Identification of Adenine Adducts Formed in Reaction of Calf Thymus

1180

Chem. Res. Toxicol. 1997, 10, 1180-1185

Identification of Adenine Adducts Formed in Reaction of Calf Thymus DNA with Mutagenic Chlorohydroxyfuranones Found in Drinking Water Frank Le Curieux, Tony Munter, and Leif Kronberg* Department of Organic Chemistry, Åbo Akademi University, Akademigatan 1, FIN-20500 Turku/Åbo, Finland Received June 23, 1997X

Calf thymus DNA was reacted with the extremely potent bacterial mutagen 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and the structurally related compounds 3,4dichloro-5-hydroxy-2(5H)-furanone (MCA) and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF). The chromatograms of the HPLC analyses of the DNA hydrolysates showed peaks that represented adducted base moieties. It was possible to establish the structures of the adducts by comparing UV spectra and chromatographical properties of the DNA adducts with known adenosine and 2′-deoxyadenosine adducts. The DNA adduct produced by MX was identified as 3-(2′-deoxyribofuranosyl-N6-adenosinyl)propenal (M1A-dR). It was calculated that 1 nucleotide/105 nucleotides was converted to M1A-dR. The same adduct was formed also in the reaction of MX with 2′-deoxyadenosine (yield 0.01%). The M1A-dR adduct may play a role in the mutational events induced by MX in Salmonella typhimurium strain TP2428. The adducts produced in the reactions of MCA and MCF with DNA were identified as 3-(2′deoxyribofuranosyl)-7-formylimidazo[2,1-i]purine (cA-dR) and 4-(2′-deoxyribofuranosyl-N6adenosinyl)-3-formyl-3-butenoic acid (fbaA-dR), respectively. The yield of cA-dR was 5 adducts/ 106 nucleotides and of fbaA-dR 4 adducts/105 nucleotides. The biological significance of these adducts is unkown.

Introduction The chlorinated hydroxyfuranone 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), is an extremely potent direct-acting mutagen in the Salmonella typhimurium assay (1-3). The compound is found in chlorine-disinfected drinking waters and is produced in reactions of chlorine with natural organic material present in the water (4-6). Besides MX, several structurally related compounds are formed, such as 3-chloro4-methyl-5-hydroxy-2(5H)-furanone (MCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA) (7-10) (Scheme 1). Also, MCF and MCA are bacterial mutagens, although their mutagenic potency is only about 10-3 that of MX (8, 11). During recent years, MX has been tested in numerous assays and has been shown to cause DNA damage in mammalian cells in vitro and in vivo (3, 12-19). In some of the studies, the genotoxicity of MCA was also determined, and in general, it was found that the compound was active but expressed lower activity than MX (13, 17, 19). In a very recent work of Komulainen et al., MX was administered to rats for about 2 years through drinking water and was found to be a multisite carcinogen in the animals (20). In studies carried out at our laboratory, it has been shown that MCA readily reacts with nucleosides and * Author for correspondence. E-mail: [email protected]. Phone: +358-2-2654186. Fax: +358-2-2654866. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: MCA, 3,4-dichloro-5-hydroxy-2(5H)-furanone; MCF, 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone; MX, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone; DNPH, 2,4-dinitrophenylhydrazine; cA-dR, 3-(2′-deoxyribofuranosyl)-7-formylimidazo[2,l-i]purine; fbaA-dR, 4-(2′-deoxyribofuranosyl-N6-adenosinyl)-3-formyl-3-butenoic acid; M1A-dR, 3-(2′-deoxyribofuranosyl-N6-adenosinyl)propenal; M1GdR, 3-(2′-deoxyribofuranosyl)pyrimido[1,2-a]purin-10(3H)-one; ESI, electrospray ionization.

S0893-228x(97)00106-9 CCC: $14.00

Scheme 1

forms etheno derivatives of adenosine, guanosine, and cytidine (21). MCA also yields adenosine derivatives where, for example, a formyl or an adenosinyl substituent is attached to the etheno bridge (22, 23). Further, it has been found that the compound produces adducts of adenosine and cytidine in which a chloropropenal group is bound at the exocyclic amino group (24). In the reaction of MCF with adenosine, two products have been identified: one with a formylbutenoic acid chain attached to the amino group and the other consisting of a methylfuranone ring where the amino group of adenosine replaces the hydroxyl group of MCF (25). Very recently, LaLonde et al. reported on the cleavage of the plasmid ΦX174 by MCA and MX (26). However, in a study performed by Alhonen-Raatesalmi et al. (27), no adducts were detected in reactions of MX with nucleosides and dinucleotides. In the current work, we report on the first adduct ever identified in reactions of MX with nucleosides, and further we show that the same adduct is formed also in calf thymus DNA following incubation with MX. In addition, we show that MCA and MCF form adducts with the adenine moiety in calf thymus DNA.

Materials and Methods Caution: The chlorohydroxyfuranones used in this study have been reported to be mutagenic in the S. typhimurium assay.

