Identification of Octenal-Related dA and dC Adducts Formed by

Article pubs.acs.org/crt

Identification of Octenal-Related dA and dC Adducts Formed by Reactions with a Hemin-ω-6-fat Peroxidation Model System Kazuaki Kawai,† Yuya Kawasaki,† Yumiko Kubota,‡ Tomoyuki Kimura,‡ Ryuichi Sawa,‡ Tomonari Matsuda,§ and Hiroshi Kasai*,† †

Department of Environmental Oncology, Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan ‡ Institute of Microbial Chemistry (BIKAKEN), Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan § Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga 520-0811, Japan S Supporting Information *

ABSTRACT: Deoxynucleosides were reacted in a lipid peroxidation model system, emulsified hemin-ethyl linoleate, and the adducts thus produced were analyzed by HPLC. Substantial amounts of stable adducts were detected in the dAand dC-reaction mixtures. The structures of the major dA and dC adducts, other than the known 4-oxo-2-nonenal adducts, were determined to be etheno-type adducts, with a C6 side chain bearing an α-hydroxyl-group. These results suggested that the substance involved in adduct formation is 2,3-epoxyoctanal. This compound showed mutagenicity in Salmonella strains TA 100 and TA 104 without the S-9 mix. In addition, based on the structure of a minor dC adduct, another possibly involved mutagen, 4-oxo-2-octenal, was proposed. These mutagens may be formed during storage and cooking of food, or during digestion, and may be involved in human cancers.

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detected in human gastric mucosa.17 We thought that many unknown mutagens may exist in lipid peroxide mixtures and that they could be trapped as adducts by DNA components because many mutagens react with DNA bases. In fact, the oxidation products from a radiolabeled ω-6 fatty acid were irreversibly associated with cellular DNA.18 In a previous study, we used a hemin-ω-6 (or ω-3) fat lipid peroxidation model system to clarify the combined effects of a high-fat and red meat diet.19 This model system is reasonable because the concomitant presence of both heme iron and ω-6 fatty acids, such as linoleic acid, has been suggested to be important for the formation of the lipid peroxidation product, 4-hydroxynonenal, in food.20 In the previous study, an etheno-type 4-oxo-2hexenal-dG adduct was identified in the reaction mixture of dG and the ω-3 model system.19 However, significant adduct formation was not observed when dG was reacted with the ω-6 model system. In this study, adduct formation between nucleosides and a hemin-ω-6 fat lipid peroxidation model system was reinvestigated with an improved method, using more lipophilic conditions and a longer reaction time, because ω-6 fat is more important in relation to human cancer.

INTRODUCTION Epidemiological studies have revealed that the diet is the major cause of human cancer.1 Therefore, it is important to identify unknown mutagens, especially in foods, to prevent cancer. A high fat diet is a risk factor for various cancers, such as breast and prostate cancer,2,3 and an elevated risk of colon cancer is associated with red meat intake.4 In an animal experiment, simultaneous feeding of a high-fat diet and heme-iron produced a significant increase in the incidence of colon cancer.5 Meat intake is reportedly also involved in esophageal and gastric cancers, as revealed in a case control study.6,7 It was also suggested that high intake of heme iron from meat is a risk factor for esophageal and stomach cancers.8 Various carbonyl compounds have been detected in auto-oxidized lipids,9 and some showed mutagenic activities.10 However, the genotoxicities and adduct formation of only a few compounds, such as malondialdehyde, 4-hydroxy-2-nonenal (4-HNE), 4-oxo-2nonenal (ONE), and various 2-alkenals (acrolein, crotonaldehyde), have been studied in detail.8 In an animal experiment, higher levels of etheno-type dC and dA adducts were detected in the colon epithelium DNA after the administration of linoleic acid.11 Various adducts, such as etheno-type-dC, -dA, and -dG adducts, 1,N2-propano-type dG adducts, and a malonaldehyde DNA adduct, induced by the lipid peroxidation products, have been measured in animal and human tissues.12−15 Chou et al. reported that 4-ONE-dA and -dC are the major types of adducts detected in human autopsy tissues.16 Lipid peroxide-derived DNA adducts were also © 2013 American Chemical Society

