorocytosine arabinoside under the same conditions as outlined above. GC-MS analysis was carried out by the method outlined above, but using the Scan mode.

Results Formation of 5-Chlorouracil from 5-Chlorocytosine. The mass spectrum of the peak at 4.25 min, taken from the total-ion chromatogram of hydrolyzed and derivatized 5-chlorocytosine arabinoside, identified it as 5-chlorouracil (Figure 1). This was confirmed by comparison with authentic material. Abundant M+ and (M - 15)+ at m/z 290 and 275, respectively, were observed

1242 Chem. Res. Toxicol., Vol. 10, No. 11, 1997

Whiteman et al.

Figure 3. Effect of increasing HOCl concentrations on the generation of purine base products. Concentration-dependent increases in hypoxanthine and 8-OH adenine are shown. Damage to guanine was not detected even at high (500 µM) HOCl concentrations. Experiments were conducted as described in the Materials and Methods section. Data points are means of three or more separate experiments plotted ( SD.

Figure 2. Effect of increasing HOCl concentrations on the generation of pyrimidine base products: increases in modified bases derived from (a) cytosine and (b) thymine. Experiments were conducted as described in the Materials and Methods section. Data points are means of three or more separate experiments plotted ( SD.

corresponding to the bis-Me3Si derivative. Highly characteristic “A+2” isotopic distribution patterns for clusters 290 and 275 amu indicate that these ions contain a single chlorine atom. Loss of Cl from M+ probably accounted for the ion at m/z 255. No peaks corresponding to 5-Cl cytosine were detected, suggesting that it converts to 5-chlorouracil during sample preparation. Action of HOCl on DNA. DNA was incubated with HOCl at pH 7.4 and the formation of base-derived products observed using gas chromatography-mass spectrometry. Treatment of DNA with HOCl led to a total of approximately 35 mmol of detectable base oxidation products/ mol of HOCl added and predominantly generated pyrimidine oxidation products in DNA, as illustrated in Figure 2a for cytosine (5-OH cytosine, 5-OH uracil, 5-OH hydantoin) and Figure 2b for thymine [thymine glycol (cis/trans)]. Significant increases were observed for all of the pyrimidine damage products at 100-150 µM HOCl, except 5-(OH,Me) hydantoin and 5-(OH,Me) uracil which were only significantly elevated at 500 µM HOCl (data not shown). There was also a marked production of 5-Cl uracil. Preliminary experiments showed that during hydrolysis and sample preparation any 5-Cl cytosine formed from the action of HOCl on cytosine residues was completely converted to 5-Cl uracil (see above).

By contrast, HOCl appeared to induce little formation of purine damage products in DNA. Small but significant increases in the adenine damage products 8-OH adenine and hypoxanthine were observed with 150 µM HOCl (Figure 3), but no significant changes in 2-OH adenine or FAPy adenine were apparent. Levels of the guanine damage products 8-OH guanine, FAPy guanine, and xanthine in calf thymus DNA were not increased even at concentrations of HOCl up to 500 µM (data not shown). The amount of DNA base damage by HOCl increased rapidly within the first minute of incubation but much more slowly subsequently (Figure 4). Formation of 5-OH cytosine, 5-OH uracil, 5-OH hydantoin, and trans-thymine glycol essentially ceased after 10 min, but 5-Cl uracil and cis-thymine glycol levels continued to rise. pH Dependence. The pH dependence of generation of products from cytosine and thymine is illustrated in Figure 5. Over the pH range studied generation of both thymine glycols was maximal at pH 7.4, as was the generation of 5-OH cytosine, 5-OH uracil, and 5-OH hydantoin. By contrast 5-Cl uracil production was optimal at pH 6.0. Hypoxanthine formation from adenine was small but appeared optimal at pH g 8.5 (Figure 5b). Effect of Metal Ions. It was of interest to investigate the effect of transition-metal ions on HOCl-dependent damage. Table 1 summarizes the damage to DNA in the presence of 500 µM HOCl and 100 µM copper or iron ions. Addition of iron or copper, either oxidized or reduced, resulted in no or only small rises (and often falls) in the yield of damaged pyrimidines measured. Of course, Cu+ rapidly disproportionates to yield Cu2+. In contrast, HOCl-induced purine damage was more influenced by the presence of metal ions. In the presence of copper ions, HOCl-dependent formation of 8-OH adenine, hypoxanthine, and 8-OH guanine was markedly increased. Iron(II) but not iron(III) increased the levels of hypoxanthine only. Effect of HOCl on Preformed DNA Base Oxidation Products. To examine whether HOCl could degrade any DNA base damage products, potentially confounding our results, DNA that had been already oxidatively damaged by an •OH-generating system (Cu2+/H2O2/ ascorbate) was exposed to HOCl for 1 h (after dialysis to