© 1997 American Chemical Society

Chlorohydroxyfuranones Form Adenine Adducts in DNA Therefore, caution should be exercized in handling and disposal of the compounds. Chemicals. The calf thymus DNA, DNase from bovine pancreas, nuclease P1 from Penicillium citrinum, alkaline phosphatase from bovine intestinal mucosa, and acid phosphatase from white potato were obtained from Sigma Chemical Co. (St. Louis, MO). 3,4-Dichloro-5-hydroxy-2(5H)-furanone (MCA) was purchased from Fluka Chemie AG (Buchs, Switzerland). Bromomalonaldehyde was prepared as described by Trofimenko (28). 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone (MX) and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) were synthesized and purified according to the method of Franze´n and Kronberg (29). MCF was crystallized from dichloromethane/n-hexane at -20 °C (mp 54-56 °C). The purity of MX and MCF was at least 98%, as estimated by 1H NMR and GC. Sodium malondialdehyde (NaMDA) was prepared by acid hydrolysis of 1,1,3,3-tetraethoxypropane as described by Stone et al. (30) and crystallized from water diluted with acetone. Chromatographic Methods. HPLC analyses were performed on a Kontron Instruments liquid chromatographic system consisting of a model 322 pump, a 440 diode-array detector (UV), a JASCO FP-920 fluorescence detector, and a KromaSystem 2000 data handling program (Kontron Instruments S.P.A., Milan, Italy). The separations were performed on a C18 analytical column, 5 µm, 4- × 125-mm (Spherisorb ODS2, phase separation; Hewlett-Packard, Espoo/Esbo, Finland). In the spiking experiments two additional columns were used: a C8 column (5 µm, 4- × 125-mm, Lichrospher 100, RP8; Hewlett-Packard) and a 250-mm long C18 column (5 µm, 4× 250-mm, Spherisorb ODS2, phase separation; HewlettPackard). The columns were eluted isocratically for 5 min with 5% acetonitrile in water and then with a gradient from 5% to 30% acetonitrile in 25 min at a flow rate of 1 mL/min. Preparative isolations of the products were carried out by column chromatography on a 2.5- × 10-cm column of preparative C18 bonded silica grade (40 µm, Bondesil; Analytichem International, Harbor City, CA). Further purification of products was performed on an HPLC system, which consisted of two Shimadzu LC-9A pumps, a variable wavelength Shimadzu SPD6A UV spectrophotometric detector (Shimadzu Europe, Germany), and a Rheodyne injector model 7120 equipped with a 2000 µL loop. The injection volume was 1 mL; the column used was the C18 analytical column (4- × 125-mm). Spectroscopic and Spectrometric Methods. The 1H NMR spectra were recorded on a JEOL JNM-A500 Fourier transform NMR spectrometer at 500 MHz. Samples were dissolved in Me2SO-d6, and TMS was used as an internal standard. The determination of the shifts and coupling constants in the ribosyl units was based on a first-order approach. The UV and fluorescence spectra of compounds were recorded as the peaks eluted from the HPLC columns. The mass spectra were recorded on a Fisions ZABSpec-oaTOF instrument (Manchester, U.K.). The ionization mode was either electron impact (EI) or electrospray (ESI). EI ionization was carried out at 70 eV, and the samples were applied through a direct inlet probe. ESI was carried out using nitrogen as both nebulizing and bath gas. A potential of 8.0 kV was applied to the ESI needle. The temperature of the pepperpot counter electrode was 90 °C. The samples were introduced by loop injection at a flow rate of 20 µL/min (H2O/CH3CN/acetic acid: 80/20/1). PFK and PEG 200 were used as standards for exact mass determinations in EI and ESI modes, respectively. The mass spectrometer was working at a resolution of 7000. Reaction of MX with 2′-Deoxyadenosine. Isolation of 3-(2′-Deoxyribofuranosyl-N6-adenosinyl)propenal (M1AdR). In the small scale reactions, 17.2 mg (0.08 mmol) of MX was reacted with 10 mg (0.04 mmol) of 2′-deoxyadenosine in 3 mL of 0.5 M phosphate buffer at pH 7.4, 6.0, and 4.6. The reactions were performed at 37 °C. The reactions were followed by HPLC analyses on the C18 analytical column of aliquots of the reaction mixtures. In the preparative scale reaction, 5 g (23.1 mmol) of MX was added to a solution of 2.91 g (11.6 mmol)

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1181 Table 1. 1H NMR Chemical Shifts (ppm) and Coupling Constants (Hz) of the Protons in M1A-dR protona

δ

multiplicity

H-8 H-2 N-H CHO Ha Hb H-1′ H-2′ H-2′′ H-3′ H-4′ H-5′ H-5′′ OH-3′ OH-5′

8.71 8.56 11.34 9.43 8.74 6.01 6.44 2.34 2.75 4.44 3.90 3.54 3.63 5.38 5.05

s s d d dd dd t m m m m dd dd d t

JH,H

11.7 8.5 13.9, 11.7 13.9, 8.5 6.9

11.9, 4.4 11.7, 4.6 4.1 5.5

a H-1′-H-5′, OH-3′, and OH-5′ ) protons and hydroxyl groups in the 2′-deoxyribosyl unit. The determination of the shifts in the 2′-deoxyribosyl unit was based on a first-order approach.