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EXPERIMENTAL PROCEDURES

Chemicals. 2′-Deoxyadenosine (dA) and 2′-deoxycytidine (dC) were purchased from Yoshitomi Pharmaceutical Industries, Ltd. (Osaka, Japan). Ethyl linoleate (purity >97.0%) and 2-octenal (purity Received: July 6, 2013 Published: September 14, 2013 1554

dx.doi.org/10.1021/tx400245a | Chem. Res. Toxicol. 2013, 26, 1554−1560

Chemical Research in Toxicology

Article

acetonitrile, at room temperature for 48 h. The major product purified from each reaction mixture, which showed the characteristic ethenodA- or etheno-dC-type UV spectrum, was used as the standard for dAONE and dC-ONE, respectively. Effect of the pH of the Lipid Peroxidation (LPO) Mixture on the Formation of dA*. Condition 1: ethyl linoleate (1 mL) and hemin (2.5 mg) were mixed with 10 mL of 200 mM phosphate buffer (pH 7.4) (A) or 50 mM phosphate buffer (pH 7.4) (B) and were vigorously shaken in an open plastic tube (50 mL) to form a homogeneous emulsion for 3 days. Two tubes of each reaction mixture were prepared for the A- and B-mixtures. Occasionally, deionized water was added to maintain the volume of each reaction mixture. After the tubes were capped, the shaking was continued for 6 days. At this stage, the pH values of the A- and B-mixtures were 6.08 and 4.30, respectively. The pH values of all of the reaction mixtures were then adjusted to 7.2 with 200 mM phosphate buffer (pH 7.3) and 15% NaOH (final volume, 14 mL). After adding 1 mL of acetonitrile and 10 mg of dA or dC to each of the A- and B-mixtures, they were incubated at 37 °C for 5 days in capped tubes, for adduct formation. Condition 2: ethyl linoleate (0.5 mL) and hemin (6.7 mg) were mixed with 5 mL of 200 mM phosphate buffer (pH 8.0) (C) or 50 mM phosphate buffer (pH 3.0) (D) and were vigorously shaken in an open plastic tube (50 mL) to form a homogeneous emulsion. Occasionally, deionized water was added to maintain the volume of each reaction mixture. After 48 h, the pH values of the C- and Dmixtures had changed to 6.06 and 2.64, respectively. The pH values of the reaction mixtures were then adjusted to 6.7 with 200 mM phosphate buffer (pH 7.3) and 15% NaOH (final volume, 10 mL). After adding 1 mL of acetonitrile and 20 mg of dA to each reaction mixture, they were incubated at 37 °C for 24 h in capped tubes for adduct formation. Effects of pH and Temperature on the Adduct Formation by 2,3-Epoxyoctanal. dA (1 mg) or dC (1 mg) was dissolved in either 50 mM phosphate buffer (pH 7.3) (1 mL) or 200 mM acetate buffer (pH 4.5) (1 mL) and was then mixed with a 2,3-epoxyoctanal solution (0.5 mg/200 μL acetonitrile). The mixtures were reacted for 64 h in capped plastic tubes with shaking at 23 °C or without shaking at 37 °C. Spectra Measurements. The mass spectra of the adducts were recorded with a Thermo Fisher Scientific LTQ Orbitrap mass spectrometer (ESI positive ion mode). The NMR spectra were measured with a JEOL JNM-ECA600 spectrometer. Chemical shifts (δ) were adjusted to the DMSO signal (1H 2.49 ppm, 13C 39.5 ppm). X-ray Crystallography of dC*-1. Single-crystal X-ray data were collected on a Rigaku VariMax with RAPID imaging plate area detector, using graphite-monochromated Cu−Kα radiation. Data collection was conducted at 93 K for dC*-1. The structure was solved by Direct Methods in SIR200825 and refined by using fullmatrix least-squares in SHELXL97.26 All non-hydrogen atoms were refined anisotropically. All hydrogen atoms were placed in the standard calculated positions and were refined isotropically. The refined crystallographic parameters are summarized in Table S1 (Supporting Information). The absolute configuration of dC*-1 is supported by the Flack parameter,27 which was 0.2(3). CCDC 940754 contains the supplementary crystallographic data of dC*-1. These data can be obtained free of charge from the Cambridge Crystallographic Data Center at www.ccdc.cam.ac.uk/data_request/cif. Mutagenicity Test. The bacterial mutagenicity test was performed according to the method of Maron and Ames.28 The Salmonella tester strain TA104 contains AT base pairs at the mutation site, in contrast to the TA100 strain, which detects mutagens damaging GC base pairs at this site.29