Hypochlorous Acid Modifies DNA Bases

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1243

Figure 5. pH dependence for formation of base products derived from cytosine and thymine: products derived from (a) cytosine and (b) thymine with hypoxanthine for comparison. Data plotted show the results obtained when DNA was exposed to 500 µM HOCl. Experiments were conducted as described in the Materials and Methods section. Data points are means of three or more separate experiments plotted ( SD.

Figure 4. Time course for the formation of DNA base products by HOCl (500 µM): accumulation of (a) products derived from cytosine, (b) products derived from thymine, (c) and products derived from adenine. Experiments were conducted as described in the Materials and Methods section. Data points are means of three or more separate experiments plotted ( SD.

remove the other reagents). In contrast to the reaction of HOCl with metal ions, the •OH-generating system produces both oxidized pyrimidines and purines. Figure 6 shows that there was a concentration-dependent loss of 5-OH cytosine, 8-OH guanine, 8-OH adenine, FAPy guanine, and FAPy adenine on treatment of the •OHexposed DNA with HOCl. Addition of up to 500 µM HOCl did not degrade these compounds below basal levels. Levels of 5-(OH,Me) hydantoin, 5-(OH,Me) uracil, and xanthine remained unchanged, while levels of thym-

ine glycol (cis and trans isomers), 5-OH uracil, 5-Cl uracil, 5-OH hydantoin and hypoxanthine increased in a concentration-dependent manner, as expected. The degradation of purine oxidation products (8-OH guanine, FAPy guanine, 2-OH adenine, 8-OH adenine, and FAPy adenine) and 5-OH cytosine on HOCl addition was rapid (Figure 7). All these products, with the exception of 2-OH adenine, were degraded within the first second of the reaction. 2-OH adenine degradation was maximal after 5 s. Losses of 8-OH guanine and FAPy guanine by 150 µM HOCl were greatest at pH 5, whereas 8-OH adenine and FAPy adenine were lost to the greatest extent at pH 8.5 (Figure 8).

Discussion HOCl has been shown to attack nucleotides and individual DNA bases in vitro (18-24) and to inactivate various DNA repair enzymes (12-14). These effects could lead to mutation and contribute to the increased risk of carcinogenesis that is associated with chronic inflammation. Of course, an important question (to be answered by future experimentation) is whether HOCl reaches the nucleus of cells exposed to it externally. Incorporation of chlorinated bases into bacterial DNA has been shown to inhibit bacterial growth (38), and such a mechanism may account for some of the antibacterial action of HOCl in vivo. HOCl may also be toxic to

1244 Chem. Res. Toxicol., Vol. 10, No. 11, 1997

Whiteman et al.

Table 1. Effect of Adding Iron or Copper Ions (100 µM) to Calf Thymus DNA Simultaneously with HOCl (500 µM)a product

no metal ions

no metal ions + HOCl

Fe3+ + HOCl

Fe2+ + HOCl

Cu2+ + HOCl

Cu+ + HOCl

5-Cl uracil 5-(OH,Me) hydantoin 5-OH hydantoin 5-OH uracil 5-(OH,Me) uracil 5-OH cytosine cis-thymine gylcol trans-thymine glycol 8-OH adenine 2-OH adenine FAPy adenine hypoxanthine 8-OH guanine FAPy guanine xanthine

0.04 ( 0.07 0.04 ( 0.00 0.06 ( 0.00 0.16 ( 0.02 0.04 ( 0.00 0.28 ( 0.03 0.54 ( 0.07 0.01 ( 0.00 0.19 ( 0.02 0.05 ( 0.01 0.06 ( 0.01 0.37 ( 0.02 0.62 ( 0.03 0.06 ( 0.02 0.29 ( 0.03

3.97 ( 0.36 0.07 ( 0.01 0.72 ( 0.07 4.98 ( 0.24 0.04 ( 0.01 6.21 ( 0.65 13.31 ( 1.10 4.05 ( 0.26 0.29 ( 0.03 0.07 ( 0.02 -0.02 ( 0.01b 1.06 ( 0.15 -0.05 ( 0.08b 0.00 ( 0.02 -0.23 ( 0.17b