of 2′-deoxyadenosine in 900 mL of 0.5 M aqueous phosphate buffer (pH 7.4). The solution was stirred at 37 °C for 8 days. The reaction mixture was filtered and passed through the preparative C18 column. The column was eluted with 250 mL of water and then with 100 mL of 5%, 6%, 10%, and 20% solutions of acetonitrile in water. Fractions of 30 mL were collected. The fractions containing M1A-dR (20% acetonitrile washes) were combined, concentrated to about 10 mL, and then further purified by successive HPLC runs on the C18 analytical column. The collected fraction was rotary evaporated to dryness, and the residue was subjected to spectroscopic and spectrometric studies: UVmax (HPLC eluent, 16% acetonitrile in water) 326, 236, 222 nm, UVmin 262, 232, 210 nm; MS [m/z (relative abundance, formation)] 305 (14, M+), 276 (13, M+ CHO), 216 (27, M+ - C4H8O2, loss from the deoxyribose unit), 188 (35, M+ - deoxyribose), 160 (100, M+ - (deoxyribose + CO)), 135 (35, adenine+). Electron impact high-resolution mass spectrometry gave the molecular formula as C13H15N5O4 (M+ 305.1121, calcd 305.112 40). The 1H NMR spectroscopic data are presented in Table 1. Synthesis of M1A-dR from Malonaldehyde. The procedure for the preparation of M1A-dR was adopted from Stone et al. (30). To a solution of 0.3 g of 2′-deoxyadenosine in 100 mL of 0.5 M phosphate buffer at pH 4.0 was added 3.96 g of tetraethoxypropane. The reaction mixture was incubated and stirred at 37 °C for 3 days. The reaction mixture was passed through the preparative C18 column. The column was eluted with 100 mL of water and then with 100 mL of 5%, 10%, 15%, 20%, 25%, and 30% acetonitrile solutions in water. Fractions of 30 mL were collected. The fractions containing M1A-dR (5% and 10% acetonitrile washes) were combined, concentrated to about 30 mL, and then once more passed through the preparative C18 column. The column was eluted with 100 mL of water and then with 100 mL of 2%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% acetonitrile solutions in water. Fractions of 20 mL were collected. The fractions containing M1A-dR (8% and 9% acetonitrile washes) were combined, concentrated to about 30 mL, and then finally purified by successive HPLC runs on the C18 analytical column. The collected fraction was rotary evaporated to dryness, and the residue was subjected to spectroscopic and spectrometric studies. The UV, 1H NMR, and mass spectra of the compound were identical to the spectra of M1A-dR produced in the reaction of MX and 2′-deoxyadenosine. Synthesis of 3-(2′-Deoxyribofuranosyl)-7-formylimidazo[2,1-i]purine (EcA-dR). 2′-Deoxyadenosine (0.8 mmol) and bromomalonaldehyde (3.2 mmol) were dissolved in 2 mL of N,Ndimethylformamide, and the solution was stirred for 4.5 h at room temperature. Water, 40 mL, was added to the reaction mixture, and cA-dR was isolated on the preparative C18 column. The column was eluted batchwise with 100 mL of 5%, 10%, 15%, and 20% solutions of acetonitrile in water. Fractions

1182 Chem. Res. Toxicol., Vol. 10, No. 10, 1997 of 30 mL were collected. The fractions containing the product (10% and 15% washes) were combined and evaporated to dryness. The residue was dissolved in warm ethanol, and cAdR precipitated as a slightly yellow powder: yield 19%; UVmax (HPLC eluent, 14% acetonitrile in water) 224, 324, 336 nm, UVmin 271 nm; 1H NMR δ 10.02 (d, 1 CHO, J ) 0.4 Hz), 9.94 (s, 1 H, H-2), 8.77 (s, 1 H, H-8), 8.60 (d, 1 H, H-a, J ) 0.4 Hz), 6.55 (t, 1 H, H-1′, J ) 6.7 Hz), 5.38 (d, 1 H, OH-3′, J ) 4.1 Hz), 4.96 (t, 1 H, OH-5′, J ) 5.5 Hz), 4.46 (m, 1 H, H-3′), 3.92 (m, 1 H, H-4′), 3.64 (dd, 1 H, H-5′, J ) 11.8, 4.8 Hz), 3.56 (dd, 1 H, H-5′′, J ) 11.8, 4.9 Hz), 2.75 (m, 1 H, H-2′), 2.42 (m, 1 H, H-2′′). Synthesis of 4-(2′-Deoxyribofuranosyl-N6-adenosinyl)3-formyl-3-butenoic Acid (fbaA-dR). MCF (150 mg, 1.0 mmol) and 2′-deoxyadenosine (127 mg, 0.5 mmol) were dissolved in 100 mL of 0.5 M aqueous phosphate buffer (pH 6.0), and the resulting solution was stirred for 6 days at 60 °C. The product was isolated from the reaction mixture on the preparative C18 column. The column was washed with 100 mL of 0%, 5%, 10%, and 20% acetonitrile solutions in 0.01 M KH2PO4 (pH 4.6). Fractions of 30 mL were collected. The fractions containing the pure product (10% and 20% washes) were combined and concentrated to about 30 mL. This solution was then desalted using the same column. The desalted solution was rotary evaporated to dryness, and the residue was subjected to spectrometric studies: UVmax (HPLC eluent, 15% ACN/0.01 M KH2PO4, pH 4.6) 328, 242, 222 nm, UVmin 272, 236, 213 nm; MS ESI [m/z (relative abundance, formation)] 364 (100, MH+), 248 (33, MH+ - deoxyribose). High-resolution mass spectrometry gave the protonated molecular formula as C15H18N5O6 (MH+ 364.1251, calcd 364.1257). 1H NMR: δ 9.25 (s, 1 H, CHO), 8.62 (s, 1 H, H-8), 8.50 (br s, 1 H, Ha), 8.49 (d, 1 H, H-2, J ) 0.6 Hz), 6.44 (t, 1 H, H-1′, J ) 7.3 Hz), 4.45 (m, 1 H, H-3′), 3.90 (dt, 1 H, H-4′, J ) 4.4 Hz), 3.64 (dd, 1 H, H-5′, J ) 11.7, 4.3 Hz), 3.55 (dd, 1 H, H-5′′, J ) 11.9, 4.4 Hz), 3.10 (s, 2 H, Hc), 2.74 (m, 1 H, H-2′), 2.34 (m, 1 H, H-2′′). Reactions with Calf Thymus DNA. MX, MCA, or MCF (18.25 mg) was reacted with double-stranded calf thymus DNA (3.75 mg) in 1.5 mL of 0.1 M phosphate buffer at pH 6.5. The mixtures were stirred and incubated at 37 °C for 2 and 4 days. During the first 12 h of reaction and then twice a day, the pH of the incubation mixture was monitored and readjusted when necessary. The modified DNA was recovered by precipitation with ethanol. To the incubation mixture were added 0.2 mL of 5 M NaCl and 3 mL of cold 96% ethanol. This mixture was centrifuged (10 min, 3000 rpm), and the supernatant containing the unreacted hydroxyfuranones was removed. The precipitated DNA was washed with 1 mL of 70% ethanol and then redissolved in 1.5 mL of water. This precipitation/washing procedure was performed (at least twice) until there was no more unreacted MX, MCA, or MCF left in the supernatant (controlled by HPLC analyses). The enzymatic hydrolysis of the DNA was carried out following essentially the procedure described by Martin et al. (31). Briefly, the modified DNA was dissolved in 3.75 mL of 0.1 M phosphate buffer, pH 7.4, containing 5 mM MgCl2. DNase I (dissolved at 10 mg of DNase/mL in 0.9% NaCl) was added to obtain 0.1 mg of DNase/mL. The mixture was incubated and stirred for 3 h at 37 °C. Nuclease P1 (dissolved at 0.5 mg of nuclease P1/mL in 1 mM ZnCl2) was added to obtain 20 µg of nuclease/mL as the final concentration. Finally, alkaline phosphatase (87 U/mL in water) and acid phosphatase (20 U/mL in water) were added to give final concentrations of 0.5 and 0.3 U/mL, respectively. The mixture was then incubated and stirred at 37 °C for 18 h. The mixture of the hydrolyzed DNA was rotary evaporated to near dryness. The residue was washed four times with 2.5 mL of ethanol/methanol (1/1). The washes were combined, and insoluble particles were removed by centrifugation (20 min, 3000 rpm). Finally, the solution was evaporated to near dryness, 0.1 mL of water was added, and 20 µL of the solution was injected on the HPLC columns. In order to ensure quantitative extraction of adducts by ethanol/ methanol, the remaining insoluble particles were dissolved in water and the solutions were subjected to HPLC analyses.

