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Articles Detection and Quantification of 1,N6-Ethenoadenine in Human Placental DNA by Mass Spectrometry Hauh-Jyun Candy Chen,*,† Li-Chang Chiang,† Ming-Chung Tseng,† Lei L. Zhang,‡ Jinsong Ni,‡,§ and Fung-Lung Chung*,‡ Department of Chemistry, National Chung Cheng University, 160 San-Hsing, Ming-Hsiung, Chia-Yi 62142, Taiwan, and Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation, Valhalla, New York 10595 Received April 29, 1999
Exocyclic DNA adducts have been reported to derive from various exogenous as well as endogenous sources, such as lipid peroxidation. Among them, 1,N6-ethenoadenine (Ade) has previously been detected in tissue DNA of untreated rodents and humans by an immunoaffinity/ 32P-postlabeling method. This study reports detection and quantification of the endogenous Ade adduct in the same human placental DNA by three independent assays, namely, GC/ MS, LC/MS, and HPLC/fluorescence. Using a recently reported gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method [Chen, H.-J. C., et al. (1998) Chem. Res. Toxicol. 11, 1474], the level of Ade in human placental DNA from a commercial source was found to be 2.3 adducts per 106 Ade bases. To confirm these findings, a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method was developed for dAdo. With this LC/MS assay, dAdo was detected at the level of 2.5 adducts per 106 dAdo nucleosides in the same human placental DNA. The stable isotopes of Ade and dAdo were added as internal standards in both GC/MS and LC/ESI/MS/MS assays, respectively, and thus provided high specificity, reproducibility, and accurate quantification. The relatively high levels of Ade in this human placental DNA detected by mass spectrometry were further verified by HPLC/fluorescence analysis. The GC/MS method was validated by the HPLC/fluorescence assay using calf thymus DNA treated with chloroacetaldehyde or by the LC/MS method with 2,3-epoxy-4-hydroxynonanal-modified calf thymus DNA. The Ade level in human placental DNA freshly isolated in the presence of an antioxidant was similar to that in DNA from the commercial source. Since Ade is a potential mutagenic lesion, analysis of Ade by the specific and sensitive GC/NICI/MS method may provide a useful biomarker in cancer risk assessment.
Introduction There is a growing interest in endogenously generated DNA damage in the multistep carcinogenesis process (14). DNA adducts detected in rodents without carcinogen treatment and in humans may originate from endogenous sources, including lipid peroxidation. Levels of endogenously generated DNA-reactive species appeared to increase with various pathophysiological conditions, such as chronic infections and inflammation (5-7), and thus might contribute to increases in the extent of DNA damage. Among endogenous DNA-reactive compounds, * To whom correspondence should be addressed. H.-J.C.C.: Department of Chemistry, National Chung Cheng University, 160 San Hsing, Ming-Hsiung, Chia-Yi 621, Taiwan; fax, (886) 5-272-1040; e-mail,
[email protected]. F.-L.C.: American Health Foundation, 1 Dana Road, Valhalla, NY 10595; fax, (914) 592-6317; e-mail,
[email protected]. † National Chung Cheng University. ‡ American Health Foundation. § Current address: Wyeth-Ayerst Research, Princeton Corporate Plaza, 9 Deer Park Dr., Building 3, Rm. 150, Monmouth Junction, NJ 08853.
