Chem. Res. Toxicol. 2000, 13, 1243-1250
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4-Hydroxy-2-nonenal and Ethyl Linoleate Form under Peroxidizing Conditions
N2,3-Ethenoguanine
Amy-Joan L. Ham,† Asoka Ranasinghe,‡ Hasan Koc,‡ and James A. Swenberg*,†,‡ Departments of Pathology and Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599 Received May 18, 2000
In these studies, we demonstrate that N2,3-ethenoguanine (N2,3-Gua) is formed from lipid peroxidation as well as other oxidative reactions. Ethyl linoleate (EtLA) or 4-hydroxy-2-nonenal (HNE) was reacted with dGuo in the presence of tert-butyl hydroperoxide (t-BuOOH) for 72 h at 50 °C. The resulting N2,3-Gua was characterized by liquid chromatography/electrospray mass spectroscopy and by gas chromatography/high-resolution mass spectral (GC/HRMS) analysis of its pentafluorobenzyl derivative following immunoaffinity chromatography purification. The amounts of N2,3-Gua formed were 825 ( 20 and 1720 ( 50 N2,3-Gua adducts/106 normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increases in the amount of N2,3-Gua compared to controls containing only t-BuOOH. Controls containing t-BuOOH but no lipid resulted in a >1000-fold increase in the level of N2,3-Gua over dGuo that was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reacted with calf thymus DNA at 37 °C for 89 h. The amounts of N2,3-Gua formed in intact ctDNA were 114 ( 32 and 52.9 ( 16.7 N2,3-Gua adducts/106 normal dGuo bases for EtLA and HNE, respectively. These compared to 2.02 ( 0.17 and 2.05 ( 0.47 N2,3-Gua adducts/106 normal dGuo bases in control DNA incubated with t-BuOOH, but no lipid. [13C18]EtLA was reacted with dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. These reactions resulted in a 89-93% level of incorporation of the 13C label into N2,3-Gua when EtLA and dGuo were in equimolar concentrations, when EtLA was in 10-fold molar excess, and when deoxyribose (thymidine) was in 10-fold molar excess. Similar reactions with ctDNA resulted in an 86% level of incorporation of the 13C label. These data demonstrate that N2,3Gua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The data also suggest, however, that N2,3-Gua is also formed by an alternative mechanism that involves some other oxidative reaction which remains unclear.
Introduction N2,3-Ethenoguanine
(N2,3-Gua,1 Figure 1) is a highly mutagenic adduct formed from a number of carcinogens, including vinyl chloride (1, 2). More recently, N2,3-Gua has been discovered in unexposed tissues of mice, rats, and humans (3). Fedtke et al. (4) originally reported that N2,3-Gua appeared to have a very long half-life in hepatic DNA following vinyl chloride exposure (>30 days). On the basis of the discovery of endogenous N2,3Gua, it is now believed that the half-life of N2,3-Gua could not be calculated accurately after exposure to vinyl chloride because baseline levels of endogenous N2,3-Gua (3) were being measured. The importance of etheno adducts (Figure 1) in carcinogenesis stems from both their formation by a number * To whom correspondence should be addressed: Department of Environmental Sciences and Engineering, CB #7400, University of North Carolina, Chapel Hill, NC 27599-7400. Telephone: (919) 9666139. Fax: (919) 966-6123. † Department of Pathology. ‡ Department of Environmental Sciences and Engineering. 1 Abbreviations: N2,3-Gua, N2,3-ethenoguanine; N2,3-Guo, N2,3ethenoguanosine; N2,3-dGuo, N2,3-ethenodeoxyguanosine; Ade, 1,N6ethenoadenine; Cyt, 3,N4-ethenocytosine; dGuo, deoxyguanosine; EtLA, ethyl linoleate; HNE, 4-hydroxy-2-nonenal; BHT, butylated hydroxytoluene; IA, immunoaffinity; EI, electron ionization; CI, chemical ionization; ctDNA, calf thymus DNA; t-BuOOH, tert-butyl hydroperoxide; SIM, selected ion monitoring; PFB, pentafluorobenzyl; PFK, perfluorokerosene; M1G, pyrimido[1,2-a]purine-10(3H)-one.