Le Curieux et al. Chart 1

A blank sample was prepared by allowing calf thymus DNA to stand for 2 days at 37 °C and then performing the precipitation, the hydrolysis, and the HPLC sample preparation exactly as described above. Identification of 3-(2′-Deoxyribofuranosyl)pyrimido[1,2-a]purin-10(3H)-one (M1G-dR) in a Reaction Mixture of Malonaldehyde and 2′-Deoxyguanosine. Malonaldehyde was reacted with 2′-deoxyguanosine according to the procedure of Seto et al. (34). The major product peak in the HPLC separation of the reaction mixture was identified as M1G-dR on the basis of the exact similarities in the characteristics of the UV and fluorescence spectra with those reported by Seto et al. (34). Determination of Malonaldehyde in Aqueous Solutions of MX. MX, 100 mg (0.46 mmol), was dissolved in 25 mL of 0.5 M phosphate buffer at pH 7.4 and 6.0. The solutions were stirred at 37 °C for 48 h and then derivatized with 2,4dinitrophenylhydrazine (DNPH) according to the method by Selim (32). The pH of the solutions was adjusted to 1.4 with 6 N HCL, and 250 µL of DNPH in 6 N HCL and 10 mL of isooctane were added. The two-phase system was stirred for 1 h at room temperature, and then the two phases were allowed to separate. The aqueous phase was extracted with 10 mL of isooctane, and the two isooctane fractions were combined. The isooctane solution was extracted twice with 10 mL of acetonitrile. The acetonitrile extract was evaporated to dryness, and the residue was redissolved in 500 µL of acetonitrile. The concentrate was analyzed by HPLC using the C18 column (4× 125-mm). The column was eluted with a gradient from 10% acetonitrile in water to 70% in 30 min at a flow rate of 1 mL/ min. A standard solution of malonaldehyde was prepared by dissolving the sodium salt in D2O. The malonaldehyde content was determined by quantitative 1H NMR using acetonitrile as an internal standard. Then an appropiate amount of the NMR sample was derivatized with DNPH. In the HPLC chromatogram, the peak due to the DNPH-malonaldehyde derivative appeared at a retention time of 15.5 min and displayed a UV absorption maximum at 306 nm. GC/MS analysis of the DNPHmalonaldehyde derivative gave the following fragment ion peaks m/z [relative abundance)] 234 (62), 217 (18), 204 (40), 177 (19), 158 (100).

Results and Discussion Reactions of MX with 2′-Deoxyadenosine. HPLC analyses of the small scale reactions of MX with 2′deoxyadenosine showed that a small product peak with a longer retention time than that of 2′-deoxyadenosine was formed. The product was obtained at all the studied pH conditions in about equal yields. In order to determine the structure of the product, a large scale reaction of MX with 2′-deoxyadenosine was performed. The product was isolated by preparative C18 column chromatography and further purified by HPLC on the 4- × 125-mm C18 column. On the basis of UV and 1H NMR spectroscopic and mass spectrometric data, the structure of the product was assigned as 3-(2′-deoxy-N6-adenosinyl)propenal (M1A-dR; Chart 1). The UV spectrum of the product showed an intense absorption maximum at 326 nm (Figure 1) and was very

Chlorohydroxyfuranones Form Adenine Adducts in DNA

Figure 1. C18 column HPLC separations of the enzymatically hydrolyzed DNA following incubation of the DNA with (a) MCA, (b) MCF, and (c) MX. The chromatograms of the spiked DNA hydrolysates are superimposed on the original chromatograms. The UV spectra of the 2′-deoxyadenosine adducts are presented on the right side of the figure.