2.37 ( 0.17 0.26 ( 0.01 0.46 ( 0.02 2.96 ( 0.23 0.03 ( 0.03 3.22 ( 0.65 9.92 ( 0.31 2.56 ( 0.10 0.09 ( 0.04 0.03 ( 0.01 -0.01 ( 0.00b 0.87 ( 0.23 -1.04 ( 0.11b 0.07 ( 0.04 -0.01 ( 0.03b

2.61 ( 0.97 0.06 ( 0.03 0.83 ( 0.12 5.31 ( 0.13 0.70 ( 0.53 5.03 ( 1.70 8.11 ( 1.10 2.31 ( 0.29 0.02 ( 0.04 0.07 ( 0.04 -0.19 ( 0.05b 2.67 ( 0.67 -0.08 ( 0.03b -0.20 ( 0.05b 0.25 ( 0.26b

1.60 ( 0.34 0.23 ( 0.02 1.12 ( 0.16 6.25 ( 0.64 0.15 ( 0.03 2.90 ( 0.37 6.52 ( 0.89 2.19 ( 0.30 3.48 ( 0.08 0.05 ( 0.09 -0.10 ( 0.10b 3.04 ( 0.32 3.01 ( 0.08 -0.43 ( 0.14b -0.59 ( 0.10b

1.62 ( 38 0.26 ( 0.03 0.95 ( 0.14 4.22 ( 0.26 0.08 ( 0.00 2.51 ( 0.31 6.51 ( 0.78 1.88 ( 0.44 4.16 ( 0.77 0.00 ( 0.02 -0.07 ( 0.03b 2.79 ( 0.51 4.50 ( 0.77 1.37 ( 0.48 0.17 ( 0.42

a Data presented are corrected for the effects on DNA of metal ions alone and expressed as nmol/mg of DNA. Data represent mean ( SD of three or more separate experiments conducted as described in the Materials and Methods section. Salt solutions were prepared immediately prior to the experiments by dissolving in water. b Addition of HOCl caused decreases in these base products when compared with metal ions added alone, accounting for a “negative”effect.

mammalian cells. Indeed, chlorinated bases have been shown to be mutagenic in the Ames test (39). Using GCMS, we have examined the pattern of DNA damage produced by HOCl. The overall DNA damage pattern consisted of extensive oxidative damage to the pyrimidines cytosine and thymine and chlorination of cytosine, but only minor oxidative damage to the purines. This pattern of damage is completely different from that produced by other reactive species studied previously by us and others, including •OH, singlet oxygen, peroxyl radicals, and peroxynitrite (ONOO-) (28, 31, 32). In particular no increases in guanine oxidation products were observed at any time during the 1 h reaction. We also observed that HOCl can cause loss of certain base-damaged species present on the DNA molecule, including all the oxidized purines: 8-OH guanine, 8-OH adenine, 2-OH adenine, FAPy guanine, and FAPy adenine. In particular the most common biomarker of in vivo DNA base damage (reviewed in ref 33), 8-OH guanine, was rapidly and extensively lost. We do not think this confounds our conclusions, since we could not detect 8-OH guanine even in the first seconds of exposing DNA to HOCl but could detect it when copper ions were present (Table 1). Consequently, previous reports of 8-OH guanine levels in inflammatory conditions where local HOCl production would be large, such as rheumatoid arthritis (34), could be underestimates of the true extent of oxidative DNA damage (depending on the extent to which HOCl reaches the nucleus). Another reactive species produced at sites of chronic inflammation is peroxynitrite (35), which has also recently been demonstrated to degrade 8-OH guanine (36). Our study reemphasizes the point that 8-OH guanine alone may not be a quantitative marker of oxidative DNA damage to measure in cases of oxidative stress at inflammatory sites (33, 37). 5-OH cytosine was unique among the other major oxidized pyrimidines measured, since it was also rapidly degraded in a concentration-dependent manner by HOCl (Figures 5b and 8). However, optimal formation at pH G 7.4 contrasted with the pH-independent degradation suggesting the actual species responsible for the two processes may be different. In other literature data, purines have been suggested to be more resistant to HOCl attack than pyrimidines (18, 21, 24), although other studies found that both purine and pyrimidine nucleosides and nucleotides were rapidly depleted by HOCl (19, 20).