Figure 1. Structures of etheno adducts.
of carcinogens and their demonstrated mutagenicity. All four etheno adducts have demonstrated miscoding potential in mutagenicity studies (2, 5-10). These studies demonstrated that Ade caused A to G transitions, Cyt caused C to A tranversions and C to T transitions, N2,3Gua caused G to A transitions, and 1,N2-Gua caused
10.1021/tx0001124 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/14/2000
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both G to A transitions and G to T transversions. These mutation studies were consistent with the mutations observed in tumors from vinyl chloride-exposed humans and rats. In five of six human liver angiosarcomas associated with vinyl chloride exposure, GC to AT transitions were observed at codon 13 of c-Ki-ras-2 gene (11). Tumors from vinyl chloride-exposed rats showed G to A transitions, A to C transversions, and C to T transitions in N-ras genes (12). Additionally, angiosarcomas from rats exposed to vinyl chloride demonstrated p53 mutations which resulted in G to A transitions as well as A to T transversions (13). All of these mutations observed in vinyl chloride-induced tumors are consistent with mutations caused by etheno adducts, suggesting an important role for these adducts in carcinogenesis. The occurrence of DNA adducts from endogenous sources has become increasingly apparent. Many of these adducts have been postulated to occur from the reaction of DNA with products of lipid peroxidation (14-20). The etheno adducts, 1,N2-ethenoguanine (1,N2-Gua) and 1,N6-ethenoadenine (Ade) (Figure 1), are formed from 2,3-epoxy-4-hydroxynonanal, the epoxide of the lipid peroxidation product trans-4-hydroxy-2-nonenal (21-24). Although these etheno adducts are formed from products of lipid peroxidation, there has been no direct evidence to show that N2,3-Gua is formed from lipid peroxidation as well. In this study, we have used a recently developed GC/HRMS method for N2,3-Gua using enrichment by immunoaffinity chromatography to determine if N2,3Gua is formed from the reaction of 4-hydroxy-2-nonenal (HNE) or ethyl linoleate (EtLA) with either deoxyguanosine (dGuo) or calf thymus DNA (ctDNA) under peroxidizing conditions. In addition, we have reacted dGuo and ctDNA with [13C18]ethyl linoleate under peroxidizing conditions to determine the extent that lipid peroxidation products form N2,3-Gua by direct alkylation by monitoring the incorporation of the 13C-labeled stable isotope into N2,3-Gua.
Experimental Procedures Materials. Spleen phosphodiesterase, alkaline phosphatase, and DNase I were purchased from Sigma Chemical Co. (St. Louis, MO). Snake venom phosphodiesterase was purchased from Worthington Biochemical (Freehold, NJ). Protein A Sepharose was purchased from Pharmacia Biotech (Piscataway, NJ). Disposable polystyrene columns were purchased from Pierce (Rockford, IL). Pentafluorobenzyl bromide was obtained from Aldrich Chemical (Milwaukee, WI). Calf thymus DNA, 2′deoxyguanosine, 4-hydroxy-2-nonenal, and tert-butyl hydroperoxide (t-BuOOH) were purchased from Sigma Chemical Co. Ethyl linoleate was obtained from NuChek Prep, Inc. (Elysian, MN). [13C18]Ethyl linoleate (>98%) was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA). All other chemicals were obtained commercially and were of the highest purity available. Centricon 10 filters were obtained from Amicon, Inc. (Beverly, MA). N2,3-Gua was synthesized by the method of Sattsangi et al. (25). Stable isotope internal standards of N2,3-Gua were synthesized by first preparing isotopically labeled guanine which was synthesized by the method of Scheller et al. (26) with the modification that for [13C4,15N2]N2,3-Gua, K13C15N and Na15NO2 were used to label the N-7 and N-9 nitrogens. Isotopically labeled N2,3-Gua was then synthesized by first protecting labeled Gua with a benzyl group at the O6 position using a two-step procedure involving acetylation at N2 and N9, according to the method of Zou and Robins (27), followed by a Mitsunobu reaction, according to the method of Khazanchi et al. (28). The etheno bridge of N2,3-Gua was then formed according to the method of Sattsangi et al. (25).
