Epoxidation of trans-4-Hydroxy-2-nonenal by Fatty Acid

In this study, we reported that fatty acid hydroperoxides and hydrogen peroxide are capable of epoxidizing 4-hydroxy-2-nonenal, a lipid peroxidation p...
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Chem. Res. Toxicol. 1996, 9, 306-312

Epoxidation of trans-4-Hydroxy-2-nonenal by Fatty Acid Hydroperoxides and Hydrogen Peroxide Hauh-Jyun Candy Chen and Fung-Lung Chung* Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Road, Valhalla, New York 10595 Received August 7, 1995X

In this study, we reported that fatty acid hydroperoxides and hydrogen peroxide are capable of epoxidizing 4-hydroxy-2-nonenal, a lipid peroxidation product, to the mutagenic epoxide. The evidence of its formation is provided (i) by trapping with [8-3H]deoxyadenosine for the formation of 7-(1′,2′-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine as a pair of diastereomers, (ii) by derivatization with (2,4-dinitrophenyl)hydrazine in acidic methanol, and (iii) by comparing its 1H-nuclear magnetic resonance and mass spectra to those of the authentic standard. After incubating 4-hydroxy-2-nonenal with 9- or 13-linoleic acid hydroperoxide at 37 °C for 24 h, the epoxide was produced in 13.4% or 12.5% yield, and with hydrogen peroxide, the yield was 21.5%. In the presence of fatty acid (linoleic acid, γ-linolenic acid, or arachidonic acid) and lipoxygenase, the epoxide of 4-hydroxy-2-nonenal was formed in 15.3%, 7.2%, or 6.2% yield, respectively. The xanthine/xanthine oxidase/superoxide dismutase system generated the epoxide in 1.2% yield. These yields are estimated on the basis of a standard curve obtained from reactions of deoxyadenosine and epoxide. These results show that 4-hydroxy-2-nonenal is epoxidized by biological oxidants, suggesting a plausible endogenous pathway for the in vivo formation of etheno adducts.

Introduction Trans-4-Hydroxy-2-nonenal (HNE)1 is an endogenous R,β-unsaturated aldehyde formed by lipid peroxidation (1, 2). It has been detected in various tissues in rodents and humans with an estimated level varying from 0.3 to 8 nmol/g, depending on the tissues and pathological conditions (3). HNE is cytotoxic at high doses and causes primarily liver and kidney toxicity in rats (3, 4). It has been shown that much of the toxicity associated with lipid peroxidation can be attributed to HNE (1). 4-Hydroxyalkenals, including HNE, induce sister chromatid exchange and chromosal aberrations and are mutagenic in cell cultures (5). Since lipid peroxidation is implicated in tumorigenesis, it has also been postulated that HNE may play a role in this process. Like other R,β-unsaturated aldehydes or enals, HNE not only conjugates with cellular proteins, but also reacts with DNA bases with the formation of adducts (6, 7). Our earlier studies showed that HNE is readily epoxidized by tert-butyl hydroperoxide (8). HNE may react with DNA bases by at least two pathways: one is initiated by Michael addition, which results in the formation of exocyclic propano adducts; and the other involves the reaction with its epoxide, 2,3-epoxy-4-hydroxynonanal (EH) (6, 7). EH appears to be more reactive toward DNA bases than the parent aldehyde, yielding 1,N6-ethenoadenine and 1,N2-ethenoguanine adducts (7). Similar adducts were detected in RNA upon incubation with *Address correspondence to this author at the Division of Chemical Carcinogenesis, American Health Foundation, 1 Dana Rd., Valhalla, NY 10595. Tel: (914) 789-7161; Fax: (914) 592-6317. X Abstract published in Advance ACS Abstracts, December 15, 1995. 1 Abbreviations: AA, arachidonic acid; dAdo, 2′-deoxyadenosine; CI, chemical ionization; EdAdo, 1,N6-ethenodeoxyadenosine; DNP, (2,4dinitrophenyl)hydrazine; EH, 2,3-epoxy-4-hydroxynonanal; ES, electrospray; FAB, fast atom bombardment; HNE, trans-4-hydroxy-2nonenal; LA, linoleic acid; LAOOH, linoleic acid hydroperoxide; γ-LNA, γ-linolenic acid; LO, lipoxygenase; FAOOH, fatty acid hydroperoxide; SOD, superoxide dismutase; X/XO, xanthine/xanthine oxidase system.