The pH dependence of the formation and degradation of other DNA base lesions suggests that both HOCl and OCl- are involved in base modification. In particular optimal degradation of guanine-derived products at acidic pH contrasted sharply with degradation of adeninederived products at basic pH, indicating that these products may be preferentially degraded by HOCl and OCl-, respectively. Purine oxidation was considerably enhanced by the addition of copper ions. It has recently been demonstrated that HOCl can react with Cu(I) ions and with Fe2+ to produce •OH or a species closely resembling it (17). This could account for increased levels of purine oxidation products in the presence of copper, but we observed little effect of iron ions. It is possible that HOCl/ OCl- reacts faster with DNA than with iron ions so that HOCl/OCl- is removed before significant OH• formation can occur. For example, HOCl has been shown to react at a slower rate with Fe2+ than it does with glutathione or ascorbate (17). Many different groups have demonstrated the reaction of HOCl with -NH2 groups on DNA bases generating chloramines (19, 21, 23). These secondary oxidants may give rise to the continuing slower formation of particular base lesions, especially 5-Cl uracil, after the initial “burst” on HOCl addition that was observed in this study. Several specific chloramines have been detected and identified (21-24), but they are usually labile intermediates that generate other products or revert to the original base. 5-Cl cytosine has previously been shown to be a major stable end product of cytosine chlorination by HOCl (19, 23). It has been established that several oxidized cytosine bases are quantitatively deaminated to form their uracil analogues during DNA hydrolysis with formic acid (40). We have demonstrated that DNA hydrolysis with formic acid at 150 °C results in complete deamination of 5-Cl cytosine to form 5-Cl uracil, which can be quantitated by GC-MS with good sensitivity (e0.01 nmol/mg of DNA). It is possible that N-chlorinated DNA bases such as 4-N-chlorocytosine may also revert to 5-Cl cytosine (23) during acid hydrolysis of DNA and contribute to the total 5-Cl uracil measured. 5-Cl uracil itself may be generated on the DNA molecule during HOCl attack, although it has been claimed that excess HOCl results in its rapid degradation (19). 5-Cl uracil is thus potentially a good marker of HOCl-induced chlorination of DNA in vitro. The instability of chloram-

Hypochlorous Acid Modifies DNA Bases

Chem. Res. Toxicol., Vol. 10, No. 11, 1997 1245

Figure 7. Time course study for the degradation of purine and cytosine oxidation products by HOCl. DNA damaged with H2O2, ascorbate, and CuCl2 was incubated with 150 µM HOCl for increasing periods of time as described in the Materials and Methods section. Data points are means of three or more separate experiments plotted ( SD.

Figure 8. pH dependence of HOCl-dependent (150 µM) degradation for base products derived from purines and cytosine. Experiments were conducted as described in the Materials and Methods section. Data points are means of three or more separate experiments plotted ( SD.

product. Measurement of this lesion (or of the overall pattern of HOCl-induced lesions) might act as a specific biomarker to examine whether HOCl-induced DNA damage occurs in vivo.

Figure 6. Degradation of purine oxidation products and 5-OH cytosine by HOCl. DNA was damaged with a mixture of H2O2, CuCl2, and ascorbate, dialyzed, and then incubated with increasing concentrations of HOCl for 1 h as described in the Materials and Methods section: degradation of (a) products derived from guanine and (b, c) products derived from adenine and cytosine. Data points are means of three or more separate experiments plotted ( SD.

ines probably explains why attempts to detect other chlorinated base products by GC-MS were unsuccessful. Our laboratory is currently attempting to identify and quantify such chloramines and to examine the extent to which they may contribute to DNA damage. In summary, we have shown that HOCl produces a unique pattern of pyrimidine-derived DNA base damage products in isolated DNA, and 5-chlorocytosine (measured after acid hydrolysis as 5-Cl uracil) is a significant

Acknowledgment. We are grateful to the Arthritis and Rheumatism Council (ARC) and the U.K. Ministry for Agriculture Fisheries and Food for their research support. M.W. particularly thanks the ARC for a research studentship.

References (1) Foote, C. S., Goyne, T. E., and Lehrer. (1983) Assessment of chlorination by human neutrophils. Nature (London) 301, 715716. (2) Weiss, S. J., Klein, P., Slivka, A., and Wei, M. (1992) Chlorination of taurine by human neutrophils. Evidence for hypochlorous acid generation. J. Clin. Invest. 70, 1341-1349. (3) Morris, J. C. (1966) The acid ionization constant of hypochlorous acid from 5 to 35 degrees. J. Phys. Chem. 70, 3798-3805. (4) Koppenol, W. H. (1994) Thermodynamic considerations on the formation of reactive species from hypochlorite, superoxide and nitrogen monoxidesCould nitrosyl chloride be produced by neutrophils and macrophages? FEBS Lett. 347, 5-8. (5) Albrich, J. M., McCarthy, C. A., and Hurst, J. K. (1981) Biological reactivity of hypochlorous acid: Implications for microbicidal mechanisms of leukocytic myeloperoxidase. Proc. Natl. Acad. Sci. U.S.A. 78, 210-214.