Ham et al. Reaction of trans-4-Hydroxy-2-nonenal, Ethyl Linoleate, and [13C18]Ethyl Linoleate with dGuo and ctDNA. The reaction of 4-hydroxy-2-nonenal and ethyl linoleate with dGuo was carried out according to the methods of Sodum and Chung (22) with minor modifications. One hundred micrograms (0.64 µmol) of trans-4-hydroxy-2-nonenal (HNE, 10 µL, 10 mg/mL in hexane) or 10 mg (32.4 µmol) of ethyl linoleate (EtLA) (100 µL, 100 mg/mL in methanol) was added to 0.2 µmol of dGuo in 0.5 mL of 50 mM sodium phosphate buffer (pH 7.4). After the addition of 10 µL of t-BuOOH (70% solution, 70 µmol), the mixture was incubated with constant mixing at 50 °C for 72 h. After the addition of 1 mL of water and 10 µL of 2% butylated hydroxytoluene (BHT) in 2-propanol (w/v) to both reaction mixtures and 0.5 mL of methanol to the EtLA reaction mixture, the samples were extracted with 1 mL of chloroform. The aqueous layer was dried under vacuum and resuspended in 3 mL of phosphate-buffered saline (pH 7.6) and stored at -80 °C until analysis was carried out. The reaction of [13C18]EtLA with dGuo was the same as described above except 10-fold less (10fold excess lipid) or 100-fold less (equimolar and dGuo) EtLA was used and the reaction was carried out for 24 h instead of 72 h. The reaction of ctDNA with HNE and EtLA was essentially the same as for dGuo (using 10-fold molar excess lipid for the [13C18]EtLA experiments) except that 300 µg of DNA in 1 mL of buffer was used (roughly equivalent to 0.2 µmol of dGuo) and the reaction was carried out at 37 °C for 89 h (24 h for the [13C18]EtLA experiments). The samples were then extracted two times with 2 mL of chloroform containing 0.5% BHT (w/v); the aqueous layer was dried under vacuum, and samples were resuspended in 1 mL of water. The EtLA samples were then extracted two additional times with a 2:1 chloroform (with BHT)/methanol mixture, and the aqueous layer was dried under vacuum and resuspended in 1 mL of water. Samples were left at 4 °C overnight to rehydrate DNA and stored at -80 °C until analysis was carried out. The resulting ctDNA was separated from depurinated product prior to analysis by Centricon 10 filtration. LC/MS of a Reaction Mixture from the Reaction of EtLA with dGuo. A Magic 2002 HPLC system (Michrom, Auburn, CA), equipped with a 718AL autosampler (Alcott Chromatography, Inc., Norcross, GA) and a Rheodyne injector, was connected to a TSQ 7000 triple mass spectrometer (ThermoQuest, San Jose, CA) with a diversion valve placed between the liquid chromatograph and the electrospray source. An Alltech (Alltech Associates, Inc., Deerfield, IL) Platinum EPS C18, 5 µm, 100 Å, 4.6 mm × 250 mm column was run at a flow rate of 0.7 mL/min with the following gradient: from 0 to 20 min, isocratic 90:10 water/methanol; from 20 to 40 min, linear gradient from 90:10 to 50:50 water/methanol; and from 40 to 65 min, isocratic 50:50 water/methanol. By using the diversion valve, only the 25-40 min portion of the LC effluent was allowed into the ESI source. The rest of the effluent was directed to the waste. Nitrogen was used as the auxiliary (40 psi) and sheath gas (80 psi). Electrospray and SRM parameters were optimized for maximum sensitivity using 5 µL loop injections of the N2,3dGuo standard (24 µM). The MS/MS detection was carried out in the selected reaction monitoring (SRM) mode. SRM transitions for N2,3-dGuo, N2,3-Gua, and [13C4,15N2]-N2,3-Gua (internal standard) were from m/z 292 to 176, 176 to 81, and 182 to 84, respectively. SRM transitions for 1,N2-dGuo, 1,N2Gua, and [13C3]-1,N2-Gua (internal standard) were from m/z 292 to 176, 176 to 148, and 182 to 150, respectively. Collisioninduced dissociation was performed in the presence of argon at about 2.5 × 10-3 mbar. The collision energy was set to 25 V. Data acquisition and processing were performed using Xcalibur version 1.0 software running under the Microsoft NT 4.0 operating system. Quantitation and GC/HRMS of the Pentafluorobenzyl Derivative of N2,3-EGua. The resulting product from the dGuo or ctDNA reactions described above was quantitated by immunoaffinity chromatography (IA) followed by GC/HRMS (IA/GC/ HRMS) by the method of Ham et al. (29), using enzymatic digestion for ctDNA. The quantitative analyses and full scan