0893-228x/96/2709-0306$12.00/0

HNE epoxide (7). Consistent with its reactivity toward nucleic acids, EH is more mutagenic in Salmonella tester strains and more tumorigenic than HNE (9). Many of these exocyclic adducts in DNA induce mutation (1014). These results suggest that epoxidation of HNE may be an activation pathway. Recently, evidence has been accumulated for the presence of relatively high levels of exocyclic adducts in tissue DNA of humans and untreated rodents, suggesting that endogenous compounds are responsible for the formation of these adducts (15-18). While the formation of malondialdehyde-derived cyclic deoxyguanosine adduct is clearly originated from lipid peroxidation, the endogenous compounds from which the exocyclic propano and etheno adducts are derived are not yet known. Since the stimulation of lipid peroxidation was shown to increase etheno adducts in microsomal incubations (19), we hypothesize that enals generated by lipid peroxidation and their subsequent epoxidation may play a role. In the present study we have shown that HNE can be epoxidized by hydrogen peroxide (H2O2) or xanthine/xanthine oxidase (X/XO) with superoxide dismutase (SOD). We have also shown that epoxidation occurred with linoleic acid hydroperoxide (LAOOH) and with linoleic (LA), γ-linolenic (γ-LNA), or arachidonic acid (AA) in the presence of lipoxygenase (LO). The epoxide formed in these reactions further reacted with deoxyadenosine (dAdo), yielding etheno dAdo adducts. These results provide evidence of HNE epoxidation by biological oxidants, a pathway which may be important in endogenous etheno adduct formation.

Materials and Methods Chemicals. HNE was synthesized by a previously described method (20). EH was synthesized from reaction with tert-butyl hydroperoxide as previously reported (8). [2,3-3H]HNE (sp act. 375 mCi/mmol) was obtained by acid hydrolysis of [2,3-3H]HNE