similar to the spectrum of 4-(N6-adenosinyl)-3-formyl-3butenoic acid (fbaA) and identical with the spectrum of 3-(N6-adenosinyl)propenal (M1A), previously identified adenosine reaction products of 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone and malonaldehyde, respectively (25, 30, 33). In the electron impact mass spectrum of the product, the molecular ion was observed at m/z 305. The fragment peaks at m/z 276 and 160 were formed by cleavage of the formyl group from the molecular ion and from the adeninylpropenal moiety, respectively. The ion peaks at m/z 188 and 216 were attributed to the loss of the deoxyribose unit and part of the deoxyribose unit, respectively. The 1H NMR spectrum of the compound displayed six signals, besides the deoxyribose protons (Table 1). The singlet signal at δ ) 8.71 ppm was assigned to the purine H-8 proton due to long-range H-H correlation to the anomeric sugar proton (H-1′). The H-2 proton in the adenine moiety gave a signal at δ ) 8.56 ppm. The aldehyde proton appeared at δ ) 9.43 ppm as a doublet (J ) 8.5 Hz) due to two-bond coupling to H-b at δ ) 6.01 ppm. The signal of H-b was split into an additional doublet because of coupling to the proton H-a at δ ) 8.74 ppm (J ) 13.9 Hz). The NH resonance appeared at δ ) 11.34, and the signal was coupled to the signal of the H-a proton (J ) 11.7 Hz). These 1H NMR data are in agreement with those published previously for M1A by Nair et al. (33) with the exception of the opposite assignment of the protons in the adenine moiety. Further, we noticed that the 2′-deoxyadenosine adduct

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1183

formed in the reaction of MX and the one formed in the reaction of malonaldehyde had identical chromatographic properties. Thus, we conclude that the product formed in the reaction of MX with 2′-deoxyadenosine is M1A-dR. The maximum yield of M1A-dR from MX was about 0.01% (calculated from the original amount of 2′-deoxyadenosine), and this yield was obtained after 8 days of reaction at 37 °C. This is the first adduct ever identified in reactions of MX with nucleosides. A likely explanation for the formation of M1A-dR would be that MX was initially broken down to malonaldehyde and M1A-dR was formed in the reaction of malonaldehyde with the adenine moiety. Since it is known that malonaldehyde reacts with guanosine and forms M1G (and the analogous 2′-deoxy derivative, M1G-dR) (34, 35), we reacted 2′-deoxyguanosine with MX and carried out a search for M1G-dR by HPLC analyses using the fluorescence detector. The result of the analyses was that MX did not form M1G-dR in detectable amounts. Since M1G has been reported to be the major adduct formed in reactions of malonaldehyde with nucleosides (34, 35), our finding strongly indicates that malonaldehyde is not the compound accounting for the formation of M1A-dR from MX. Further support for this statement was obtained from the finding that a DNPH-malonaldehyde derivative could not be detected by HPLC analyses of aqueous solutions of MX held at pH 6.0 and 7.4 at 37 °C for 48 h. We calculated that if 2 × 10-6 mol % of MX would have been transformed to malonaldehyde this would have been observed. Thus we conclude that malonaldehyde was not formed from MX, at least not in quantities high enough to account for the production of M1A-dR. It seems likely that M1A-dR was formed by an attack of MX or a MX degradation product (other than malonaldehyde) on 2′deoxyadenosine, and subsequently the initial adduct underwent degradation to the propenal adduct. Reactions with Calf Thymus DNA. A blank sample of calf thymus DNA was treated in exactly the same way as the DNA samples reacted with the chlorohydroxyfuranones. HPLC analysis of the blank sample showed no peaks that could represent the adducts formed by the chlorohydroxyfuranones. Following the reaction of the chlorohydroxyfuranones with DNA, enzymatic hydrolyses of the DNA, and evaporation of the hydrolysates to near dryness, the adducts in the residues were extracted with ethanol/methanol. No adduct peaks were observed upon HPLC analyses of the redissolved residues, and thus the extraction of the adducts was considered to be quantitative. 1. Reaction of MX with Calf Thymus DNA. Calf thymus DNA was reacted with MX at pH 6.5 for 4 days. Following hydrolysis of the DNA, HPLC analyses showed the presence of an adduct peak at exactly the same retention time as that of M1A-dR. Following spiking the DNA hydrolysate with pure M1A-dR, it was found that the compounds coeluted when chromatographed on a short (125-mm) and a longer (250-mm) C18 column and on a C8 column (Figure 1). Since also the UV spectra of the adduct in the hydrolysate and of M1A-dR were in all essential features identical, we conclude that M1A-dR is formed in the reaction of MX with calf thymus DNA. The extent of adduct formation in DNA was about 1 adduct/ 105 nucleotides. Also a search for M1G-dR was carried out, but the adduct could not be observed. M1A-dR is the first adduct ever shown to be formed in the reaction of MX with nucleosides and DNA. MX is an extremely potent direct-acting mutagen in the S.