>95%) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Hemin was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). 2,3-Epoxyoctanal was a gift from Nagaoka Perfumery Co., Ltd. (Osaka, Japan). We are grateful to the company. Reactions of Nucleosides in Model Systems of Lipid Peroxidation. The nucleosides, dA (10 mg) or dC (10 mg), were mixed with hemin (2.5 mg), in 50 mM phosphate buffer (pH 7.3) (10 mL), and were vigorously shaken with 1 mL of ethyl linoleate in an open plastic tube (50 mL) to form a homogeneous emulsion. Occasionally, deionized water was added to maintain the volume of each reaction mixture. The reaction was continued for 3 days at room temperature. After adding 1 mL of acetonitrile, the tubes were capped and vigorously shaken for another 19 days. At various time points, a 50 μL aliquot of the aqueous layer was injected into an HPLC column (CAPCELL PAK C18 MG, 5 μm, 4.6 × 250 mm, Shiseido Fine Chemicals, Japan; elution speed, 1 mL/min) connected with a photodiode array UV detector (Hewlett-Packard 1100 HPLC Detection System). The following linear gradients of acetonitrile concentrations in water were used: 0 to 30 min, linear gradient of acetonitrile (10% to 37%); 30 to 40 min, 37% acetonitrile. In addition, the pH of the reaction mixture was measured at each point. The pure adducts for mass spectra measurements were obtained by the following HPLC conditions [column, CAPCELL PAK C18 MG, 5 μm, 10 × 250 mm; elution speed, 3.5 mL/min elution, 0 to 60 min, linear gradient of acetonitrile (10% to 37%). The retention times of dA*, dC*-1, dC*-2, and dC*-3 were 46.5−47.1 min, 44.4−45.0 min, 50.5−51.1 min, and 51.5−52 min, respectively. Preparation of dA* [11-(1-Hydroxyhexyl)-1,N6-etheno-dA] .21 dA (30 mg) was mixed with 0.34 mL of 30% hydrogen peroxide in 100 mM phosphate buffer (pH 7.3) (30 mL) and 2-octenal (444 μL in 15 mL of methanol) in a plastic tube (50 mL), and was reacted at 50 °C for 16 h. The product (retention time, 29.9−31.2 min) was separated by repeated rounds of HPLC (column, CAPCELL PAK C18 MG, 5 μm, 10 × 250 mm; elution speed, 3.5 mL/min). The following linear gradients of acetonitrile concentrations in water were used: 0 to 30 min, linear gradient of acetonitrile (10% to 37%); 30 to 50 min, 37% acetonitrile. The yield was 7.9 mg. MS data for synthetic dA*: positive ion mode, m/z 376.1979, (M + H)+ 376.1979 calcd. for C18H26N5O4. Preparation of dC*-1 [8-(2-Oxohexyl)-3,N4-etheno-dC]. One milliliter of 2-octenal was dissolved in 6 mL of methanol in a plastic tube (50 mL). Two milliliters of tert-butyl hydroperoxide and 0.96 mL of 0.1 M NaOH were then added, and the mixture was shaken for 90 min at room temperature.22 The reaction mixture (6 mL) was then mixed with 10 mg of dC dissolved in 10 mL of 200 mM acetate buffer (pH 4.5). The reaction was continued for 5 days at 50 °C in a capped tube. The same reaction was performed five times. The product (retention time, 28.4−28.9 min) was separated by repeated rounds of HPLC (same conditions as for dA*). The total yield was 8.1 mg. The product, dC*-1, crystallized in 15% ethanol. MS data for synthetic dC*-1: positive ion mode, m/z 372.1532, (M + H)+ 372.1530 calcd. for C17H23N3O5 Na. Preparation of dC*-2 and dC*-3 [8-(1-Hydroxyhexyl)-3,N4etheno-dC]. dC (50 mg) was dissolved in 10 mL of 50 mM phosphate buffer (pH 6.0). After nitrogen substitution, 2,3epoxyoctanal (8.8 mg in 2 mL of methanol) was added, and the reaction was incubated at 50 °C for 72 h in a capped tube. The same reaction was performed three times. The products (retention time of dC*-2, 32.2−32.7 min; retention time of dC*-3, 32.7−33.1 min) were separated by repeated rounds of HPLC (same conditions as for dA*). The total yields of dC*-2 and dC*-3 were 9.7 and 9.6 mg, respectively. MS data for synthetic dC*-2: (positive ion mode, m/z 374.1676, (M + Na)+ 374.1686 calcd. for C17H25N3O5Na). MS data for synthetic dC*3 (positive ion mode, m/z 374.1677, (M + Na)+ 374.1686 calcd. for C17H25N3O5Na). Preparation of 11-(2-Oxoheptyl)-1,N6-etheno-dA (dA-ONE) and 8-(2-Oxoheptyl)-3,N4-etheno-dC (dC-ONE). dA-ONE and dC-ONE were prepared according to the previously published methods.23,24 Five milligrams of dA or dC was reacted with 20 μL of 4-ONE, in 1 mL of 50 mM phosphate buffer (pH 7.4) with 10%