© 1996 American Chemical Society

Epoxidation of 4-Hydroxy-2-nonenal diethyl acetal (Chemsyn Science Laboratories, Lenexa, KS). Briefly, [2,3-3H]HNE diethyl acetal (0.39 mCi) was hydrolyzed with 0.017 M HCl (60 µL) at room temperature for 2 h, followed by purification with reversed phase high performance liquid chromatography (HPLC) using system 1. [2,3-3H]EH (sp act. 25.3 mCi/mmol) was obtained by mixing [2,3-3H]HNE (1.0 mCi, sp act. 25.3 mCi/mmol) with 90% aqueous tert-butyl hydroperoxide (10 µL) in 2-propanol (50 µL) with 1 N NaOH (20 µL) and then vortexing for 10 min. After the solvent was removed under a stream of air, the reaction mixture was treated with 1 N HCl (10 µL) and extracted with chloroform (1.0 mL × 2). The chloroform extract was dried over anhydrous sodium sulfate. The chloroform was then evaporated in vacuo to afford the crude product. The crude product was purified by silica gel TLC eluting with dichloromethane/methanol (97:3), and the product was detected by spraying with 2% H2SO4 in ethanol followed by heating. 7-(1′,2′-Dihydroxyheptyl)-1,N6-ethenodeoxyadenosine [7-(l′,2′-dihydroxyheptyl)-EdAdo, 1] was synthesized as described previously (7). [8-3H]dAdo (10 Ci/mmol) was obtained from ICN Biomedicals, Inc. (Costa Mesa, CA). Hydrogen peroxide (30% in water by weight), dAdo, acetic anhydride, 4-(dimethylamino)pyridine, and (2,4-dinitrophenyl)hydrazine (DNP) were purchased from Aldrich Chemical Co., Inc. (Milwaukee, WI). Linoleic acid (LA), γ-linolenic acid (γ-LNA), arachidonic acid (AA), xanthine (X), xanthine oxidase (XO) from buttermilk, catalase from mouse liver, superoxide dismutase (SOD) from human erythrocytes, Tween-20, and soybean lipoxygenase-1S (LO) were obtained from Sigma Chemical Co. (St. Louis, MO). 13(S)-LAOOH and 9(S)-LAOOH were from Cayman Chemical Co. (Ann Arbor, MI). Instrumentation. 1H-NMR was carried out on a Brucker AM 360 WB instrument (Billerica, MA). The chemical ionization mass spectra (CI-MS) were obtained on a Hewlett-Packard Model 5988A spectrometer (Wilmington, DE). The electrospray (ES) mass spectroscopy was performed on a VG Quattro by Fisions Instruments (Danvers, MA). The fast atom bombardment (FAB) mass spectrum was obtained with a VG 707E double focusing mass spectrometer. Chromatography. The radioflow-HPLC was performed on a Waters system (Waters Associates, Milford, MA) with two Model 501 pumps, a Model 660 solvent programmer, a Reodyne injector, a Shimadzu SPD-10A UV-vis detector (Shimadzu Scientific Instruments, Inc., Braintree, MA), and a β-RAM radioflow detector (Inus Systems, Inc., Tampa, FL). The photodiode array-HPLC was performed on a Waters system equipped with two Model 510 pumps, a Model 660 solvent prorammer, a Reodyne injector, and a Waters 994 programmable photodiode array detector. HPLC conditions are as follows: System 1: a Supelcosil LC-18-DB 250 × 4.6 mm 5 µm column (Supelco, Inc., Bellefonte, PA) eluted with a H2O and CH3CN gradient: 0-5 min, 5% CH3CN; 5-60 min, 5-50% CH3CN, at a flow rate of 1.0 mL/min with UV detection at 222 nm. System 2: a reversed phase B&J OD5 octadecyl 250 × 4.6 mm 5 µm column (Baxter Diagnostics Inc., Murskegon, MI) eluted with a H2O and CH3CN gradient: 0-5 min, 5% CH3CN; 5-60 min, 5-35% CH3CN, at a flow rate of 1.0 mL/min with UV detection at 230 nm. System 3: a Supelcosil SPL C-18-DB 250 × 10 mm 5 µm column eluted with a H2O and CH3CN gradient: 0-5 min, 5% CH3CN; 5-60 min, 5-50% CH3CN, at a flow rate of 4.0 mL/min with UV detection at 210 nm. System 4: a Supelcosil LC-18-DB 250 × 4.6 mm 5 µm column eluted with a H2O and CH3CN gradient: 0-5 min, 5% CH3CN; 5-60 min, 5-100% CH3CN, at a flow rate of 1.0 mL/min with UV detection at 370 nm. Standard Curve of Formation of 1 from Reactions with EH. To 5.0 uL of potassium phosphate buffer (1.0 M, pH 7.4) were added 2.0 µL of [8-3H]dAdo (2.0 µCi), 25.0 µL of dAdo (10 mM in 0.1 M KPi, pH 7.4), 2.5 µL of 27.1 mM aqueous EH (final concentration 1.35 mM), and 15.5 µL of H2O in a 500 µL Eppendorf tube. Reactions containing EH with final concentrations of 813, 542, 270, 135, 27.0, 13.5, 10.8, 8.1, and 5.4 µM were also carried out. The reaction mixture was incubated at 37 °C for 24 h and quenched by freezing in dry ice. 7-(1′,2′-Dihydroxyheptyl)-EdAdo (1) was added to the final incubation