Lipid Peroxidation Forms N2,3-Ethenoguanine
Chem. Res. Toxicol., Vol. 13, No. 12, 2000 1245
Figure 3. Chromatogram resulting from the LC/ESI-MS/MS analysis of the reaction between EtLA and dGuo using selected reaction monitoring. Stable isotopically labeled internal standards for N2,3-Gua ([13C4,15N2]-N2,3-Gua) and 1,N2-Gua ([13C3]-1,N2-Gua) were added to the reaction mixture prior to analysis. Figure 2. Full scan mass spectral analysis of the pentafluorobenzyl derivative of the product from the reaction of HNE with deoxyguanosine. The product was isolated by immunoaffinity chromatography prior to derivatization and mass spectral analysis. The top panel is the mass spectrum using positive electron impact mode, and the bottom panel is the mass spectrum using negative chemical ionization mode. mass analyses of the resulting pentafluorobenzyl derivatives were performed on an HP 5890 gas chromatograph interfaced with a VG70-250SEQ mass spectrometer at mass resolving powers of 10 000 and 1000 (full scan), respectively. A DB-5MS fused silica capillary column (30 m × 0.32 mm, 0.1 µm thickness; J&W Scientific) with a helium (Sunox, 99.999% pure) head pressure of 10 psi was used in all experiments. The samples were introduced using the direct injection mode utilizing Restek Uniliner 20355. The injection port temperature was 290 °C, and the initial column temperature was 100 °C for 30 s, increasing at a rate of 25 °C/min to 150 °C and at a rate of 15 °C/min to 300 °C, and held for 3 min. Selected ion monitoring (SIM) experiments at a mass resolving power of 10 000 were performed by monitoring the fragment ions, (M - PFB)- [m/z 354.0413 (N2,3-Gua), 356.0481 ([13C2]-N2,3-Gua), 358.0549 (13C4-labeled internal standard), and 360.0489 (13C4- and 15N2-labeled internal standard)]. The perfluorokerosene (PFK) lock mass and the dwell time were 354.9792 amu and 50 ms/ion, respectively. The methane (Airco, 99.99% pure) pressure was adjusted to give an ion gauge reading of 3 × 10-5 mbar. The CI slit in the EI/CI source was selected, and the instrument was tuned using the PFK mass (m/z 293) under CI negative ion mode. The electron energy and emission current were also optimized such that the highest ion abundance of m/z 293 from PFK (PCR Inc.) was displayed on the oscilloscope. After the tuning, the electron energy and emission current ranged between 60 and 100 eV and between 0.5 and 1.0 mA, respectively. EI full scan mass spectra were obtained using the EI slit and with an electron energy of 70 eV. The ion source temperature was set at 250 °C during all the experiments.
Results The reaction mixtures resulting from the reaction of dGuo with EtLA or HNE were purified by IA chromatography, hydrolyzed to the nucleobase, and derivatized with pentafluorobenzyl bromide. The resulting derivative (PFB2-N2,3-Gua) was subjected to positive EI and negative CI full scan mass spectral analysis. The resulting full scan spectra are shown in Figure 2. The positive EI analysis resulted in peaks at m/z 535, 516, and 181
that corresponded with the molecular ion (M+), a molecular ion minus a fluorine (M - F), and a PFB fragment, respectively, consistent with that of the PFB2-N2,3-Gua standard. Additionally, negative CI analysis resulted in a mass spectral peak at m/z 354, consistent with a (M PFB) fragment. These data matched those of a synthetic standard for PFB2-N2,3-Gua as well as previously published data (30). The reaction mixture dGuo with EtLA was also analyzed by LC/ESI-MS using selected reaction monitoring (SRM), to confirm the GC/MS results. The resulting chromatogram is pictured in Figure 3. From this experiment, we were able to confirm that N2,3-Gua, 1,N2-Gua, and 1,N2-ethenodeoxyguanosine (1,N2-dGuo) were formed. 