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Communications 4,5-Epoxy-2(E)-decenal-Induced Formation of 1,N6-Etheno-2′-deoxyadenosine and 1,N2-Etheno-2′-deoxyguanosine Adducts Seon Hwa Lee, Tomoyuki Oe, and Ian A. Blair* Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6160 Received September 6, 2001
trans-4,5-Epoxy-2(E)-decenal reacted with 2′-deoxyadenosine to give 1,N6-etheno-2′-deoxyadenosine as well as other 2′-deoxyadenosine adducts. It also reacted with 2′-deoxyguanosine to give 1,N2-etheno-2′-deoxyguanosine and other 2′-deoxyguanosine adducts. Synthetic trans4,5-epoxy-2(E)-decenal was quite stable under the reaction conditions that were used. It was not contaminated with 2,3-epoxyoctanal, a potential precursor to the formation of unsubstituted etheno adducts. Furthermore, using a sensitive LC/MS assay, it was possible to show that no 2,3-epoxyoctanal was formed during prolonged incubations of trans-4,5-epoxy-2(E)-decenal. Therefore, trans-4,5-epoxy-2(E)-decenal, a primary product of lipid peroxidation, is a precursor to the formation of 1,N6-etheno-2′-deoxyadenosine and 1,N2-etheno-2′-deoxyguanosine. There is no need for an additional oxidation step such as would be required if trans,trans-2,4decadienal or 4-hydroxy-2-nonenal were the lipid hydroperoxide decomposition products that initiated the formation of unsubstituted etheno adducts. These findings provide an important link between a primary product of lipid peroxidation and a mutagenic DNA lesion that has been detected in human tissues.
Introduction Lipid peroxidation of polyunsaturated fatty acids (PUFAs)1 is thought to play an important role the degenerative diseases of aging such as cancer (1, 2). The formation of lipid hydroperoxides from PUFAs by free radical processes is a complex process, which leads to a number of different regioisomers and stereoisomers (3). LOX-mediated oxidation of PUFAs results in the formation of lipid hydroperoxides with much greater stereoselectivity (4). Lipid hydroperoxides undergo homolytic decomposition to bifunctional electrophiles (5) that can react with DNA (2). In previous studies, we examined the homolytic decomposition of 13-[S-(Z,E)]-9,11-hydroperoxyoctadecadienoic acid (13-HPODE; a prototypic ω-6 PUFA lipid hydroperoxide) in the presence of the DNA bases 2′-deoxyadenosine (dAdo) and 2′-deoxyguanosine (dGuo). From structures of the resulting DNA adducts, we proposed that the major covalent modifications arose * Correspondence should be addressed to this author at the Center for Cancer Pharmacology, University of Pennsylvania School of Medicine, 1254 BRB II/III, 421 Curie Blvd., Philadelphia, PA 191046160. Fax: (215) 573-9889. E-mail:
[email protected]. 1Abbreviations: APCI, atmospheric pressure chemical ionization; brs, broad singlet; CID, collision-induced dissociation; dAdo, 2′deoxyadenosine; d, doublet; dd, doublet of doublets; dGuo, 2′-deoxyguanosine; Gua, guanine; 4-HNE, 4-hydroxy-2-nonenal; 4-HPNE, 4-hydroperoxy-2-nonenal; 13-HPODE, 13-[S-(Z,E)]-9,11-hydroperoxyoctadecadienoic acid; LC/MS, liquid chromatography/mass spectrometry; LOX, lipoxygenase; MH+, protonated molecular ion; MSn, multiple tandem mass spectrometry; 4-ONE, 4-oxo-2-nonenal; PUFA, polyunsaturated fatty acid; s, singlet; td, triplet of doublets; SIM, selected ion monitoring; TIC, total ion current.
Figure 1. Formation of 4-oxo-2-nonenal (4-ONE), 4-hydroperoxy-2-nonenal (4-HPNE), 4-hydroxy-2-nonenal (4-HNE), and 4,5-epoxy-2(E)-decenal (4,5-EDE) from 13-HPODE.