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 307 mixture as a UV marker prior to analysis with radioflow-HPLC using system 2. Reaction of HNE with LAOOH. (i) Trapping with dAdo. Typically, to 5.0 µL of potassium phosphate buffer (1.0 M, pH 7.4) were added 2.0 µL of [8-3H]dAdo (2.0 µCi), 25.0 µL of dAdo (10 mM in 0.1 M KPi, pH 7.4), 2.5 µL of HNE (54 mM in H2O as a suspension), 2.0 µL of 13- or 9-LAOOH (100 mM in 2.0 mM Tween-20), and 13.5 µL of H2O in a 500 µL Eppendorf tube. The reaction mixture was incubated at 37 °C for 24 h and stopped by freezing in dry ice. For the control experiments, LAOOH was omitted. 7-(1′,2′-Dihydroxyheptyl)-EdAdo (1) and 3 were added to the final incubation mixture as UV markers prior to analysis using HPLC system 2. (ii) Derivatization with DNP. To an Eppendorf tube, containing 20 µL of potassium phosphate buffer (1.0 M, pH 7.4), 5.0 µL of [2,3-3H]-HNE (3.1 µCi, 572 µM), and 135 µL of H2O, was added 40 µL of LAOOH (20 mM in 2.0 mM Tween-20). The reaction mixture was incubated at 37 °C for 24 h with the cap open. To a portion of the incubation mixture (100 µL) was added EH as UV marker and it was analyzed by HPLC system 1 with UV detection at 210 nm. The fraction corresponding to EH was collected, evaporated to dryness in vacuo, and reconstituted with 70 µL of MeOH. A portion (10 µL) of this solution was analyzed by radioflow-HPLC using system 1. To the remaining portion (60 µL) were added 2% (w/v) DNP methanol solution (60 µL) and 1 N HCl (25 µL). The mixture was incubated at 37 °C for 2 h, and then analyzed by HPLC system 4. Reaction of HNE with Fatty Acids and LO. To 5.0 µL of potassium phosphate buffer (1.0 M, pH 7.4) in a 500 µL Eppendorf tube were added LO (13.5 µL, 1647 units) and 2.0 µL of fatty acids (0.1 M in 2.0 mM Tween-20). After incubation at 37 °C for 1 h with the cap open, the following were added: 2.0 µL of [8-3H]dAdo (2.0 µCi), 25 µL of dAdo (10 mM in 0.1 M KPi, pH 7.4), and 2.5 µL of HNE (54 mM in H2O as a suspension). The reaction mixture was incubated at 37 °C for 24 h and then quenched by addition of ethanol (50 µL) and centrifuged at 13000g for 10 min. For the control experiments, the incubation was carried out under identical conditions except that LO was boiled for 10 min before use. To the supernatant were added 7-(1′,2′-dihydroxyheptyl)-EdAdo (1) and 3 as UV markers, and it was analyzed by HPLC system 2. Reaction of HNE with H2O2. (i) Trapping with dAdo. To 5.0 µL of potassium phosphate buffer (1.0 M, pH 7.4) were added 2.0 µL of [8-3H]dAdo (2.0 µCi), 25.0 µL of dAdo (10 mM in 0.1 M KPi, pH 7.4), 2.5 µL of HNE (54 mM in H2O as a suspension), 2.0 µL of H2O2 (100, 25, or 2.5 mM), and 13.5 µL of H2O. The reaction mixture was incubated at 37 °C for 24 h and then quenched by freezing in dry ice. The reaction mixture (50 µL) was spiked with 7-(1′,2′-dihydroxyheptyl)-EdAdo (1) as UV marker and analyzed by using HPLC system 2. For the control experiments, H2O2 was omitted. (ii) Derivatization with DNP. To an Eppendorf tube, containing 20 µL of 1.0 M potassium phosphate buffer (pH 7.4), 5.0 µL of [3H]HNE (3.1 µCi, 572 µM), and 135 µL of H2O, was added 40 µL of 20 mM H2O2. The reaction mixture was incubated at 37 °C for 24 h with the cap open. To a portion of the incubation mixture (100 µL) was added EH as UV marker, and it was analyzed by HPLC system 1. The fraction corresponding to EH was collected and evaporated to dryness in vacuo. The residue was reconstitued in 70 µL of MeOH. A portion (10 µL) of collected EH was analyzed by radioflow-HPLC in system 1. The remaining portion (60 µL) was derivatized with 2% (w/v) DNP in methanol solution (60 µL) and 1 N HCl (25 µL) by incubating at 37 °C for 2 h. The mixture was then analyzed by HPLC system 4. Reactions of HNE with X/XO with and without Superoxide Dismutase. To a 500 µL Eppendorf tube containing 5.0 µL of 1.0 M potassium phosphate (pH 7.4) were added 2.0 µL of xanthine (0.1 M in H2O) and 6.0 µL of xanthine oxidase (0.1 unit) with superoxide dismutase (7.5 µL, 50 units) or with 7.5 µL of H2O. After incubation at 37 °C for 1 h with the cap open, the following were added: 2.0 µL of [8-3H]dAdo (2.0 µCi), 25 µL of dAdo (10 mM in 0.1 M KPi, pH 7.4), and 2.5 µL of aqueous