1184 Chem. Res. Toxicol., Vol. 10, No. 10, 1997

typhimurium strain TA100. The mutational events in this strain take place mainly at the hisG46 allele, where the target sequence is CCC and GGG (36). It has been shown that MX induces primarily GC f TA transversions in TA100 (37, 38). These transversions cannot be due to the formation of adducts at the adenine moiety in DNA, and therefore, it seems unlikely that M1A-dR would be an important premutagenic lesion in strain TA100. On the other hand, in strain TP2428, Knasmu¨ller et al. (37) found in the MX mutational spectra a moderate increase in intragenic transversions (TAA f AAA), and this alteration could be due to misincorporation opposite an adenosine adduct. Malonaldehyde has been shown to induce base-pair substitutions, among them A f G transitions, when incubated with single-stranded DNA (39). This implies that 2′-deoxyadenosine adducts, for example, M1A-dR, may be important premutagenic lesions (40). 2. Reaction of MCA with Calf Thymus DNA. HPLC analyses of the extracts of the hydrolysate of the calf thymus DNA incubated with MCA showed the occurrence of product peaks at longer retention times than those of the unmodified nucleosides (Figure 1). The UV spectrum of one of these compounds was identical with the spectrum of the previously identified cA-R formed in the reaction of MCA and adenosine (Figure 1). The spectrum was characteristic for the etheno carbaldehyde adduct of adenine; two maxima of about equal intensity were observed at 324 and 336 nm, and a shoulder was found at about 300 nm. The deoxy analogue (cA-dR) was prepared by reacting bromomalonaldehyde with 2′-deoxyadenosine in N,N-dimethylformamide (24). The retention time of the synthesized compound was the same as for the product in the DNA hydrolysate, and following spiking of the hydrolysate, the compounds were found to coelute on the C18 and C8 columns. Collectively, these findings strongly indicate that MCA forms cA-dR in reactions with calf thymus DNA. The yield of the adduct was 5 adducts/106 nucleotides. In the HPLC chromatogram of the DNA hydrolysate, additional peaks were found at retention times between 10 and 15 min (Figure 1). These peaks may represent DNA adducts of MCA, but since the UV spectrum of none of the peaks was identical with previous identified MCA adducts (21-24), the identities of the products remain unknown. In an earlier report from this laboratory, it was shown that MCA formed etheno adducts in reactions with adenosine, guanosine, and cytidine (21). In the current work, we could not observe any etheno adducts in the DNA hydrolysate, even when we used the fluorescence detector. We have proposed that MCA is broken down to mucoxychloric acid and chloroacetaldehyde in water and that the etheno carbaldehyde adduct is produced from the acid and the etheno adducts are produced from the aldehyde (24). The formation of cA-dR, but not the etheno-2′-deoxyadenosine adduct, may be explained by higher reactivity of mucoxychloric acid than of chloroacetaldehyde toward the nitrogens in the adenine moieties in double-stranded DNA. In studies carried out by Kimura et al. (41) and Kayasuga et al. (42), it was reported that chloroacetaldehyde and bromoacetaldehyde are single-strand specific and do not form etheno adducts in reactions with double-stranded DNA. The mutational spectrum of MCA in S. typhimurium (37), strain TA100, showed that the compound caused 2

Knasmu¨ller et al., unpublished results.

Le Curieux et al.

mainly GC f AT transitions in the hisG46 allele (target sequence CCC). The most likely explanation for the transition is the formation of adducts with the guanine and cytosine moieties in the DNA and not by the formation of an adenine adduct. Thus, it is likely that the cA-dR lesion does not account for the mutagenicity of MCA in strain TA100. 3. Reaction of MCF with Calf Thymus DNA. The chromatographic analyses of the hydrolysate of calf thymus DNA reacted with MCF revealed the formation of a major product peak with a retention time of 17.8 min (Figure 1). The UV spectrum of this adduct was very similar to the spectum of M1A-R (and M1A-dR); the major difference was a slight red shift of the absorbance maximum to 328 nm. In a previous work of Munter et al. (25), it was found that MCF produced in reactions with adenosine the derivative 4-(N6-adenosinyl)-3-formyl-3butenoic acid (fbaA-R). The UV spectrum of fbaA-R was identical, in all essential features, to the UV spectrum of the major peak in the DNA hydrolysate (Figure 1). The 2′-deoxyadenosine derivative (fbaA-dR) of fbaA-R was prepared, and its structure was confirmed by mass spectrometry and 1H NMR and UV spectroscopy. Following spiking of the DNA hydrolysate with the synthesized fbaA-dR, the compounds were found to coelute on the C18 and C8 columns. Thus, we conclude that the compound formed when MCF was incubated with calf thymus DNA was fbaA-dR. The yield of the fbaA-dR was about 4 adducts/105 nucleotides. In the work of Munter et al., a second MCF adduct was also identified (25). This adduct could not be detected in the hydrolysate of calf thymus DNA. The mutational spectrum of MCF in the S. typhimurium strain TA100 has been preliminarily examined by Knasmu¨ller et al.2 The spectrum showed that the mutational specificity of MCF hardly can be explained by an adducted adenine moiety. Thus, the biological impact of fbaA-dR remains to be determined.

Conclusions The current work shows that the potent bacterial mutagen MX reacts with 2′-deoxyadenosine and with the adenine moiety in calf thymus DNA. The reaction results in the addition of a propenal unit to the amino group in adenine, i.e., the same adduct as the one produced in reaction of malonaldehyde with the adenine moiety. Further, the work shows that also the hydroxyfuranones, MCA and MCF, attack the adenine base in DNA and each compound produces one previously identified adenosine adduct. However, in the light of the mutational specificity in S. typhimurium strain TA100 of MX, MCA, and MCF, it seems unlikely that the adenine adducts in DNA would be important premutagenic lesions. On the other hand, M1A-dR may provide an explanation for the transversion observed in the MX mutational spectra in S. typhimurium strain TP2428.

Acknowledgment. We thank Dr. Rainer Sjo¨holm for the 1H NMR spectra and Mr. Markku Reunanen for the mass spectra. Financial support for the work was obtained from the European Science Foundation, the Magnus Ehrnrooth Foundation (Dr. Frank Le Curieux), and the Maj and Tor Nessling Foundation (Mr. Tony Munter).

References (1) Tikkanen, L., and Kronberg, L. (1990) Genotoxic effects of various chlorinated butenoic acids identified in chlorinated drinking water. Mutat. Res. 240, 109-116.