through generation of 4-oxo-2-nonenal from 13-HPODE. The same adducts were formed when DNA bases were treated with synthetic 4-oxo-2-nonenal (6, 7). Subsequently, 4-oxo-2-nonenal was confirmed as a major breakdown product of homolytic lipid hydroperoxide decomposition (Figure 1) (8), a finding that was recently confirmed by Spiteller et al. (9). Surprisingly, 4-hydroperoxy-2-nonenal was also characterized as a product of 13-HPODE decomposition by our laboratory and by Schneider et al. (8, 10). 4-Hydroperoxy-2-nonenal was subsequently shown to be a precursor in the formation of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal (11) (Figure 1). The other major bifunctional electrophile identified in homolytic 13-HPODE decomposition was 4,5epoxy-2(E)-decenal (11) (Figure 1), a recently identified product from the autoxidation of arachidonic acid (12). trans,trans-2,4-Decadienal is an environmental contaminant in water and food (see ref 13 for discussion). It is also a product of lipid peroxidation through R-cleavage
10.1021/tx010147j CCC: $22.00 © 2002 American Chemical Society Published on Web 02/12/2002
Communications
of the alkoxy radicals derived from 9-hydroperoxy-(E,E)10,12-octadecadienoic acid or 11-hydroperoxy-(Z,Z,E,E)eicosa-5,8,12,14-tetraenoic acid (9, 12). Recent studies have shown that the reaction of peroxide-treated trans,trans-2,4-decadienal with dAdo or dGuo results in the formation of 1,N6-etheno-dAdo (13) and 1,N2-ethenodGuo (14), respectively. We reasoned that 4,5-epoxy-2(E)decenal could have been formed when trans,trans-2,4decadienal was treated with peroxides (12, 15) and that this bifunctional electrophile was in fact the precursor to the formation of etheno adducts from lipid hydroperoxides.
Materials and Methods Materials. Ammonium acetate, trifluoroacetic acid, dAdo, dGuo, 1,N6-etheno-dAdo, O6-benzylguanine, chloroacetaldehyde, (triphenylphosphoranylidene)acetaldehyde, deuterated NMR solvents, and hydrogen peroxide were purchased from SigmaAldrich. (St. Louis, MO). 3-Morpholinopropanesulfonic acid (MOPS) was obtained from Fluka BioChemika (Milwaukee, WI). Chelex-100 chelating ion-exchange resin (100-200 mesh size) was purchased from Bio-Rad Laboratories (Hercules, CA). HPLC grade water, methanol, acetonitrile, hexane, and 2-propanol were obtained from Fisher Scientific Co. (Fair Lawn, NJ). Gases were supplied by BOC Gases (Lebanon, NJ). Mass Spectrometry. Mass spectrometry was conducted with a Thermo Finnigan LCQ ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) equipped with an APCI source. The mass spectrometer was operated in the positive ion mode with a discharge current of 5 µA applied to the corona needle. Nitrogen was used as the sheath (80 units) and auxiliary (10 units) gas to assist with nebulization. The vaporizer and heated capillary temperatures were set at 450 and 150 °C, respectively. Full scanning analyses were performed in the range of m/z 50 to m/z 800. Collision-induced dissociation (CID) experiments coupled with multiple tandem mass spectrometry (MSn) employed helium as the collision gas. The relative collision energy was set at 20% of maximum (1 V). Liquid Chromatography. Analysis and purification of synthetic DNA adducts was conducted on a Hitachi L7100 module (Hitachi, San Jose, CA) equipped with a Waters 996 photodiode array detector. System 1 employed a Beckman Ultrasphere-Si column (250 × 10 mm i.d., 5 µm; Alltech, Deerfield, IL). Systems 2 and 3 employed a YMC basic column (150 × 2.0 mm i.d., 5 µm); system 4 employed a YMC-Pack ODSAQ column (250 × 10 mm i.d., 5 µm). For normal phase system 1, solvent A was hexane/2-propanol (197:3, v/v), and solvent B was hexane/2-propanol (7:3, v/v). The linear gradient for system 1 was as follows: 1% B at 0 min, 2% B at 18 min, 100% B at 20 min, 100% B at 24 min with a flow rate of 2 mL/min. For systems 2 and 3, solvent A was 5 mM aqueous ammonium acetate containing 0.01% (v/v) trifluoroacetic acid, and solvent B was 5 mM ammonium acetate containing 