R,β-unsaturated aldehydes, such as malondialdehyde (MDA), acrolein, and trans-4-hydroxy-2-nonenal (HNE),1 are major products of lipid peroxidation known to modify DNA bases by forming exocyclic adducts (8-10). This endogenous DNA damage could cause mismatched base pairing, leading to chromosomal instability and mutation, and thus contribute to cancer development (11). Since lipid peroxidation is implicated in tumorigenesis, it has been postulated that DNA damage caused by these aldehydic products plays an important role in this process (10, 12, 13). Our previous studies showed that HNE can be epoxidized to 2,3-epoxy-4-hydroxynonanal (EH) by biological oxidants and, thus, could contribute to the endogenous formation of the etheno DNA adduct (14-16). 1 Abbreviations: Ade, adenine; BHT, butylated hydroxytoluene; CAA, chloroacetaldehyde; CEO, chloroethylene oxide; DHH-Ade, 7-(1′,2′-dihydroxyheptyl)-3H-imidazo[2,1-i]purine; iPr2EtN, diisopropylethylamine; , etheno; Ade, 1,N6-ethenoadenine; Cyt, 3,N4-ethenocytosine; Gua, N2,3-ethenoguanine; dAdo, 1,N6-etheno-2′-deoxyadenosine; EH, 2,3-epoxy-4-hydroxynonanal; ESI, electrospray ionization;
10.1021/tx990074s CCC: $18.00 © 1999 American Chemical Society Published on Web 11/04/1999
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In addition to endogenous sources, exogenous chemicals known to form etheno adducts include carcinogens such as mucochloric acid, vinyl chloride (VC), ethyl carbamate (urethane), and their metabolites, chloroethylene oxide (CEO) and chloroacetaldehyde (CAA). Mucochloric acid is found in chlorinated drinking water (17). VC is widely used in the plastics industry (18, 19), and urethane is a natural contaminant in fermented foods and alcoholic beverages (20, 21). The relative levels of three etheno adducts, 1,N6-ethenoadenine (Ade), 3,N4ethenocytosine (Cyt), and N2,3-ethenoguanine (Gua), formed in tissue of rats treated with CEO were in the order Ade > Gua > Cyt (22), the same order as observed in in vitro CEO-treated DNA (23). The repair efficiencies of these etheno adducts by human DNA glycosylases are different, with a decreased order of Ade ≈ Cyt . Gua (24, 25). Using the immunoaffinity/32Ppostlabeling method, Ade and Cyt were detected in tissue DNA of VC-treated (22) or urethane-treated animals (27). Recently, Ade, a promutagenic lesion of DNA (28-30), was detected in the urine of CEO-treated rats (31) as well as in untreated rats (32). Because of their potential roles in carcinogenesis, DNA adducts have been used as biomarkers for cancer risk assessment (33, 34). The development of quantitative assays for DNA adducts is important not only for providing risk biomarkers in molecular epidemiology studies but also for understanding mechanisms of carcinogenesis (2, 35-38). Different methods of detection developed for endogenous exocyclic DNA adducts in animals and humans include 1,N2-propanoguanine from acrolein and crotonaldehyde, the MDA-guanine adduct (M1G), Ade, Cyt, and Gua (28, 29, 39-46). 1,N2-Propanoguanine adducts from acrolein and crotonaldehyde were detected by a 32P-postlabeling/HPLC assay (39). M1G has been detected by 32P-postlabeling (40), gas chromatography/ electron capture negative ion chemical ionization mass spectrometry (GC/ECNCI/MS) (42), liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) (43), and immunoslot blot assay (46). Among etheno adducts, Gua was detected by GC/ ECNCI/MS (26) and by LC/ESI/MS/MS (44), whereas Ade and Cyt were both detected by the immunoaffinity/ 32 P-postlabeling assay (30, 45). In this study, we report detection and quantitation of Ade in human placental DNA using a recently developed GC/NICI/MS assay with selective ion monitoring (SIM) (47) and by a new LC/ESI/MS/MS method with selective reaction monitoring (SRM). The levels of the Ade adduct in human placental DNA were found to be higher than those reported previously in other human tissues (45). Because of the unusually high levels of Ade, we further verified our results by HPLC/fluorescence analysis. Our study shows high levels of the Ade adduct in human placental DNA by these assay methods. These results further emphasize the advantage of using stable isotopes of the adduct as internal standards in the mass spectrometry-based assays which provide the specificity and more accurate quantification of DNA adducts in vivo. HNE, trans-4-hydroxy-2-nonenal; MS, mass spectrometry; MS/MS, tandem mass spectrometry; NICI, negative ion chemical ionization; PDA, photodiode array; PFB, pentafluorobenzyl; SIM, selective ion monitoring; SPE, solid-phase extraction; SRM, selective reaction monitoring; VC, vinyl chloride.
Chen et al.