Chlorohydroxyfuranones Form Adenine Adducts in DNA (2) LaLonde, R. T., Cook, G. P., Pera¨kyla¨, H., and Dence, C. W. (1991) Effect on mutagenicity of stepwise removal of hydroxyl group and chlorine atoms from 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone: 13C NMR chemical shifts as determinants of mutagenicity. Chem. Res. Toxicol. 45, 35-40. (3) Meier, J. R., Blazak, W. F., and Knohl, R. B. (1987) Mutagenic and clastogenic properties of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone: a potent bacterial mutagen in drinking water. Environ. Mol. Mutagen. 10, 411-424. (4) Hemming, J., Holmbom, B., Reunanen, M., and Kronberg, L. (1986) Determination of the strong mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]furanone in chlorinated drinking water and humic waters. Chemosphere 15, 549-556. (5) Horth, H. (1990) Identification of mutagens in water. J. Fr. Hydr. 21, 135-145. (6) Meier, J. R., Knohl, R. B., Coleman, W. E., Ringhand, H. P., Munch, J. W., Kaylor, W. H., Streicher, R. P., and Kopfler, F. C. (1987) Studies on the potent bacterial mutagen 3-chloro-4(dichloromethyl)- 5-hydroxy-2(5H)-furanone: Aqueous stability, XAD recovery and analytical determination in drinking water and in chlorinated humic acid solutions. Mutat. Res. 189, 363-373. (7) Kronberg, L., Christman, R. F., Singh, R., and Ball, L. (1991) Identification of oxidized and reduced forms of the strong bacterial mutagen (Z)-2-chloro-3-(dichloromethyl)-4-oxobutenoic Acid (MX) in extracts of chlorine-treated water. Environ. Sci. Technol. 25, 99-104. (8) Kronberg, L., and Franze´n, R. (1993) Determination of chlorinated furanones, hydroxyfuranones, and butenedioic acids in chlorinetreated water and in pulp bleaching liquor. Environ. Sci. Technol. 27, 1811-1818. (9) Franze´n, R., and Kronberg, L. (1994) Determination of chlorinated 5-methyl-5-hydroxyfuranones in drinking water, in chlorinated humic water, and in pulp bleaching liquor. Environ. Sci. Technol. 28, 2222-2227. (10) Smeds, A., Vartiainen, T., Ma¨ki-Paakkanen, J., and Kronberg, L. (1997) Concentrations of Ames mutagenic chlorohydroxyfuranones and related compounds in drinking water. Environ. Sci. Technol. 31, 1033-1039. (11) Ishiguro, Y., Santodonato, J., and Neal, M. W. (1988) Mutagenic potency of chlorofuranones and related compounds. Environ. Mol. Mutagen. 11, 225-234. (12) Brunborg, G., Holme, J. A., So¨derlund, E. J., Hongslo, J. K., Vartiainen, T., Lo¨tjo¨nen, S., and Becher, G. (1991) Genotoxic effects of the drinking water mutagen 3-chloro-4-(dichloromethyl)5-hydroxy-2(5H)-furanone (MX) in mammalian cells in vitro and in vivo. Mutat. Res. 260, 55-64. (13) Chang, L. W., Daniel, F. B., and DeAngelo, A. B. (1991) DNA strand breaks induced in cultured human and rodent cells by chlorohydroxyfuranones - mutagens isolated from drinking water. Teratogen. Carcinogen. Mutagen. 11, 103-114. (14) Jansson, K., Ma¨ki-Paakkanen, J., Vaittinen, S. L., Vartiainen, T., Komulainen, H., and Tuomisto, J. (1993) Cytotoxic effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat peripheral lymphocytes in vitro and in vivo. Mutat. Res. 299, 2528. (15) Jansson, K., and Hyttinen, J. M. T. (1994) Induction of gene mutation in mammalian cells by 3-chloro-4-(dichloromethyl)-5hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water. Mutat. Res. 322, 129-132. (16) Furihata, C., Yamashita, M., Kinae, N., and Matsushima, T. (1992) Genotoxicity and cell proliferative activity of 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat grandular stomach. Water Sci. Technol. 25, 341-345. (17) Daniel, F. B., Olson, G. R., and Stober, J. A. (1991) Induction of gastrointestinal tract nuclear anomalies in B6C3F1 mice by 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]furanone and 3,4(dichloro)-5-hydroxy-2[5H]furanone, mutagenic by-products of chlorine disinfection. Environ. Mol. Mutagen. 17, 32-39. (18) Ma¨ki-Paakkanen, J., Jansson, K., and Vartiainen, T. (1994) Induction of mutations, sister-chromatid exchanges and chromosome aberrations by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone in Chinese hamster ovary cells. Mutat. Res. 310, 117123. (19) Fekadu, K., Parzefall, W., Kronberg, L., Franze´n, R., SchulteHermann, R., and Knasmu¨ller, S. (1994) Induction of genotoxic effects by chlorohydroxyfuranones, byproducts of water disinfection, in E. coli K-12 cells recovered from various organs of mice. Environ. Mol. Mutagen. 24, 317-324. (20) Komulainen, H., Kosma, V.-M., Vaittinen, S.-L., Vartiainen, T., Kaliste-Korhonen, E., Lo¨tjo¨nen, S., Tuominen, R. K., and Tuomisto, J. (1997) Carcinogenicity of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in rat. J. Natl. Cancer Inst. 89, in press. (21) Kronberg, L., Sjo¨holm, R., and Karlsson, S. (1992) Formation of 3,N4-ethenocytidine, 1,N6-ethenoadenosine, and 1,N2-ethenogua-

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1185

(22) (23)

(24)

(25)