308 Chem. Res. Toxicol., Vol. 9, No. 1, 1996 suspension of 54 mM HNE. The reaction mixture was incubated at 37 °C for 24 h. After incubation, the reaction mixture was quenched by ethanol (50 µL) and then centrifuged at 13000g for 10 min. For the control experiments, xanthine oxidase and superoxide dismutase were boiled for 10 min before use. To the supernatant was added 7-(1′,2′-dihydroxyheptyl)-EdAdo (1) as UV marker, and it was analyzed by HPLC system 2. Derivatization of EH with DNP to 3,4-Dihydroxy-2methoxynonanal (2′,4′-Dinitrophenyl)hydrazone (2). The DNP derivative of EH was obtained from reaction of EH (20 mg, 0.12 mmol) with 2% (w/v) DNP in methanol (330 µL) and 1 N HCl (30 µL) at 37 °C for 2 h. After purification on a preparative silica gel TLC plate (20 cm × 20 cm, 1000 µm) (Analtech, Inc., Newark, DE) eluting with dichloromethane/ methanol (99:1), the product was obtained in 21% yield (9.2 mg). CI-MS (m/z): 163, 182, 350, and 384 (M). ES-MS (negative ion mode) (m/z): 251, 253, 383 (M - 1). FAB-MS (positive ion mode) (m/z): 253, 385 (M + 1). 1H-NMR (CDCl3) δ 0.89 (t, J ) 6.6 Hz, 3H, C9-H), 1.31 (m, 6H, C6-H, C7-H, C8-H), 1.60 (m, 2H, C5-H), 3.45 (s, 3H, CH3O), 3.70 (dd, J ) 5.6, 2.7 Hz, 1H, C3-H), 3.80 (dt, J ) 5.0, 2.7 Hz, C4-H), 4.03 (dd, J ) 6.7, 5.6 Hz, 1H, C2-H), 7.49 (d, J ) 6.7 Hz, 1H, C1-H), 7.96 (m, 1H, C6′-H), 8.34 (m, 1H, C5′-H), 9.14 (m, 1H, C3′-H), 11.2 (s, 1H, NH). 13C-NMR (CDCl3) δ 14.0 (C-9), 22.6 (C-8), 25.3 (C-6), 31.7 (C-7), 33.6 (C-5), 57.5 (CH3O), 70.4 (C-4), 74.1 (C-3), 81.8 (C-2), 116.6 (C-6′), 123.4 (C-3′), 130.0 (C-5′), 148.7 (C-1). 3,4-Diacetoxy-2-methoxynonanal (2′,4′-Dinitrophenyl)hydrazone. To a round-bottom flask containing 2 (4.6 mg, 12.0 µmol) were added acetic anhydride (100 µL) and 4-(dimethylamino)pyridine (5.0 mg, 40.9 µmol). The reaction mixture was stirred at room temperature for 1.5 h, followed by quenching with water (10 mL) and 1% aqueous citric acid (10 mL). It was then extracted with CHCl3 (2.0 mL × 3). The combined extract was dried over anhydrous sodium sulfate and evaporated in vacuo to afford the crude product (5.9 mg). The crude product was purified on a preparative TLC plate (20 cm × 20 cm, 500 µm) eluting with dichloromethane/methanol (99:1) to afford the acetyl derivative in 50% yield (2.8 mg). 1H-NMR (CDCl3) δ 0.85 (t, J ) 6.7 Hz, 3H, C9-H), 1.26 (m, 6H, C6-H, C7-H, C8-H), 1.59 (m, 2H, C5-H), 2.06 (s, 3H, OC(dO)CH3), 2.08 (s, 3H, OC(dO)CH3), 3.33 (s, 3H, OCH3), 3.85 (dd, J ) 7.3, 7.1 Hz, 1H, C2-H), 5.22 (dd, J ) 7.1, 3.0 Hz, 1H, C3-H), 5.29 (dt, J ) 5.8, 3.0 Hz, 1H, C4-H), 7.33 (d, J ) 7.3 Hz. 1H, C-1H), 7.94 (d, J ) 9.7 Hz, 1H, C6′-H), 8.33 (dd, J ) 9.7, 2.5 Hz, 1H, C5′-H), 9.12 (d, J ) 2.5 Hz, 1H, C3′-H), 11.09 (s, 1H, NH). CI-MS (m/z): 468 (M), 469 (M + 1). Isolation and Identification of EH from Reaction of HNE with LA/LO or H2O2. (i) LA/LO. To 1.50 mL of 0.2 M potassium phosphate buffer (pH 7.4) containing LO (3.23 mg, 197 000 units) was added LA (28.0 mg, 0.1 mmol) in 0.3 mL of 2.0 mM Tween-20. The reaction mixture was bubbled with oxygen for 2.5 h with stirring at room temperature, then quenched with 2.0 mL of methanol, and centrifuged at 13000g for 10 min. The supernatant obtained was incubated with HNE (25.0 mg, 0.16 mmol) at 37 °C for 19 h. The solvent was then removed in vacuo to afford the crude product. The crude product was purified by preparative TLC (20 × 20 cm, 1000 µm) developed twice with dichloromethane/methanol (100:3) and was further purified by HPLC system 3 to afford 2.4 mg (14% yield) of the epoxy aldehyde. (ii) H2O2. To 1.40 mL of 0.42 M potassium phosphate buffer (pH 7.4) containing HNE (16.6 mg, 0.106 mmol) was added 1.0 mL of 1.0 M H2O2. The reaction mixture was incubated at 37 °C for 24 h. Then sodium chloride was added to saturation and the mixture was extracted with chloroform (15 mL × 3). The chloroform extract was dried over anhydrous sodium sulfate and evaporated to afford 15 mg of the crude product. The crude product was purified by HPLC system 3 to afford 2.7 mg of the epoxy aldehyde in 15% yield.