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RESULTS Detection of Nucleoside Adducts Formed in Lipid Peroxidation Model Reactions. When 4 nucleosides, dC, dT, dA, and dG, were reacted with the hemin-ethyl linoleate lipid peroxidation model system, substantial amounts of stable adducts were detected in the dA and dC-reaction mixtures. 1555



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to 3.9. In the meantime, the amounts of dA*, dC*-2, and dC*3 continually increased until the 22nd day, while those of dCONE, dA-ONE, and dC*-1 initially increased but decreased at the final stage, with a concomitant shift to the lower pH. These results suggested that both the adduct formation rates and stabilities influence the final production levels. As discussed below, although adduct formation is enhanced under acidic pH conditions, the exact mechanisms of dC-ONE, dA-ONE, and dC*-1 degradation are not known. In the dG reaction mixture, many adducts were detected; however, most of them gradually decomposed to dG upon an incubation under physiological conditions (pH 7.4, 37 °C), except for a small amount of an etheno-type adduct. Therefore, we did not study the dG adducts further. No adduct formation was detected with dT. Structure Determination of the dA and dC Adducts. The structures of dA*, dC*-1, dC*-2, and dC*-3 were determined, as shown in Figure 4, mainly by the MS, NMR,

Typical HPLC profiles of the dA and dC adduct analyses are shown in Figures 1 and 2, respectively. New adducts, dA* in

Figure 1. HPLC profile of the dA-reaction mixture at 15 days and online measurements of UV spectra of prominent peaks.