1,N2-dGuo was analyzed by monitoring the transition from m/z 292 to 176. The respective nucleobases, N2,3Gua and 1,N2-Gua, were analyzed by monitoring the transitions from m/z 176 to 81 and from m/z 176 to 148, respectively. The presence of N2,3-Gua and 1,N2-Gua was confirmed by the addition of authentic internal standards ([13C4,15N2]-N2,3-Gua and [13C3]-1,N2-Gua) that coeluted with the identified N2,3-Gua and 1,N2Gua peaks, respectively (Figure 3). The amounts of N2,3Gua formed were approximately 10-fold lower than the amount of 1,N2-Gua and 1,N2-dGuo combined (estimated on the basis of the relative response to the internal standards [13C4,15N2]-N2,3-Gua and [13C3]-1,N2-Gua). Further analysis of the reactions of dGuo with HNE and EtLA was achieved by IA/GC/HRMS of the pentafluorobenzyl derivative of N2,3-Gua, which utilized IA chromatography and selected ion monitoring mass spectroscopy with a resolving power of 10 000 based on a previously described method (29). These methods allow for a very high selectivity, thereby increasing the confidence in the formation of N2,3-Gua. It is also important to note that the antibodies used for the IA chromatography do not cross react with 1,N2-Gua and that the pentafluorobenzyl derivative of 1,N2-Gua can be separated from N2,3-Gua by GC under the conditions used for analysis (data not shown). A representative chromatogram after the reaction of dGuo with HNE is shown in Figure 4. In addition to confirmation of the presence of N2,3Gua, the IA/GC/HRMS method allowed us to quantitate
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Ham et al. Table 1. N2,3-Ethenoguanine Formed from the Reaction of EtLA and HNE with dGuo or ctDNA under Peroxidizing Conditions EtLA reaction conditionsb (N2,3-Gua adducts/ 106 dGuo bases)
Figure 4. Representative GC/HRMS chromatogram of N2,3Gua resulting from the reaction of HNE with deoxyguanosine.
N2,3-Gua
the amount of formed in these reaction mixtures. The results for the formation of N2,3-Gua from reactions of dGuo with either HNE or EtLA are shown in Table 1. The amount of EtLA used in these reactions was greater than the amount of HNE since it was hypothesized that the epoxide of HNE was the ultimate reactant and this would have to be formed from EtLA in a low yield. The reaction of 200 nmol of dGuo with EtLA in the presence of t-BuOOH resulted in the generation of 825 ( 20 N2,3-Gua adducts/106 normal dGuo bases. The same reaction with HNE resulted in the generation of 1720 ( 50 N2,3-Gua adducts/106 normal dGuo bases. This corresponded to yields of 0.08 and 0.17%, based on deoxyguanonsine, for EtLA and HNE, respectively. Additionally, the control reaction of dGuo with t-BuOOH but no lipid resulted in the formation of 21 ( 5 and 21 ( 6 N2,3-Gua adducts/106 normal dGuo bases for the reaction conditions for EtLA and HNE, respectively. These numbers for control incubations with t-BuOOH corresponded to a 0.002% yield. Unreacted dGuo was also analyzed for the presence of N2,3-Gua, and although a small amount was detected, this number was below the limit of quantitation and it was at least 1000-fold lower than that in the control incubations containing t-BuOOH. Similar reactions were performed using ctDNA instead of dGuo. The results of these experiments are also shown in Table 1. The amounts of N2,3-Gua in ctDNA after reaction with EtLA and HNE were 114 ( 32 and 52 ( 17 N2,3-Gua adducts/106 normal dGuo bases, from EtLA and HNE, respectively. These numbers corresponded to 0.01 and 0.005% yields, based on deoxyguanosine, from the EtLA and HNE reactions, respectively. The reactions with EtLA and HNE resulted in 56- and 25-fold increases in the level of N2,3-Gua, respectively, over the control reactions of dGuo with t-BuOOH but no lipid. These control incubations corresponded to 2.02 ( 0.17 and 2.05 ( 0.47 N2,3-Gua adducts/106 normal dGuo bases using the reaction conditions for EtLA and HNE, respectively. Additionally, N2,3-Gua was detected in ctDNA that was either not incubated or incubated but not exposed to either lipid or t-BuOOH. The amount of N2,3-Gua in control DNA that was incubated but not exposed to t-BuOOH was 2-4-fold lower than the amount of DNA incubated with t-BuOOH only. The amount of N2,3-Gua in control DNA that was not incubated (0.26 ( 0.05 N2,3Gua adduct/106 normal dGuo bases) was approximately 20-fold lower than the amount of N2,3-Gua detected in control incubations in the presence of t-BuOOH. These control levels of N2,3-Gua in ctDNA were very high in
deoxyguanosinea lipid and t-BuOOH t-BuOOH only (no lipid) control (no incubation) ctDNAa lipid and t-BuOOH t-BuOOH only (no lipid) control (incubation only) control (no incubation)
HNE reaction conditionsb (N2,3-Gua adducts/ 106 dGuo bases)
825 ( 20c 21 ( 5