Results We have demonstrated that HNE is converted to its epoxide by fatty acid hydroperoxides (FAOOH) and H2O2

Chen and Chung

or under conditions in which these oxidants are produced. [8-3H]dAdo was used as a trapping agent, which reacts with EH forming 7-(1′,2′-dihydroxyheptyl)-EdAdo (1) as a pair of diastereomers 1a and 1b (7). Typical HPLC chromatograms obtained from these reaction mixtures are shown in Figure 1. The chemistry underlying the formation of 1 from EH had been previously described (7) (Scheme 1). 1a and 1b are stereoselectively formed from the two isomers of EH. In the reactions examined here, no significant quantitative differences in the formation of 1a and 1b were observed. These results suggest that fatty acid hydroperoxides or H2O2 epoxidizes HNE with no apparent stereoselectivity. Table 1 summarizes the yields of 1 from the reactions examined. Based on the amount of 1 in these reactions, yields of epoxide were estimated from a standard curve obtained from reactions of dAdo and EH, carried out under identical conditions with EH concentrations ranging from 5.4 µM to 1.35 mM (r2 ) 0.97) (Figure 2). The percent yields of EH are shown in Figure 3. The yields of 1 from reactions with 13-LAOOH or 9-LAOOH were similar, indicating that both isomers are equally effective in epoxidizing HNE. Reactions of HNE with fatty acids in the presence of LO which generate fatty acid hydroperoxides in situ yielded comparable quantities of 1 as those from reactions with LAOOH. The LA/LO system produced 1 in 2.02% yield, whereas γ-LNA/LO and AA/LO gave 1 in 0.96% and 0.83% yield, respectively. The LA/LO system appeared to be a more efficient epoxidizing agent, producing a 2-fold greater yield of 1 than the other two systems under the same conditions. These values correspond to 15.3% of EH in LA reactions and 7.2% and 6.2% in γ-LNA and AA reactions. Although a small amount of 1 was detected in the control experiments in which either LAOOH was omitted or the boiled lipoxygenase was used, those yields are significantly lower than those found in the reactions with oxidants. The background level is probably derived from autoxidation of HNE and/or fatty acids. We found that H2O2 was relatively effective in converting HNE to its epoxide. Under the same conditions as those with fatty acid hydroperoxides (except that Tween20 was omitted), it produced 2.8% of 1, corresponding to a 21.5% yield of EH. A substantial amount of EH was also produced with H2O2 at the lower concentrations (1.0 and 0.1 mM). In fact, a sufficient quantity of EH was isolated from reaction mixtures of both LA/LO and H2O2. Its 1H-NMR and mass spectra were obtained and shown to be identical to those of the authentic standard. This provides direct evidence for EH formation in these reactions. In the xanthine/xanthine oxidase/superoxide dismutase (X/XO/SOD) system which generates H2O2 in situ, EH was produced in 1.2% yield. We did not detect any EH when boiled enzymes were used. No detectable amount of EH was found without SOD, indicating that superoxide anion was not an epoxidizing agent. Addition of catalase to these reactions resulted in undetectable amounts of 1 in the reaction mixture, further supporting the involvement of H2O2. Upon incubation of [2,3-3H]EH at 37 °C with the X/XO system, only 22% of EH remained after 24 h. In the reaction of EH (0.27 mM) with X/XO in the presence of [8-3H]dAdo as trapping agent, only 0.13% of 1 was obtained as compared to 0.95% under the same conditions without X/XO. These results suggest that EH decomposed at a faster rate in the presence of superoxide anion, possibly via a nucleophilic ring-opening of epoxide.