1246 Chem. Res. Toxicol., Vol. 10, No. 11, 1997 (6) Barette, W. C., Hannum, D. M., Wheeler, W. D., and Hurst, J. K. (1989) General mechanism for the bacterial toxicity of hypochlorous acid: Abolition of ATP production. Biochemistry 28, 91729178. (7) Schraufstatter, I. U., Browne, K., Harris, A., Hyslop, P. A., Jackson, J. H., Quehenberger, O., and Cochrane, C. G. (1990) Mechanisms of hypochlorite injury of target cells. J. Clin. Invest. 85, 554-562. (8) Aruoma, O. I., Halliwell, B., Hoey, B., M., and Butler, J. (1989) The antioxidant action of N-acetylcysteinesIts reaction with hydrogen peroxide, hydroxyl radical, superoxide and hypochlorous acid. Free. Radical Biol. Med. 6, 593-597. (9) Halliwell, B., Wasil, M., and Grootveld, M. (1987) Biologically significant scavenging of the myeloperoxidase-derived oxidant hypochlorous acid by ascorbic acid: Implications for antioxidant protection in the inflamed rheumatoid joint. FEBS Lett. 213, 1518. (10) Folkes, L. K., Candeias, L. P., and Wardman, P. (1995) Kinetics and mechanisms of hypochlorous acid reactions. Arch. Biochem. Biophys. 323, 120-126. (11) Prutz, W. A. (1996) Hypochlorous acid interactions with thiols, nucleotides, DNA and other biological substrates. Arch. Biochem. Biophys. 332, 110-120. (12) Van Rensberg, C. E. J., Van Staden, A. M., and Anderson, R. (1991) Inactivation of poly(ADP-ribose) polymerase by hypochlorous acid. Free Radical Biol. Med. 1, 285-291. (13) Van Rensberg, C. E. J., Van Staden, A. M., Anderson, R., and Van Rensberg, E. J. (1992) Hypochlorous acid potentiates hydrogen peroxide-mediated DNA-strand breaks in human mononuclear leucocytes. Mutat. Res. 265, 255-261. (14) Pero, R. W., Sheng, Y., Olsson, A., Bryngelsson, C., and LundPero, M. (1996) Hypochlorous acid/N-chloramines are naturally produced DNA repair inhibitors. Free Radical Biol. Med. 17, 1318. (15) Carr, A., VanderBerg, J. J. M., and Winterbourn, C. (1996) Chlorination of cholesterol in cell membranes by hypochlorous acid. Arch. Biochem. Biophys. 332, 63-69. (16) Candeias, L. P., Patel, K. B., Stratford, M. L., and Wardman, P. (1993) Free hydroxyl radicals are formed on reaction between the neurophil-derived species superoxide anion and hypochlorous acid. FEBS Lett. 333, 151-153. (17) Candeias, L. P., Stratford, M. L., and Wardman, P. (1994) Formation of hydroxyl radicals on reaction of hypochlorous acid with ferrocyanide, a model iron (II) complex. Free Radical Res. 20, 241-249. (18) Dennis, W. H. (1979) The reaction of nucleotides with aqueous hypochlorous acid. Water Res. 13, 357-362. (19) Gould, J. P., Richards, J. T., and Miles, M. G. (1984) The kinetics and primary products of uracil chlorination. Water Res. 18, 205212. (20) Gould, J. P., and Hay, T. R. (1982) The nature of the reactions between chlorine and purine and pyrimidine bases: Products and kinetics. Water Sci. Technol. 14, 629-640. (21) Itoh, N., Yoshikazu, I., and Yamada, H. (1987) Haloperoxidasecatalyzed halogenation of nitrogen containing aromatic heterocyclics represented by nucleic bases. Biochemistry 26, 282-289. (22) Gould, J. P., Richards, J. T., and Miles, M. G. (1984) The formation of stable organic chloramines during the aqueous chlorination of cytosine and 5-methylcytosine. Water Res. 18, 991-999. (23) Patton, W., Bacon, V., Duffield, A. M., Halpern, B., Hoyano, Y., Pereira, W., and Lederberg, J. (1972) Chlorination studies I: The reaction of aqueous hypochlorous acid with cytosine. Biochem. Biophys. Res. Commun. 48, 880-884. (24) Hoyano, Y., Bacon, V., Summons, R. E., Pereira, B., Halpern, B., and Duffield, A. M. (1973) Chlorination studies IV: The reaction

Whiteman et al.