0.01% (v/v) trifluoroacetic acid in methanol. The linear gradient for system 2 was as follows: 1% B at 0 min, 91% B at 18 min, 91% B at 20 min with a flow rate of 0.25 mL/min. The linear gradient for system 3 was as follows: 10% B at 0 min, 100% B at 18 min, 100% B at 20 min with a flow rate of 0.25 mL/min. System 4 employed an isocratic mobile phase of methanol/water (1:1, v/v; 2 mL/min). All separations were performed at ambient temperature. Liquid Chromatography/Mass Spectrometry. Chromatography for LC/MS studies was performed using a Waters Alliance 2690 HPLC system (Waters Corp., Milford, MA). System 5 employed two silica columns in series (250 × 4.6 mm i.d., 5 µm; Regis, Morton Grove, IL). Systems 6 and 7 employed a YMC C18 ODS-AQ column (250 × 4.6 mm i.d., 5 µm; Waters, Milford, MA). System 5 used normal phase solvents. Solvent A was hexane/2-propanol (197:3, v/v), and solvent B was hexane/ 2-propanol (7:3, v/v). Isocratic elution was performed with 50% B at a flow rate of 0.8 mL/min. For system 6, solvent A was 5 mM aqueous ammonium acetate containing 0.01% (v/v) trifluo-
Chem. Res. Toxicol., Vol. 15, No. 3, 2002 301 roacetic acid, and solvent B was 5 mM ammonium acetate in methanol containing 0.01% (v/v) trifluoroacetic acid. The linear gradient was as follows: 30% B at 0 min, 30% B at 3 min, 55% B at 13 min, 55% B at 16 min, 70% B at 24 min, 100% B at 26 min, 100% B at 30 min with a flow rate of 1.0 mL/min. For system 7, solvent A was 5 mM ammonium acetate in water, and solvent B was 5 mM ammonium acetate in acetonitrile. The linear gradient was as follows: 6% B at 0 min, 6% B at 3 min, 20% B at 9 min, 20% B at 13 min, 60% B at 21 min, 80% B at 22 min, 80% B at 24 min with a flow rate of 1.0 mL/min. All separations were performed at ambient temperature. 1H NMR. Spectra (500 MHz) were determined at 25 °C using a Varian UNITY 500 instrument. Samples were dissolved in 750 µL of CDCl3 [trans-4,5-epoxy-2(E)-decenal] or 750 µL of DMSO-d6 (1,N2-etheno-Gua and N2,3-etheno-Gua) and introduced into a Kontes (Vineland, NJ) NMR tube (200 mm × 5 mm o.d.). The CHCl3 (7.25 ppm) or DMSO (2.50 ppm) residual signal was used as the reference signal. Coupling constants are shown as hertz values. Acquisition conditions were as follows: spectral width of 6000 Hz [trans-4,5-epoxy-2(E)-decenal] or 7000 Hz (1,N2-etheno-Gua and N2,3-etheno-Gua), 30° pulse flip angle, 32K data points, and 32 transients. Synthesis of trans-4,5-Epoxy-2(E)-decenal. trans-2Octenal was subjected to epoxidation with hydrogen peroxide under alkaline conditions as described by Lin et al. (16). The crude trans-2,3-epoxyoctanal was recrystallized from ethyl acetate to remove 5% of contamination by cis-2,3-epoxyoctanal, and then subjected to a Wittig reaction with (triphenylphosphoranylidene)acetaldehyde. After silica gel column chromatography with hexane/diethyl ether/triethylamine (350:150:0.2; v/v/v), the crude trans-4,5-epoxy-2(E)-decenal was isolated and purified by distillation under vacuum (60 °C, 2 mmHg) as a colorless oil (UV λmax ) 229 nm; hexane/2-propanol) with a purity of >98% as determined by HPLC analysis using system 1 [tR ) 13.5 min; cis-4,5-epoxy-2(E)-decenal < 0.1%]. The 1H NMR (CDCl3) spectrum was identical with that reported by Lin et al. (16): δ 0.90 [3H, t, C(10)-H3], 1.33 [4H, m, C(8)-H2 and C(9)-H2], 1.47 [2H, m, C(7)-H2], 1.63 [2H, m, C(6)-H2], 2.96 [1H, td, J56 ) 5.5 Hz, J45 ) 2 Hz, C(5)-H], 3.32 [1H, dd, J34 ) 7 Hz, J45 ) 2 Hz, C(4)H], 6.54 [1H, dd, J23 ) 16 Hz, J34 ) 7 Hz, C(3)-H], 6.38 [1H, dd, J23 ) 16 Hz, J12 ) 7.5 Hz, C(2)-H], 9.56 [1H, d, J12 ) 7.5 Hz, C(1)HO]. APCI/MS: m/z 169 (MH+), m/z 151 (MH+-H2O). APCI/MS/MS of m/z 169 (MH+): m/z 151 [MH-H2O]+, m/z 133 [MH-2H2O]+, m/z 123 [MH-H2O-CO]+, m/z 113 [MH-C4H8]+, m/z 109 [MH-H2O-C3H6]+, m/z 99 [MH-C5H10]+, m/z 95 [MHH2O-C4H8]+, m/z 85 [MH-CO-C4H8]+, m/z 81 [MH-H2OCO-C3H6]+, m/z 67, [MH-H2O-CO-C4H8]+. Synthesis of 1,N2-Ethenoguanine (Gua). 