(26) (27)

(28) (29) (30) (31)

(32)

(33) (34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

nosine in reactions of mucochloric acid with nucleosides. Chem. Res. Toxicol. 5, 852-855. Kronberg, L., Karlsson, S., and Sjo¨holm, R. (1993) Formation of ethenocarbaldehyde derivatives of adenosine and cytidine in reactions with mucochloric acid. Chem. Res. Toxicol. 6, 495-499. Asplund, D., Kronberg, L., Sjo¨holm, R., and Munter, T. (1995) Reaction of mucochloric acid with adenosine: Formation of 8-(N6adenosinyl)ethenoadenosine derivatives. Chem. Res. Toxicol. 8, 841-846. Kronberg, L., Asplund, D., Ma¨ki, J., and Sjo¨holm, R. (1996) Reaction of mucochloric and mucobromic acids with adenosine and cytidine: Formation of chloro- and bromopropenal derivatives. Chem. Res. Toxicol. 9, 1257-1263. Munter, T., Kronberg, L., and Sjo¨holm, R. (1996) Identification of adducts formed in reaction of adenosine with 3-chloro-4-methyl5-hydroxy-2(5H)-furanone, a bacterial mutagen present in chlorinedisinfected drinking water. Chem. Res. Toxicol. 9, 703-708. LaLonde, R., and Ramdayal, F. (1997) Mucochloric acid action on ΦX174 DNA: A comparison to other chlorine-substituted 2(5H)-furanones. Chem. Res. Toxicol. 10, 205-210. Alhonen-Raatesalmi, A., and Hemminki, K. (1991) Assay for nucleoside and nucleotide binding of a potent mutagen, 3-chloro4-(dichloromethyl)-5-hydroxy-2(5H)-furanone. Toxicol. Lett. 56, 167-172. Trofimenko, S. (1963) Dihalomalonaldehydes. J. Org. Chem. 28, 3243-3245. Franze´n, R., and Kronberg, L. (1995) Synthesis of chlorinated 4-methyl-5-hydroxy-2(5H)-furanones and mucochloric acid. Tetrahedron Lett. 36, 3905-3908. Stone, K., Ksebati, M. B., and Marnett, L. J. (1990) Investigation of the adducts formed by reaction of malonaldehyde with adenosine. Chem. Res. Toxicol. 3, 33-38. Martin, C. N., Beland, F. A., Roth, R. W., and Kadlubar, F. F. (1982) Covalent binding of benzidine and N-acetylbenzidine to DNA at the C-8 atom of deoxyguanosine in vivo and in vitro. Cancer Res. 42, 2678-2696. Selim, S. (1977) Separation and quantitative determination of traces of carbonyl compounds as their 2,4-dinitrophenylhydrazones by high-pressure liquid chromatography. J. Chromatogr. 136, 271-277. Nair, V., Turner, G. A., and Offerman, R. J. (1984) Novel adducts from the modification of nucleic acid bases by malondialdehyde. J. Am. Chem. Soc. 106, 3370-3371. Seto, H., Okuda, T., Takesue, T., and Ikemura, T. (1983) Reaction of malonaldehyde with nucleic acid. I. Formation of fluorescent pyrimido[1,2-a]purin-10(3H)-one nucleosides. Bull. Chem. Soc. Jpn. 56, 1799-1802. Basu, A., O’Hara, S. M., Valladier, P., Stone, K., Mols, O., and Marnett, J. M. (1988) Identification of adducts formed by reaction of guanine nucleosides with malondialdehyde and structurally related aldehydes. Chem. Res. Toxicol. 1, 53-59. Cebula, T. A., and Koch, W. H. (1990) Analysis of spontaneous and psoralen-induced Salmonella typhimurium hisG46 revertants by oligodeoxyribonucleotide colony hybridization: use of psoralens to cross-link probes to target sequences. Mutat. Res. 229, 79-87. Knasmu¨ller, S., Zo¨hrer, E., Kronberg, L., Kundi, M., Franze´n, R., and Schulte-Hermann, R. (1996) Mutational spectra of Salmonella typhimurium revertants induced by chlorohydroxyfuranones, byproducts of chlorine disinfection of drinking water. Chem. Res. Toxicol. 9, 374-381. DeMarini, D. M., Abu-Shakra, A., Felton, C. F., Patterson, K. S., and Shelton, M. L. (1995) Mutational spectra in Salmonella of chlorinated, chloraminated, or ozonated drinking water extracts: Comparison to MX. Environ. Mol. Mutagen. 26, 270-285. Benamira, M., Johnson, K., Chaudhary, A., Bruner, K., Tibbetts, C., and Marnett, L. J. (1995) Induction of mutations by replication of malondialdehyde-modified M13 DNA in Escherichia coli: Determination of the extent of DNA modification, genetic requirements for mutagenesis, and types of mutations induced. Carcinogenesis 16, 93-99. Chaudhary, A. K., Reddy, G. R., Blair, I. A., and Marnett, L. J. (1996) Characterization of an N6-oxopropenyl-2′-deoxyadenosine in malondialdehyde-modified DNA using liquid chromatography/ electrospray ionization tandem mass spectrometry. Carcinogenesis 17, 1167-1170. Kimura, K., Nakanishi, M., Yamamoto, T., and Tsuboi, M. (1977) A correlation between the secondary structure of DNA and the reactivity of adenine residues with chloroacetaldehyde. J. Biochem. 81, 1699-1703. Kayasuga-Mikado, K., Hashimoto, T., Negishi, T., Negishi, K., and Hayatsu, H. (1980) Modification of adenine and cytosine derivatives with bromoacetaldehyde. Chem. Pharm. Bull. 28, 932-938.

TX970106G