Experimental Procedures Caution: CAA, HNE, and EH are potential mutagens and tumorigens. Precautions should be taken in handling these compounds, e.g., wearing protective gloves, performing experiments under a fume hood, and disposing of waste according to appropriate safety guidelines. Materials. Calf thymus DNA, human placental DNA (D3287, lot no. 67H3912), nuclease P1, phosphodiesterase I, alkaline phosphatase, adenosine deaminase, Ade, and dAdo were purchased from Sigma Chemical Co. (St. Louis, MO). [15N5]Adenosine ([15N5]Ado) triethylamine salt and [15N5]-2′-deoxyadenosine ([15N5]dAdo) were purchased from Cambridge Isotope Laboratories (Andover, MA). CAA, R-bromo-2,3,4,5,6-pentafluorotoluene (PFB-Br), diisopropylethylamine (iPr2EtN), anhydrous methanol, and anhydrous phosphorus pentoxide were obtained from Aldrich Chemical Co. (Milwaukee, WI). HNE was synthesized by a previously described method (48). EH was synthesized from the reaction of HNE with tert-butyl hydroperoxide as previously reported (14). Bond Elut C18 and Si solid-phase extraction (SPE) columns were from Varian (Harbor City, CA). Liquid Chromatography. Characterization and purification of the isotope adducts were performed by photodiode array (PDA) HPLC on a Waters system equipped with two model 510 pumps, a model 660 solvent programmer, a Rheodyne injector, and a Waters 994 programmable photodiode array detector. (1) System 1. A Prodigy ODS (3) 250 mm × 4.6 mm, 5 µm column (Phenomenex, Torrance, CA) was eluted with a linear H20 and CH3CN gradient of 0% CH3CN from 0 to 5 min and 0 to 34% CH3CN from 5 to 40 min at a flow rate of 1.0 mL/min. (2) System 2. A Prodigy ODS (3) 250 mm × 2.0 mm, 5 µm column was used with the following isocratic conditions: 10 mM ammonium acetate (pH 5.5)/0.05% acetic acid in methanol (75/25) at a flow rate of 0.2 mL/min. (3) System 3. A Prodigy ODS (3) 250 mm × 4.6 mm, 5 µm column was eluted with a linear solvent A and B gradient of 0% B from 0 to 5 min and 5 to 50% B from 5 to 60 min [solvent A being 10 mM NaH2PO4 (pH 3.5) and solvent B being methanol] at a flow rate of 1.0 mL/min. (4) System 4. A Prodigy ODS (3) 250 mm × 4.6 mm, 5 µm column was eluted with a linear gradient of 0 to 23% CH3CN from 0 to 29 min at a flow rate of 1.0 mL/min. Synthesis of Isotope Standards. (1) [15N5]EAde. [15N5]Adenosine triethylamine salt (3.61 mg) was heated in 1 N HCl at 110 °C for 2 h to give [15N5]adenine. The pH of the reaction mixture was adjusted to 4.5, and 12 µL of CAA was added. The reaction mixture was heated at 37 °C for 48 h while it was being stirred. [15N5]Ade was collected from 18.5 to 21.0 min using HPLC system 1 according to its UV spectrum and retention time as compared with those of Ade at 19.6 min. (2) [15N5]EdAdo. [15N5]dAdo (4.0 mg) was dissolved in potassium phosphate buffer (0.1 M, pH 6.0) and reacted with CAA (12 µL) at 37 °C for 18 h and collected from 21.2 to 23.0 min by HPLC system 1. [15N5]Ade and [15N5]dAdo were quantitated using the same HPLC system on the basis of the molar UV absorbance of the nonisotope standards, and their identities were further confirmed by LC/ESI/MS/MS. Isolation of Fresh Human Placental DNA. Placental tissue of a 35-year-old Taiwanese woman was obtained after Caesarian operation in the Veteran General Hospital (Taipei, Taiwan). The tissue was immediately frozen in dry ice and stored below -80 °C. DNA isolation was performed 4 days later, based on modified Marmur’s method (49), and 4.5 mM BHT was included in all the buffer solutions. The tissue was homogenized and centrifuged. The pellet was redissolved and digested with proteinase K. After two phenol/chloroform/isoamyl alcohol extraction and one chloroform extraction, DNA was precipitated with sodium acetate and cold ethanol. The precipitated DNA was redissolved and incubated with RNase A and RNase T1, followed by chloroform/isoamyl alcohol extraction. The yield and purity of DNA were determined by measuring the UV absorption at 230, 260, and 280 nm.