Figure 2. HPLC profile of the dC-reaction mixture at 15 days and online measurements of UV spectra of prominent peaks. Figure 4. Structures of the dA and dC adducts.

the dA reaction and dC*-1, dC*-2, and dC*-3 in the dC reaction, were detected in addition to the known adducts, dAONE and dC-ONE. The UV spectra of these dA and dC adducts were very similar to those of dA-ONE and dC-ONE. The time course of adduct formation and the pH changes of the reaction mixtures during the 22 days are shown in Figure 3. We found that the pH of the reaction mixture changed from 7.4

and X-ray crystallographic data of the isolated or synthetic materials. The molecular formula of dA*, isolated from the reaction mixture of dA and hemin-ethyl linoleate model system, is consistent with C18H25N5O4, as determined by highresolution electrospray ionization mass spectrometry (HRESIMS) (positive ion mode, m/z 376.1979, (M + H)+ 376.1979 calcd. for C18H26N5O4). The UV spectrum of dA* was similar to that of the dA-ONE adduct. The UV λmax values in 10% acetonitrile were 232, 269, and 278 nm. These data suggested that dA* is an etheno-dA type adduct, which has an imidazopyrimidine skeleton and a hydroxyl-hexyl side chain. It was highly possible that dA* is formed by the reaction of dA with 2,3-epoxyoctanal,21 which may be generated by the oxidation of 2-octenal, a major linoleate peroxidation product.30 A comparison of the elution positions in the HPLC, the UV spectra, and the high-resolution mass spectra, between dA* and an adduct prepared by the reaction of dA with 2-octenal in the presence of hydrogen peroxide,21 revealed that they are identical. The 1H and 13C NMR data for the synthetic dA* are provided in Table 1. Further detailed structure determination of dA* was accomplished by various two-dimensional NMR techniques, such as correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond correlation (HMBC), as summarized in Figure S1 (Supporting Information). As an example, the

Figure 3. Time courses of dA and dC adduct formation and pH change of the reaction mixtures. 1556



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Table 1. NMR Data of dA*

Table 2. NMR Data of dC*-1

δC (ppm)

multiplicity

δH (ppm)

2 4 5 6 8 10 11 1′ 2′

135.8 137.9 123.0 140.9 139.7 129.5 127.5 83.9 39.6

d s s s d d s d t

9.18

3′ 3′-OH 4′ 5′

70.6

d

87.9 61.6

d t

5′-OH 1″ 1″-OH 2″ 3″

63.5

d

34.8 25.2

t t

4″ 5″ 6″

31.1 22.1 13.9

t t q

δC (ppm)

multiplicity

2 4 5 6 7 8 1′ 2′ 3′ 3′-OH 4′ 5′ 5′-OH 1″

146.8 145.1 98.8 127.8 132.7 123.0 84.7 40.0 70.4

s s d d d s d t d

87.7 61.2

d t

39.0

t

2″ 3″ 4″ 5″ 6″

206.2 41.0 25.2 21.7 13.8

s t t t q

multiplicity s

8.53 7.42

s s

6.48 2.36 2.73 4.43 5.35 3.89 3.54 3.62 4.96 5.02 5.48 1.93 1.3 1.49 1.3 1.3 0.86

t 6.7 ddd 3.4, 6.2, 13.3 m m d 4.1 m m dt 5.0, 11.6 dt 2.1, 5.7 brq 5.3 dd 1.7, 4.4 m m m m m t 6.8

δH (ppm)

multiplicity

6.62 7.61 7.11

d 7.8 d 7.9 s

6.29 2.13 4.26 5.27 3.82 3.58 5.04 4.11 4.15

t 6.6 m m d 3.9 dd 3.6, 6.7 m t 5.1 d 17.7 d 17.7

2.52 1.47 1.27 0.86

t 7.3 m m t 7.3

data, the structure of dC*-2 was proposed to be an etheno-dC derivative with a hydroxy-hexyl side chain. It was quite possible that dC*-2 has the same side chain as dA*. Thus, a candidate compound was prepared by reacting dC with crystalline 2,3epoxyoctanal, and the products were compared with dC*-2. The elution positions in HPLC, the UV spectra, and the highresolution mass spectra of dC*-2 and one of the synthetic products were identical. The 1H and 13C NMR data of the synthetic dC*-2 are shown in Table 3. Further structure determination was accomplished by various two-dimensional NMR techniques, such as COSY, HMQC, and HMBC, as summarized in Figure S1 (Supporting Information). As an