Epoxidation of 4-Hydroxy-2-nonenal

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 309

Figure 1. HPLC chromatograms obtained from reactions of dAdo with HNE in the presence of (a) 13-LAOOH (similar chromatogram was obtained with 9-LAOOH); (b) LA/LO; (c) H2O2; (d) X/XO/SOD. Upper panels are obtained by UV detection of reaction mixtures spiked with EdAdo 1 standards as UV markers. Bottom panels show that two radioactive peaks comigrate with 1 in each reaction.

Additional evidence of EH formation is provided by derivatization with its (2,4-dinitrophenyl)hydrazone. These reactions were carried out with [2,3-3H]HNE and LAOOH or H2O2. The fraction collected from the reaction mixture which corresponds to EH was derivatized with DNP. Comigration of a radioactive peak with the UV marker of the EH-DNP derivative indicated formation of EH (Figure 4). The product of DNP derivatization was characterized as 3,4-dihydroxy-2-methoxynonanal (2′,4′dinitrophenyl)hydrazone (2) by its mass and 1H- and 13CNMR spectra and by being further converted to its acetyl derivative. Acidic methanol solution rather than acidic aqueous solution of DNP was used to facilitate solubility and increase the yield of derivatization. Both NMR and MS data indicated that a methoxy group was introduced to the hydrazone via ring-opening of epoxide by methanol. From coupling constants (J1,2 ) 6.7 Hz, J2,3 ) 5.6 Hz, J3,4 ) 2.7 Hz, J4,5 ) 5.0 Hz) and COSY experiments, the doublet of a doublet at 4.03 ppm, the doublet of a doublet

at 3.70 ppm, and the doublet of a triplet at 3.80 ppm were unambiguously assigned to C2-H, C3-H, and C4-H, respectively. The position of the methoxy group (C-2 or C-3) was determined from its acetyl derivative. The large downfield shifts of both C3-H (from 3.70 to 5.22 ppm) and C4-H (from 3.80 to 5.29 ppm) and their characteristic patterns (C2-H and C3-H are doublets of doublets, whereas C4-H is a doublet of a triplet) in the acetylated derivative clearly demonstrate that the methoxy substitution is at C-2.

Discussion In this study we have shown that HNE, a product of lipid peroxidation, can be epoxidized by biological oxidants and subsequently modifies dAdo to give etheno adducts. We recently described autoxidation of HNE to an intermediate which reacted with dAdo to give 1 (21); however, this intermediate was chromatographically

310 Chem. Res. Toxicol., Vol. 9, No. 1, 1996

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Scheme 1. Formation of 7-Substituted EdAdo 1 and Unsubstituted EdAdo 3 from EHa

a Compound 1 appeared as a pair of diastereomers (1a and 1b) which were separated by reversed phase HPLC (see Figure 1). EH was also derivatized with DNP to 2.

Table 1. Percentage Yields of 1 in the Reaction of HNE with FAOOH or H2O2 [3H]EdAdo 1 (%)

b

13-LAOOH (4.0 mM) 9-LAOOH (4.0 mM) control

1.66 ( 0.12 1.78 ( 0.09 0.05 ( 0.00

LA/LOa control γ-LNA/LOa control AA/LOa control

2.02 ( 0.11 0.09 ( 0.02 0.96 ( 0.08 0.18 ( 0.02 0.83 ( 0.04 0.09 ( 0.01

H2O2 (4.0 mM) H2O2 (1.0 mM) H2O2 (0.1 mM) control

2.82 ( 0.20 1.32 ( 0.02 0.13 ( 0.02 0.04 ( 0.02

X/XO control

NDb ND

X/XO/SOD control

0.17 ( 0.00 ND

X/XO/SOD/catalase control

ND ND

a The final concentration of LA, γ -LNA, and AA was 4.0 mM. ND: not detected.

different from EH and was not fully characterized. Results of the present study suggest that HNE generated by oxidation of membrane lipids may be epoxidized by endogenous fatty acid hydroperoxides or H2O2. Fatty acid hydroperoxides can be produced by Fe or Cu as well as by lipoxygenase-catalyzed lipid peroxidation. Mammalian lipoxygenases have been found in cells such as platelets and polymorphonuclear leukocytes (22). Among the reactive oxygen species produced in the aerobic cells, H2O2 (produced in the stimulated neutrophils and macrophages) is perhaps the most long-lived, readily penetrating cell membranes, and has a steady-state concentration of 0.2 nM in red blood cells (23). It is conceivable that HNE can be epoxidized in the proximity of these