(25) (26) (27)

(28)

(29)

(30) (31)

(32)

(33) (34)

(35) (36)

(37)

(38) (39)

(40)

of aqueous hypochlorous acid with pyrimidine and purine bases. Biochem. Biophys. Res. Commun. 53, 1195-1199. Dizdaroglu, M., and Bertgold. (1986) Characterisation of freeradical induced base damage in DNA at biologically relevant levels. Anal. Biochem. 156, 182-188. Cavelieri, L. F., and Bendich. A. (1950) The ultraviolet absorption spectra of pyrimidines and purines. J. Am. Chem. Soc. 72, 25872592. Dizdaroglu, M., Holwitt. E., and Hagen, M. P. (1986) Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide. Biochem. J. 235, 531536. Spencer, J. P. E., Jenner, A., Aruoma, O. I., Evans, P. J., Kaur, H., Dexter, D. T., Jenner, P., Lees, A. J., Marsden, D. C., and Halliwell, B. (1994) Intense oxidative DNA damage promoted by L-DOPA and its metabolites. Implications for neurodegenerative disease. FEBS Lett. 353, 246-250. Aruoma, O. I., Halliwell, B., and Dizdaroglu, M. (1989) Iron iondependent modification of bases in DNA by the superoxide radical generating system hypoxanthine/xanthine oxidase. J. Biol. Chem. 264, 13024-13028. Aruoma, O. I., Halliwell, B., Gajewski, E., and Dizdaroglu, M. (1991) Copper-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem. J. 273, 601-604. Spencer, J. P. E., Wong, J., Jenner, A., Aruoma, O. I., Cross, C. E., and Halliwell, B. (1996) Base modification and strand breakage in isolated calf thymus DNA and in DNA from human skin epidermal keratinocytes exposed to peroxynitrite or 3-morpholinosydnonimine. Chem. Res. Toxicol. 9, 1152-1158. Spencer, J. P. E., Jenner, A., Chimel, K., Aruoma, O. I., Cross, C., Wu, R., and Halliwell, B. (1995) DNA damage in human respiratory tract epithelial cells: Damage by gas phase cigarette smoke apparently involves attack by reactive nitrogen species in addition to oxygen radicals. FEBS Lett. 375, 179-182. Halliwell, B., and Dizdaroglu, M. (1992) Commentary: The measurement of oxidative damage to DNA by HPLC and GC/MS techniques. Free Radical Res. Commun. 16, 75-87. Bashir, S., Harris, G., Denman, M. A., Blake, D. R., and Winyard, P. G. (1993) Oxidative damage and cellular sensitivity to oxidative stress in human autoimmune diseases. Ann. Rheum. Dis. 52, 659-666. Koppenol, W. H., Moreno, J. J., Pryor, W. A., Ishiropolous, H., and Beckman, J. S. (1992) Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol. 5, 834-842. Uppo, R. M., Cueto, R., Squadrito, G. L., Salgo, M. G., and Pryor, W. A. (1996) Reactions of peroxynitrite with 2′-deoxyguanosine, 7,8-dihydro-8-oxo-2′-deoxyguanosine, and calf thymus DNA. Free Radical Biol. Med. 21, 407-41. Alam, Z. I., Jenner, A., Daniel, S. E., Lees, A. J., Cairns, N., Marsden, C. D., Jenner, P., and Halliwell, B. (1997) Oxidative DNA damage in the parkinsonian brain: An apparent increase in 8-hydroxyguanine levels in substantia nigra. J. Neurochem. 69, 1196-1203. Dunn, D. R., and Smith, J. D. (1957) Effects of 5-halogenated uracils on the growth of Escherichia coli and their incorporation into deoxyribonucleic acids. Biochem. J. 67, 494-506. Cumming, R. B. (1975) The potential for increased mutagenic risk to the human population due to the products of water chlorination. Proc. First Conf. Chlor. Environ. Impact. Hlth. Effects 1, 247258. Dizdaroglu, M. (1990) Gas chromatography-mass spectrometry of free radical-induced products of pyrimidines and purines in DNA. Methods Enzymol. 193, 842-857.

TX970086I