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H-13C HMQC NMR spectrum of dA* is shown in Figure S2 (Supporting Information). The 1-hydroxy-hexyl side chain was attached to C-11 of the imidazole ring, as confirmed by the cross-peaks observed between H-1″ and N-1 by 1H-15N HMBC. The molecular formula of dC*-1, isolated from the dChemin-ethyl linoleate mixture, is consistent with C17H23N3O5, as determined by HRESIMS (positive ion mode, m/z 372.1528 (M + Na)+ 372.1530 calcd. for C17H23N3O5Na). Its UV spectra were similar to those of the dC-ONE adduct. The UV λmax in 10% acetonitrile was 278 nm. On the basis of these data, the structure of dC*-1 was proposed to be an etheno-dC derivative with an oxo-hexyl side chain. To characterize the structure of dC*-1 in detail, a candidate compound was prepared by the reaction of dC with 2-octenal, in the presence of t-butylhydroperoxide. The elution positions in HPLC, the UV spectra, and the high-resolution mass spectra of dC*-1 and the major synthetic product were identical. Therefore, the structure determination of dC*-1 was accomplished using the synthetic sample. The 1H and 13C NMR data are shown in Table 2. The 1 H-13C HMQC NMR spectrum of dC*-1 is shown in Figure S3 (Supporting Information). dC*-1 generated a highly crystalline product, and thus, further structure elucidation was performed by X-ray crystallography. The compound was recrystallized by an ethanol/water (15:85) solvent system and yielded colorless needles. An ORTEP drawing of dC*-1 is shown in Figure S4 (Supporting Information). The sugar of dC*-1 adopts the C(3′)-endo conformation, and the glycosidic torsion is in the anti form. The molecular formula of dC*-2, isolated from the dChemin-ethyl linoleate mixture, is consistent with C17H25N3O5, as determined by HRESIMS (positive ion mode, m/z 374.1687, (M + Na)+ 374.1686 calcd. for C17H25N3O5Na). It also showed the UV spectra characteristic of an etheno type dC adduct. The UV λmax in 10% acetonitrile was 278 nm. On the basis of these

Table 3. NMR Data of dC*-2 and dC*-3

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δC (ppm)

multiplicity

2 4 5 6 7 8 1′ 2′

146.7 145.0 98.8 127.5 129.7 134.0 85.1 40.1

s s d d d s d t

3′ 3′-OH 4′ 5′ 5′-OH 1″ 1″-OH 2″

70.3

d

87.7 61.2

d t

64.5

d

37.1

t

3″

25.1

t

4″ 5″ 6″

31.1 22.1 13.9

t t q

δH (ppm)

multiplicity

6.62 7.64 7.21

d 7.8 d 7.8 brs

6.37 2.16 2.20 4.27 5.28 3.84 3.60 5.05 5.28 4.98 1.62 1.80 1.34 1.42 1.26 1.26 0.85

t 6.5 m m m m q 3.6 m brt 5.1 m d 5.3 m m m m m m t 6.7



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phosphate buffer, pH 7.4), the final pH of the lipid peroxidation mixture was regulated to 6.08 versus 4.30 under condition 1 and 6.06 versus 2.64 under condition 2 (with a higher amount of hemin). Under conditions 1 and 2, the formation of the lipid peroxidation-derived mutagen 2,3-epoxyoctanal was 1.2 and 1.4 times higher in the acidic pH range than under neutral conditions, as revealed by the amount of dA* formation (Table 4).