oxidants. EH may be sufficiently stable that it is transported through cellular compartments and eventually reacts with DNA. The chemical mechanism by which the etheno adducts are formed from EH is discussed in our previous papers (7, 8). We have observed that both the substituted and unsubstituted etheno adducts were formed upon reactions with epoxide (Scheme 1). The yields of the unsubstituted etheno adduct 3 increased with basicity of the reaction medium.2 Similar results from reactions of EH with deoxyguanosine were observed (7). In the present study, reactions with dAdo at pH 7.4 yielded predominantly the substituted etheno adducts 1. The unsubstituted EdAdo (3) was also detected, which appeared as a peak at 28 min (Figure 1a,b) as identified by its UV and comigration with the authentic standard of 3. However, the amount was too low for quantitation. On the basis of these observations, one would predict that the substituted etheno adducts 1 may be present in DNA at greater levels in vivo than the unsubstituted adducts, if both adducts are repaired with similar efficacy. Furthermore, EH exists as a pair of diastereomers which stereoselectively modified dAdo, yielding the corresponding isomeric etheno adducts. While epoxidation of HNE by tert-butyl hydroperoxide gave predominantly one of the EH isomers (7), the stereoselectivity was not evident with the oxidants studied. Dix and Marnett reported that lipid peroxidation and its peroxyl products contributed to the epoxidation of polycyclic hydrocarbon metabolites (24, 25). Hughes et al. showed that epoxidation of benzo[a]pyrene 7,8-diol also occurred with AA and γ-LNA in the presence of LO, presumably mediated by a peroxy radical (26). Other enzyme systems which involve epoxidation have been identified. For example, microsomal enzymes epoxidize substrates with diverse structures, including acrolein, a 2

F.-L. Chung and H.-J. C. Chen, unpublished results.

Epoxidation of 4-Hydroxy-2-nonenal

Chem. Res. Toxicol., Vol. 9, No. 1, 1996 311

Figure 2. Standard curve of % yields of 1 vs EH concentration under the same reaction conditions as those studied (r2 ) 0.97).

Figure 4. HPLC chromatograms showing comigration of a radioactive peak (bottom) with a UV standard of 3,4-dihydroxy2-methoxynonanal (2′,4′-dinitrophenyl)hydrazone (top) after derivatization with DNP of the collected fraction corresponding to EH in the reaction with H2O2. Similar results were obtained with LAOOH.

Figure 3. Percentage yields of EH in (a) fatty acid hydroperoxide and (b) H2O2 reactions as estimated based on standard curve in Figure 2.

homolog of HNE, and prostaglandin synthase mediates epoxidation of aflatoxin β and benzo[a]pyrene 7,8-diol (27-31). However, our attempts to use these systems have been unsuccessful. It is possible that HNE readily undergoes Michael addition with protein sulfhydryl groups and consequently inhibits the enzymes at the

concentration used. Alternatively, HNE may not be epoxidized by a peroxyl radical-dependent mechanism which appears to predominate in prostaglandin synthasemediated oxidation (32). It has been reported that HNE is metabolized in subcellular liver fractions or in cell cultures and in vivo to the corresponding alcohol and carboxylic acid, as well as by conjugation with glutathione (33, 34). However, the lipophilic character of HNE may facilitate its transportation through cell membranes, and this process may subject it to epoxidation. Moreover, our initial study showed that EH appeared to be a poor substrate for epoxide hydrolase.2 Ultimately, in order to demonstrate in vivo production of EH, one has to detect either the epoxide or its products with DNA or protein.

312 Chem. Res. Toxicol., Vol. 9, No. 1, 1996

Our results demonstrate that in vitro epoxidation of HNE by cellular oxidants can lead to the formation of etheno adducts. This pathway provides a plausible mechanistic basis for the recent observation of the background EdAdo 3 and maybe other etheno adducts in tissue DNA of humans and untreated rodents (18). However, if the substituted EdAdo 1 is detected in vivo, it is an ultimate proof for the production of EH. We are at present developing a method for the in vivo detection of this adduct. Since this DNA adduct can only be derived from HNE in the presence of peroxides, it could serve as an additional specific indicator of the in vivo oxidative process.

Acknowledgment. We would like to thank Professor Charles Iden in the Department of Chemistry of the State University of New York at Stony Brook for fast atom bombardment mass spectra. We also thank Dr. Ding Jiao for performing some of the 1H- and 13C-NMR spectrometry. This work was supported by Grant CA 43159 from National Cancer Institute.

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