Ade in DNA Calibration Curves. (1) GC/NICI/MS. The stock solutions of Ade and [15N5]Ade (1 mg/mL) in methanol were stored at -80 °C. Sample solutions for calibration were freshly prepared by diluting the stock solutions in H2O for each analysis. [15N5]Ade (10.0 ng) was added to each sample as an internal standard. Various amounts of Ade ranging from 0, 10, 50, 100, 200, 400, 600, 800, 1000, 2500, and 5000 pg were added. The samples were pentafluorobenzylated, purified with a Si SPE column, and analyzed by GC/MS (47). (2) LC/ESI/MS/MS. Various amounts of dAdo ranging from 0, 0.2, 0.4, 0.6, 1.0, 2.5, 5.0, 7.5, 10, 15, and 20 ng were added to a solution containing 10 ng of [15N5]dAdo, applied to a methanol-preconditioned C18 SPE column, washed with 9 mL of water, and eluted with 6 mL of 30% methanol. These fractions were combined, evaporated to dryness, reconstituted in 100 µL of water, and analyzed by LC/ESI/MS/MS. (3) HPLC/Fluorescence. Solutions containing 0, 10, 100, 1000, 5000, and 10 000 pg of Ade were hydrolyzed, applied to a C18 SPE column, and analyzed by procedures described in HPLC/Fluorescence Assay. The equations of the calibration curves were obtained by linear regression. Modification of Calf Thymus DNA with CAA or EH. Calf thymus DNA (1.0 mg/mL) was incubated with 20 µM CAA in 0.1 M phosphate buffer (pH 7.0) at 25 °C for 1 h. The DNA reaction mixtures were then extracted with chloroform (2 × 3 mL) to remove unreacted CAA. Calf thymus DNA (1.0 mg/mL) was incubated with 5.8 µM EH in 0.1 M phosphate buffer (pH 7.0) at 37 °C for 50 h. The DNA reaction mixtures were then extracted with chloroform (2 × 3 mL) to remove unreacted EH. Hydrolysis of EdAdo and Stability of EAde in Acid. dAdo (200 µg, 0.57 µmol) was dissolved in 1.0 mL of 0.1 N HCl and heated at 70 °C. An aliquot (100 µL) was removed and analyzed for remaining dAdo (retention time of 22.0 min) and Ade formation (retention time of 19.6 min) by HPLC system 1 after 10, 20, 30, and 45 min. Another dAdo solution was hydrolyzed at 100 °C and analyzed after 30, 45, and 60 min. GC/NICI/MS Analysis of PFB-EAde. GC/NICI/MS spectra were recorded on a Hewlett-Packard model 5988A spectrometer with a Hewlett-Packard 5890 gas chromatograph (Wilmington, DE) or a VG Trio-2000 spectrometer with a Fison 8000 GC (Taiwan Micromass). GC/NICI/MS experiments were performed as previously reported (47). Briefly, [15N5]Ade (10 ng) was added to DNA and hydrolyzed in 0.1 N HCl at 70 °C for 45 min. The DNA hydrolysate was applied to a preconditioned C18 SPE column to enrich it with Ade. The fractions containing Ade were combined, evaporated under a vacuum, dried over phosphorus pentoxide, and derivatized by PFB-Br. The pentafluorobenzylated adduct was purified by a Si SPE. After the eluant was evaporated, the residue was dissolved in 10 µL acetonitrile, and a 1 µL aliquot was analyzed by GC/NICI/MS with SIM at m/z 158 and 163. The quantitation of Ade in the sample was based on the ratio of the peak areas of PFB-Ade to [15N5]PFBAde in the calibration curve. Enzymatic Digestion of DNA and C18 SPE Purification. Fifty nanograms of [15N5]dAdo was added to DNA. Enzymatic digestion of DNA was performed according to a published method (50). For each milligram of DNA in 10 mM ammonium acetate (pH 5.3), 80 units of nuclease P1 was added and the mixture incubated at 45 °C for 2 h. To the reaction mixture was then added phosphodiesterase I (0.08 unit) in 0.1 M Tris buffer (pH 7.4) and the mixture incubated at 37 °C for 2 h, followed by addition of alkaline phosphatase I (20 units) and adenosine deaminase (30 units), and the mixture was incubated for an additional 1 h. The reaction mixture was filtered through a 0.22 µm nylon filter, and the filtrate was purified by C18 SPE. After the volume of the hydrolysate was eluted, the column was washed with 9 mL of water and eluted with 6 mL of 30% methanol. These fractions were combined, evaporated to dryness, reconstituted in 100 µL of water, and analyzed by LC/ ESI/MS/MS. LC/ESI/MS/MS Analysis of EdAdo. Mass spectrometry was performed on a Finnigan TSQ 700 triple-quadrupole mass spectrometer (Finnigan, San Jose, CA) with electrospray ioniza-