example, the 1H-13C HMQC NMR spectrum of dC*-2 is shown in Figure S5 (Supporting Information). The 1-hydroxyhexyl side chain was presumed to be attached to C-8 of the imidazo-pyrimidine ring, based on the 1H-13C HMBC data. Especially, the presence of the three bond 1H-13C HMBC between the 7.21 ppm proton and C-4 and the absence of a cross-peak between the 7.21 ppm proton and C-2 support this structure. The same HMBC method was used to determine the position of the side chain in the structure of dC-4-oxo-2pentenal.31 The molecular formula of dC*-3, isolated from the dChemin-ethyl linoleate mixture, is consistent with C17H25N3O5, as determined by HRESIMS (positive ion mode, m/z 374.1690, (M + Na)+ 374.1686 calcd. for C17H25N3O5Na). The UV spectrum of dC*-3 was identical to that of dC*-2 (λmax in 10% acetonitrile, 278 nm), suggesting that dC*-2 and dC*-3 are isomers. The elution positions in HPLC, the UV spectra, and the high-resolution mass spectra of dC*-3 and one of the synthetic products formed by the reaction of dC with crystalline 2,3-epoxyoctanal were identical. The 1H and 13C NMR spectra were identical to those of dC*-2 (Table 3). Therefore, dC*-2 and dC*-3 seem to be stereoisomers in relation to the 1hydroxy-hexyl side chain. Mutagenic Activity of 2,3-Epoxyoctanal in Salmonella Strains. 2,3-Epoxyoctanal showed mutagenic activity in the Salmonella typhimurium TA 100 and TA 104 strains without S9 mix (Figure 5). The specific mutagenic activities of 2,3-

Table 4. Acidic pH during Lipid Peroxidation (LPO) Enhances 2,3-Epoxyoctanal Formationa LPO

pH

4.3

6.1

condition 1 LPO

yieldb of dA* pH

11.5 ± 1.0 2.6

9.5 ± 0.5 6.1

condition 2

yieldb of dA*

7.5 ± 0.7

5.4 ± 0.6

a

Generation of 2,3-epoxyoctanal was estimated by the formation of dA*. bdA*/dA × 103; mean values (±deviation) of 2 independent experiments.

Effect of pH on Adduct Formation. To examine the second possibility that efficient adduct formation of dC and dA with 2,3-epoxyoctanal occurs under acidic conditions, the yields of dA*, dC*-2, and dC*-3 were compared between the reactions at pH 4.5 and pH 7.3. At both 37 and 23 °C, the dA adduct (dA*) and the dC adduct (dC*-2 plus dC*-3) were both produced with higher yields under acidic conditions. dA* was partially decomposed to the free base after a 64 h reaction under the pH 4.5 and 37 °C conditions (Figure 6). This is

Figure 5. Mutagenicity of 2,3-epoxyoctanal in TA 100 and TA 104. Each value represents the mean ± SD, N = 3. *, P < 0.05; **, P < 0.01 (significant difference from the negative control (dose 0) by Student ttest).

epoxyoctanal to TA 100 and TA 104 were 138 and 37 revertants/μg, calculated from the data at a concentration of 30 μg/plate, respectively. TA 100 was more responsive than TA 104 to the mutagenic activity. Effect of pH on 2,3-Epoxyoctanal Formation during Lipid Peroxidation. The formation of dA*, dC*-2, and dC*3, induced by 2,3-epoxyoctanal was accompanied by a pH shift from neutral to acidic (Figure 3). There are two possible ways to explain this higher adduct formation under acidic conditions: (i) acidic conditions enhance lipid peroxidation, leading to the formation of 2,3-epoxyoctanal. (ii) Acidic conditions enhance the adduct formation reaction or increase the stability of the adduct. To distinguish between these possibilities, we first examined the effect of pH on lipid peroxidation. During lipid peroxidation, the reaction mixture gradually becomes acidified. Using a different buffer concentration (0.2 M versus 50 mM

Figure 6. Effect of pH on the formation of adducts by 2,3epoxyoctanal.