1,N2-Etheno-Gua was prepared by a modification of the method of Sattsangi et al. (17) from the reaction of dGuo with chloroacetaldehyde at 37 °C at pH 6.4 for 5 days in water. After purification by reversed-phase HPLC using system 2 (tR ) 12 min) followed by system 4 (tR ) 8 min), 1,N2-etheno-dGuo was obtained in poor yield (5%) as a white solid (UV λmax ) 226, 285 nm in methanol/ water containing 0.1% trifluoroacetic acid) with >97% purity by HPLC system 2 (tR ) 12 min). APCI/MS: m/z 292 (MH+), m/z 176 (BH2+). MS2 (m/z 292 f): m/z 176 [BH2]+. MS3 (m/z 292 f m/z 176 f): m/z 148 [MH-CO]+, m/z 121 [MH-COCNH]+. 1,N2-Etheno-dGuo was depurinated by dissolving in aqueous 1 N HCl and allowing to stand for 4 h at 60 °C. The aqueous HCl was evaporated under nitrogen and the resulting solid dried under vacuum at 60 °C. 1,N2-Etheno-Gua was obtained in essentially quantitative yield from 1,N2-etheno-dGuo as a white amorphous solid (UV λmax ) 221, 268, 295 nm; 0.1 N HCl) with >97% purity as determined by reversed-phase HPLC analysis on system 2 (tR ) 10 min). The 1H NMR spectrum was essentially identical with that we reported previously for [13C3]etheno-Gua (18) (DMSO-d6): δ 7.38 (1H, d, Jolefinic ) 2.5 Hz, etheno), 7.56 (1H, d, Jolefinic ) 2.5 Hz, etheno), 7.82 [1H, s, C(8)H]. The assignment of C-8 as δ 9.37 by Sattsangi et al. (17) appears to be incorrect. APCI/MS: m/z 176 (MH+). MS2 (m/z 176 f): m/z 148 [MH-CO]+, m/z 121 [MH-CO-CNH]+.
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Synthesis of N2,3-Etheno-Gua. N2,3-Ethenoguanine was also prepared by a modification of the method of Sattsangi et al. (17). O6-Benzylguanine was reacted with chloroacetaldehyde in 75% aqueous ethanol at 37 °C for 5 days at pH 3.5. The solvent was evaporated under vacuum, and the residue was crystallized from ethanol/water (1:1, v/v). After drying under vacuum, O6-benzyl-N2,3-etheno-Gua was isolated as a white solid with >98% purity (system 4, tR ) 17 min) in 80% yield. The benzyl moiety was removed by heating at 100 °C in aqueous 1 N HCl for 4 h. The aqueous solution was neutralized with aqueous sodium hydroxide and washed with ether, the water was evaporated, and the resulting white solid was dried under vacuum. After crystallization from ethanol/water, N2,3-ethenoGua was obtained in quantitative yield as a white amorphous powder with >99% purity (UV λmax ) 215, 256 nm; 0.1 M HCl) on HPLC system 3 (tR ) 6 min). The 1H NMR (DMSO-d6) spectrum was similar to that reported previously (17): δ 7.09 (1H, d, Jolefinic ) 2 Hz, etheno), 7.60 (1H, d, Jolefinic ) 2 Hz, etheno), 8.14 [1H, s, C(8)-H], 12.3 (1H, brs, NH or OH), 13.8 (1H, brs, NH or OH). However, reported chemical shifts for the etheno protons were at lower field (δ 7.61 and 8.02) than these values, although the coupling constants were identical (2 Hz). APCI/MS: m/z 176 (MH+). MS2: no product ions were detected. LC/MS Analysis of Epoxides. LC/APCI/selected ion monitoring (SIM)/MS analysis of the two epoxides employed MH+ for 2,3-epoxyoctanal and trans-4,5-epoxy-2(E)-decenal at m/z 143 and m/z 169, respectively. 2,3-Epoxyoctanal and trans-4,5-epoxy2(E)-decenal eluted with retention times of 8.34 and 9.04 min, respectively, on HPLC system 5. Under these conditions, there was a clear separation of the two epoxides with no interfering signals at the retention times of either analyte. Quantitation of 2,3-Epoxyoctanal in Synthetic trans4,5-Epoxy-2(E)-decenal. Calibration standards containing known amounts of 2,3-epoxyoctanal (0.02, 0.10, 0.20, 1, 2, 10, and 20 µg) were prepared as an ether solution (100 µL) containing trans-4,5-epoxy-2(E)-decenal (20 µg). A portion of the standard solution (10 µL) was subjected to LC/APCI/SIM/MS analysis using normal phase system 5 by monitoring m/z 143 (MH+) for 2,3-epoxyoctanal and m/z 169 (MH+) for trans-4,5epoxy-2(E)-decenal. A typical regression line was y ) 6E+06x + 194 385, r2 ) 0.9995. The amount of 2,3-epoxyoctanal in the authentic trans-4,5-epoxy-2(E)-decenal (20 µg, 2 µg on column) was then determined. The 2,3-epoxyoctanal was below the detection limit of the assay (