Chem. Res. Toxicol., Vol. 12, No. 12, 1999 1121 tion. A voltage of 4.5 kV was applied to the electrospray needle. N2 was used as the sheath gas at a pressure of 65 psi to help nebulization and as the auxiliary gas at a pressure of 20 psi to help desolvation and to stabilize the spray. The stainless steel capillary was heated to 220 °C to obtain optimal desolvation. Full scan data were obtained by scanning from m/z 100 to 600 in 1 s. Argon was used as the collision gas in MS/MS experiments, and the collision voltage was -16 V. To maximize the analyte concentration for optimum electrospray response (52), the splitless mode was used with a small internal diameter LC column (0.2 mm) with a slow flow rate (0.2 mL/min). In selective reaction monitoring (SRM) experiments, precursor [M + H]+ ions for dAdo and [15N5]dAdo were generated in the ESI source and focused in quadrupole 1 (Q1). The ions were dissociated in a collision cell (quadrupole 2), yielding product [M + H]+ ions for Ade and [15N5]Ade which were analyzed in quadrupole 3 (Q3). For dAdo, analyses were performed at m/z 276 and 281 for [15N5]dAdo in Q1 and at m/z 160 and 165 for [15N5]Ade in Q3. The mass spectrometer was interfaced with a Waters 610 liquid chromatography system eluting with LC system 2. Quantification of Ade was accomplished by measuring the ratio of the peak areas of dAdo to Ade (m/z 276 f 160) to [15N5]dAdo to [15N5]Ade (m/z 281 f 165). HPLC/Fluorescence Assay. DNA was hydrolyzed in 0.1 N HCl at 70 °C for 45 min. The mixture was applied to a preconditioned C18 SPE column and washed with 9 mL of H2O. Ade was eluted with 6 mL of 25% methanol. When large amounts of DNA were used, several SPE columns were used so that the limit of 2.5 mg of DNA per column was not exceeded. The fractions were combined, treated with adenosine deaminase (4 units/mg of DNA) at 37 °C and pH 7.0 for 4 h, and filtered through a 0.22 µm nylon filter. The filtrate was dried in a 4 mL silanized vial with a centrifugal concentrator (Pan Chum), reconstituted in 250 µL water, and analyzed. Duplicate aliquots of 100 pL were analyzed with a Hitachi L-7000 pump system with a D-7000 interface, a Rheodyne injector, and an L-7450A photodiode array (PDA) detector with an on-line L-7480 fluorescence detector (Hitachi) using HPLC system 3. The excitation wavelength was 275 nm, and the emission wavelength was 400 nm. Recoveries of Ade in DNA samples were based on the calibration curves that were constructed. The amount of adenine in DNA samples was determined by hydrolysis with 88% formic acid at 100 °C for 2 h, followed by HPLC analysis using system 4. Fluorescence Spectrum of EAde in Human Placental DNA. Human placental DNA (10.0 mg) was hydrolyzed by 0.1 N HCl, and the hydrolysate was applied to a preconditioned C18 SPE column as described previously (47). The fractions containing Ade were collected, dried, and reconstituted in 145 µL of water, and 60 µL was analyzed by HPLC/fluorescence using HPLC system 3 as described above. Ade was collected from 19.0 to 21.0 min, and its fluorescence spectrum was obtained from a Hitachi F-4500 fluorescence spectrometer (Hitachi) with excitation at 275 nm. The spectrum was compared with that of standard Ade (1.5 ng) dissolved in 10 mM NaH2PO4 (pH 3.5).
Results Analysis of EAde by GC/NICI/MS/SIM. The assay was performed as previously reported (47). To reach high specificity and accurate quantification, stable isotope of Ade, i.e., [15N5]Ade, was synthesized and quantified according to the UV extinction coefficient of the nonisotope standard. The purity of [15N5]Ade was 98% (U-15N5), meaning that for each 15N, the purity was at least 98%. The content of 14N5 should be (0.2)5 ) 3.2 × 10-7% or 0.032 fg in 10 ng of [15N5]Ade, which is far below the detection limit of PFB-Ade (10 fg) injected on-column. Furthermore, in samples containing 10 ng of [15N5]Ade only, no peaks corresponding to PFB-Ade at m/z 158 could be detected after pentafluorobenzylation. Prolonged
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Figure 3. Calibration curve for the LC/ESI/MS/MS analysis for dAdo at m/z 276 f 160 and [15N5]dAdo at m/z 281 f 165. Figure 1. Stability of Ade in 0.1 N HCl at 70 ([) and 100 °C (b).
Scheme 1. LC/ESI/MS/MS Analysis of EdAdo in DNA
Figure 2. Calibration curve for the GC/NICI/MS analysis for PFB-Ade at m/z 158 and [15N5]PFB-Ade at m/z 163.