reasonable, based on the fact that glycosidic bond hydrolysis in purine deoxynucleosides occurs under weakly acidic conditions.32 From these results, we concluded that the enhancement of adduct formation, by the lipid peroxide model system accompanied with acidic conditions, is mainly due to the higher reactivities of 2,3-epoxyoctanal with dA and dC at an acidic pH. 1558



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Scheme 1. Possible Mechanisms of 2,3-Epoxyoctanal Formation by Omega-6 Linoleate Peroxidation

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DISCUSSION The detection and structural characterization of nucleoside adducts formed by a mutagenic complex mixture, such as heated sugar and oxidized fatty acids, is a successful approach to identify mutagens.33,19 In the present study, we applied this method to a mixture of linoleate peroxidation products. We improved the previous procedure19 in two ways: (i) The reaction time was extended from 3 days to 22 days, and (ii) 10% acetonitrile was added to the reaction mixture for efficient adduct formation of lipophilic products with nucleosides. As a result, an unknown adduct dA* was detected in the dA-reaction with a higher yield than dA-ONE, while in the dC-reaction, three unknown adducts, dC*-1, -2, and -3, were detected with considerable yields. On the basis of the structures of the three adducts, dA*, dC*-2, and dC*-3, the mutagen involved in the adduct formation was determined to be 2,3-epoxyoctanal. However, the mutagen involved in dC*-1 formation is proposed to be 4-oxo-2-octenal, by analogy to the formation of the 4-ONE and 4-OHE adducts. Regarding DNA adduct formation by epoxy-type lipid peroxidation products, ethenotype dG and dA adduct formation by 4,5-epoxy-2-decenal and 2,3-epoxy-4-hydroxynonenal has been reported.34−37 It is worth mentioning that Carvalho et al. isolated the same adduct (adduct III in ref 21) as dA*, formed between dA and oxidized 2,4-decadienal via a 2,3-epoxyoctanal intermediate. Various pathways are possible for the formation of 2,3-epoxyoctanal. For example, it can be generated from 9-hydroperoxy-10,12octadecadienoic acid (9-HPODE) via 2,4-decadienal and 2octenal38,39 or from 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE) via 12,13-epoxy-9-HPODE39 (Scheme 1). We observed the tendency of more 2,3-epoxyoctanal formation under the acidic lipid peroxidation conditions (Table 4). This is compatible with the report by Kanner and Lapidot,40 who found that the accumulation of linoleate hydroperoxides such as 9-HPODE and 13-HPODE, which are possible precursors of 2,3-epoxyoctanal, are amplified in a linoleate peroxidation emulsion at acidic pH in the presence of metmyoglobin or iron ions. These results suggested that this mutagen could be efficiently formed during storage and cooking under acidic conditions or in the stomach during digestion at low pH and may be involved in human cancers.

Further studies on the detection of these mutagens in foods and their carcinogenic potentials will be necessary to clarify the roles of these mutagens in human cancer.

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ASSOCIATED CONTENT

S Supporting Information *

Selected 2D NMR results for the dA and dC adducts; 1H−13C HMQC NMR spectra of dA*, dC*-1, dC*-2, and crystal structure of dC*-1; and refined crystallographic parameters of dC*-1. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-93-691-7469. Fax: +81-93-601-2199. E-mail: [email protected]. Funding

This work was supported by grants from the Ministry of Health, Labor and Welfare of Japan. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS 4-OHE, 4-oxo-2-hexenal; 4-ONE, 4-oxo-2-nonenal; 4-HNE, 4hydroxy-2-nonenal; dA, 2′-deoxyadenosine; dC, 2′-deoxycytidine; ORTEP, Oak Ridge Thermal Ellipsoid Program; COSY, Correlation Spectroscopy; HMQC, Heteronuclear Multiple Quantum Correlation; HMBC, Heteronuclear Multiple Bond Correlation; HRESIMS, High Resolution Electrospray Ionization Mass Spectrometry

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REFERENCES

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