acid (0.1 N HCl) treatment or hydrolysis at higher temperatures leads to substantial decomposition of Ade (Figure 1). The C18 SPE conditions were optimized to remove all the pyrimidine nucleotides not cleaved by acid, almost all of the guanine, and about 88% of the adenine. Most of the remaining adenine was separated from PFBAde after pentafluorobenzylation followed by purification with a Si SPE column. One-tenth of the sample solution was analyzed. The amount of DNA samples can be reduced if all the samples were injected. Levels of Ade in DNA were quantified according to the standard curve, which was linear from 1 pg with a correlation coefficient (γ2) of 0.999 (Figure 2). Analysis of EdAdo by LC/ESI/MS/MS. The assay procedure is outlined in Scheme 1. The stable isotope of the analyte, [15N5]dAdo (50 ng), was added to DNA as an internal standard, and the mixture was hydrolyzed by enzymes. The hydrolysate was purified by C18 SPE columns to enrich it with dAdo, followed by LC/ESI/MS/ MS analysis. The enzyme hydrolysis employed in this assay ensured complete hydrolysis within 5 h at pH 5.3 or 7.4 in which Ade was stable. The detection limit for dAdo was 0.28 ng injected on-column. The calibration curve was linear ranging from 0.6 to 20 ng (Figure 3) with recovery of 40.7 ( 7.0%. Selected reaction monitoring (SRM) was used to monitor fragmentation of dAdo to Ade. Precursor [M + H]+ ion of the nucleoside adduct, dAdo, was generated in the ESI source and focused in quadrupole 1. This ion was dissociated in a collision cell (quadrupole 2) with the loss of a neutral deoxyribosyl fragment of 116 mass units, yielding the defined product
ion, Ade, which was analyzed in quadrupole 3. dAdo was analyzed at m/z 276 f 160 and [15N5]dAdo at m/z 281 f 165. Blank samples with water only were included to ensure no carryover peaks before sample runs. This assay is highly specific to the analyte as a result of the incorporation of stable isotopes and SRM. However, the sensitivity is not comparable with those of the other methods described here, mainly due to the broad LC peak shape. Analysis of EAde by HPLC/Fluorescence. The acidhydrolyzed DNA was purified by C18 SPE, incubated with adenosine deaminase, and analyzed by HPLC/ fluorescence detection at acidic pH. Because of the relatively low sensitivity of the method, a large amount of DNA was used and a number of SPE columns were used to avoid overloading the SPE column so that no more than 2.5 mg of DNA was loaded onto each column. The adduct-enriched fraction was collected and treated with adenosine deaminase for conversion of adenine to the nonfluorescent hypoxanthine. The limit of detection was 500 fg (3.2 fmol). Again, blank samples with water only were included to ensure that no carryover peaks were observed before sample runs. The amount of analyte in DNA samples was obtained by intrapolation of the
Ade in DNA
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Figure 4. Calibration curve for the HPLC/fluorescence assay.
calibration curve with various amounts of Ade ranging from 10 pg to 10 ng (Figure 4). Recovery was 28.5% on average. EAde in DNA Treated with CAA or EH. CAA is a stable metabolite of vinyl chloride, and it is very reactive with respect to DNA, forming etheno adducts (52). Ade levels in DNA treated with CAA were determined by both GC/NICI/MS and HPLC/fluorescence to validate the accuracy in quantification by these two methods. The adduct levels determined by these two methods appeared to be quite similar and reasonably reproducible after adjusting recoveries for the latter method. The Ade level was 1.03 ( 0.01 adducts/103 Ade bases as determined by GC/NICI/MS and 1.44 ( 0.08 adduct/103 Ade bases as determined by HPLC/fluorescence. We previously reported 2,3-epoxy-4-hydroxynonanal (EH) as an epoxidation product of HNE (14, 16). Ade was detected as a minor product in EH-treated calf thymus DNA (47). To determine Ade levels, the same DNA sample was analyzed by LC/ESI/MS/MS. The level of Ade was 1.10 ( 0.11 adducts/103 Ade bases as determined by GC/NICI/MS, and the level of dAdo was 5.25 adducts/104 dAdo bases as determined by LC/ESI/ MS/MS in EH-treated DNA. EAde in Human Placental DNA. Ade in human placental DNA obtained from a commercial source was assessed by all three methods described in this study, GC/NICI/MS, LC/ESI/MS/MS, and HPLC/fluorescence. A 2.5 mg sample of DNA analyzed by GC/NICI/MS contained 2.32 ( 0.02 Ade per 106 Ade bases (Figure 5). Due to the low sensitivity of LC/ESI/MS/MS, analysis of 2.5 mg of DNA showed a dAdo peak that was too small to be quantified. Using 4.4, 5.4, and 25.1 mg of human placental DNA, the dAdo levels in these samples were 2.76, 2.32, and 2.35 adducts/106 dAdo nucleosides, respectively, as analyzed by LC/ESI/MS/MS (Figure 6). The dAdo eluted at 10.3 min. The identity of the peak at 6.0 min, also seen in EH-treated DNA, was not known. Finally, the Ade level was verified by HPLC/fluorescence using 2.0 mg of DNA, giving 2.77 ( 0.24 adducts/106 Ade bases. The identity of the peak was confirmed by coinjection with standard Ade and by comparison of the fluorescence spectrum of Ade in human placental DNA hydrolysate collected from HPLC with that of standard Ade (not shown). To exclude the possibility that the high level of Ade was due to phenol/chloroform extraction during DNA isolation procedures and/or lipid peroxidation due to improper storage of tissue, another human
Figure 5. GC/NICI/MS analysis with selective ion monitoring chromatogram of PFB-Ade in 2.5 mg of human placental DNA. The peak at 19.76 min represents 0.16 pmol (55 pg) of PFBAde. One-tenth of entire sample was injected.
Figure 6. Selected reaction monitoring (SRM) LC/ES/MS/MS analysis of dAdo at m/z 276 f 160 and [15N5]dAdo at m/z 281 f 165 in 25.1 mg of human placental DNA. The peak at 10.25 min represents 14.5 pmol (4.00 ng) of dAdo.
placental DNA was freshly obtained and isolated in the presence of butylated hydroxytoluene (BHT). The donor was a 35-year-old nonsmoking Taiwanese woman in healthy condition without diabetes, hypertension, or other systemic diseases. The level of Ade in this placental tissue was determined to be 2.63 adducts/106 Ade bases by HPLC/fluorescence using 2.0 mg of DNA (Figure 7). The peaks eluting at 9.4 and 11.2 min were guanine and adenine, respectively. The peak at 18.9 min represented 204 pg of Ade and was confirmed by co-injection
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Figure 7. HPLC/fluorescence detection of Ade in 2.0 mg of fresh human placental DNA. The peak at 18.9 min represents 1.28 pmol (204 pg) of Ade. Table 1 sample
Ade/Ade
internal standards only NDa [[15N5]Ade (10 ng)] internal standards only NDa [[15N5]dAdo (50 ng)] human placental DNAb,c 2.32 ( 0.02/106 human placental DNAb,d 2.48 ( 0.25/106 human placental DNAb,e 2.77 ( 0.24/106 human placental DNAf,g 2.63 ( 0.12/106 CAA-treated calf thymus 1.03 ( 0.01 /103 DNAh,i CAA-treated calf thymus 1.44 ( 0.08 /103 DNAh,j EH-treated calf thymus 1.10 ( 0.11/103 DNAk,l EH-treated calf thymus 5.25/104 DNAk,m
method GC/MS LC/MS/MS GC/MS LC/MS/MS HPLC/fluorescence HPLC/fluorescence GC/MS HPLC/fluorescence GC/MS LC/MS/MS
a ND, not detectable. b From Sigma (catalog no. D3287, lot no. 67H3912). c Mean and standard deviation from duplicate experiments with 2.5 mg of DNA. d Mean and standard deviation from triplicate experiments with 4.4, 5.4, and 25.1 mg of DNA. e Mean and standard deviation from duplicate experiments with 4.1 mg of DNA. f Freshly isolated (see the text for details). g Mean and standard deviation from duplicate experiments with 2.0 mg of DNA. h CAA (20 µM) at 25 °C and pH 7.0 for 1 h. i Mean and standard deviation from duplicate experiments with 0.25 µg of DNA. j Mean and standard deviation from duplicate experiments with 1.25 µg of DNA. k EH (5.8 µM) at 37 °C and pH 7.0 for 50 h. l Mean and standard deviation from duplicate experiments with 0.1 mg of DNA. m Single experiment with 0.25 mg of DNA.
with 200 pg of standard Ade. The identity of the peak at 17.0 min was not known. Results of these measurements are summarized in Table 1.
Discussion Using a GC/NICI/MS/SIM method we recently described (47), Ade was detected in human placental DNA and its level in DNA appeared to be several adducts per 106 Ade bases. These levels were much higher (102-103fold) than those previously reported in livers of rodents and humans by an immunoaffinity/32P-postlabeling method (30, 45). This observation led us to investigate the validity of this assay with other methods. Using a newly developed LC/ESI/MS/MS method, which involves enzymatic hydrolysis under mild conditions and without chemical derivatization, dAdo was detected at levels comparable to those obtained from the GC/MS assay. The important features of these two assays are the addition of the stable isotopes of the adducts analyzed for monitoring the recovery of each step and the use of disposable C18 solidphase extraction (SPE) chromatography for obtaining fractions enriched with the adducts. Internal standards
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that were added also serve as carriers for the ultratrace amount of endogenous adducts present in tissue DNA, which can be lost substantially during multistep procedures. The high Ade levels in human placental DNA were confirmed by a HPLC/fluorescence assay. Quantification of these three assays was obtained by intrapolation of the calibration curves constructed from various amounts of standards going through the entire assay procedures. The isotopic [15N5]Ade and [15N5]dAdo were quantified by HPLC using the molar UV absorbance of commercially available Ade and dAdo, respectively. Positive control experiments with isotopes only were carried out to ensure the purity of isotopes and to ensure that the assay systems were free of contamination. Solvent blank runs were performed before each run of biological samples. The electron rich pentafluorobenzyl moiety of PFBAde gives very high molar response in electron capture NICI/MS, and provides high sensitivity. The detection limit for the entire assay was 1 pg (47). Since this isotope is chemically identical to the analyte, it monitors the reaction and recovery of each step in the entire procedure. The addition of internal standard before acid hydrolysis of DNA is especially important since Ade is acid labile. The possible discrepancies in levels of DNA adduct measured by different methodologies have been reported and discussed (2, 53, 54). The hydrolysis, workup, derivatization, and purification procedures, the stability of the analyte, the availability of appropriate internal standards, and the limits of detection are important factors in quantification of DNA adducts. The 32Ppostlabeling assay is probably one of the most sensitive methods available; however, it lacks proper internal standards for accurate quantification. Immunoaffinity chromatography for adduct enrichment can be poorly reproducible due to batch-to-batch preparation of antibody and the age of antibody (32). In the immunoaffinity/ 32P-postlabeling technique, variation in sample recovery between columns or between experiments can result in significant quantitative discrepancy. This is especially important since a very small amount of analyte can be easily lost in the multistep procedures. Besides, since Ade is acid sensitive, its degradation in steps involving acidic conditions such as development on polyethylene cellulose TLC plates at pH 3.5 in the 32P-postlabeling assay should be cautioned. In our isotope dilution mass spectrometry, stable isotope internal standards, which exist in the nanogram quantities, can serve as carriers for trace amounts of analytes. The analytes can be accurately quantified from the ratio to the internal standards since their chemical behaviors are virtually identical. The reproducibility in isotope dilution mass spectrometric methods is also much higher compared to those of other assays mentioned above. Mass spectrometry also provides the chemical identity of the analytes and offers advantage in characterization of DNA adducts that may be present in the complex mixture of DNA hydrolysates. In this study, Ade was detected in human placental DNA and its level was determined by three independent assay methods, GC/NICI/MS, LC/ESI/MS/MS, and HPLC/ fluorescence. The levels were in the range of 2-4 adducts per 106 Ade bases. Levels of M1G were found to be 9 adducts per 107 bases and 6 adducts per 108 bases in human liver and leukocyte DNA, respectively (41, 42). Gua in human liver DNA was at a level of 2 adducts
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per 108 bases (0.06 pmol/mg of DNA) by an immunoaffinity-LC/ESI/MS assay (32). The limit of detection of and that of Gua by LC/ESI/MS were 270 fmol (43 pg) and 50 fmol (8.8 pg) (32, 44), respectively. In our LC/ESI/MS/ MS method, the limit of detection of dAdo was relatively high (2.2 pmol or 0.6 ng) compared with that for Ade in the immunoaffinity-LC/ESI/MS assay (32). Although tandem mass spectrometry is highly specific, sensitivity of the assay is sacrificed due to fragmentation of the parent ion dAdo to the daughter ion Ade. Therefore, our LC/ESI/MS/MS method is useful in adduct characterization and quantification, but it is not practical in analyzing in vivo samples with low adduct levels when only milligram quantities of DNA samples are available. On the other hand, our GC/MS method for Ade is at least 1000-fold more sensitive than our LC/MS assay and is thus appropriate for analyzing large numbers of samples in epidemiological studies. The levels of Ade in human placental DNA we found were quite high. Although Ade can be formed from chemicals in the environment and as food contaminants (35, 36), endogenous sources of this adduct might be even more important. The detection of HNE as a local product of placental metabolism (55), the high lipid peroxidation index, and lower glutathione levels in placenta (56) all indicate that human placenta isolated after pregnancy is a tissue with high oxidative stress. Evidence suggests that pre-eclampsia in the third trimester is associated with decreased placental antioxidant enzyme protection leading to uncontrolled lipid peroxidation in placenta (57). Although human glycosylases isolated from placenta are capable of repairing Ade (58, 59), with the high oxidative stress, the steady state level of Ade can still be high. These might explain our finding of high levels of Ade in human placental DNA. With three different assay methods, including the highly specific mass spectrometry, the levels of Ade are in good agreement. Our results suggest that the GC/NICI/MS assay is a useful tool in quantification of Ade in biological samples.
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Acknowledgment. This work was supported by Grants NSC 87-2119-M-194-009 (to H.-J.C.C.) and NSC 88-2113-M-194-010 (to H.-J.C.C.) from the National Science Council of Taiwan, Grant 86-NEW-07 (to H.J.C.C.) from National Chung Cheng University, and Grant CA 43159 from the National Cancer Institute (to F.-L.C.). We thank Dr. Der-Cherng Tarng of Veterans General Hospital at Taipei for help in obtaining fresh human placental tissue and Professor Kuen-Yu Wu of Chinese Medical College, Taiwan, for help in DNA isolation.
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