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National Institute for Occupational Health Safety, Centers for Disease Control and Prevention,. Morgantown, West Virginia 26505. Received March 7, 200...
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High-Performance Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantitation of Benzo[a]pyrene-DNA Adducts Frederick A. Beland,*,† Mona I. Churchwell,† Linda S. Von Tungeln,† Shoujun Chen,† Peter P. Fu,† Sandra J. Culp,† Bernadette Schoket,‡ Erika Gyo _rffy,‡ Ja´nos Mina´rovits,# Miriam C. Poirier,§ Elise D. Bowman,§ Ainsley Weston,| and Daniel R. Doerge† Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, National Institute of Environmental Health, Jo´ zsef Fodor National Center for Public Health, Budapest, H-1097, Hungary, Be´ la Johan National Center for Epidemiology, Budapest, H-1529, Hungary, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, and National Institute for Occupational Health Safety, Centers for Disease Control and Prevention, Morgantown, West Virginia 26505 Received March 7, 2005

A method, using HPLC combined with electrospray tandem mass spectrometry (ES-MS/ MS), was developed and validated to detect and quantify the major DNA adduct resulting from exposure to the ultimate tumorigenic benzo[a]pyrene (BP) metabolite, trans-7,8-dihydroxyanti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Calf thymus DNA was reacted with BPDE, digested enzymatically to nucleosides, and the major DNA adduct, 10-(deoxyguanosinN2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE), was purified by HPLC. Similar procedures were applied to prepare dG-BPDE-d8 from [1,2,3,4,5,6,11,12-2H8]BPDE for use as an internal standard. The HPLC-ES-MS/MS method was validated using a mixture of hydrolyzed salmon testis DNA (82 µg) and 10 pg dG-BPDE (analogous to 6.9 adducts/108 nucleotides). The results indicated an inter- and intraday accuracy of 99-100% and precision of 1.6-1.7% (relative standard deviation). When applied to a calf thymus DNA sample modified in vitro with [1,3-3H]BPDE, the method gave a value very similar to those obtained by radiolabeling, 32P-postlabeling, and immunoassay. HPLC-ES-MS/MS analysis of hepatic DNA from mice treated intraperitoneally with 0.5 and 1.0 mg of [7,8-3H]BP gave values comparable to those determined by 32P-postlabeling and immunoassay. Lung DNA from mice fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for one month had 0.6 ( 0.04 dGBPDE adducts/108 nucleotides. This value is much lower than the 102 ( 14 total DNA adducts/ 108 nucleotides determined by 32P-postlabeling, which suggests that dG-BPDE makes only a minor contribution to the DNA adducts formed in lung tissue of mice administered coal tar. The HPLC-ES-MS/MS method was used to assess human lung DNA samples for the presence of dG-BPDE. Based upon a limit of detection of 0.3 dG-BPDE adducts/108 nucleotides, when using 100 µg of DNA, dG-BPDE was detected in only 1 out of 26 samples. These observations indicate that HPLC-ES-MS/MS is suitable to assess the contribution of BP to DNA damage caused by exposures to polycyclic aromatic hydrocarbon (PAH) mixtures. The results further suggest that dG-BPDE may contribute only a small fraction of the total DNA adducts detected by other DNA adduct methodologies in individuals exposed to PAHs.

Introduction Benzo[a]pyrene (BP)1 is a polycyclic aromatic hydrocarbon (PAH) found in coal tar, tobacco smoke, broiled foods, automotive exhausts, and various polluted environments (1). Due to its ubiquitous occurrence and the fact that it is carcinogenic in laboratory animals and humans, BP has been studied extensively as a model for PAH carcinogenesis (2). The metabolic activation of BP involves a series of steps including an initial oxidation, followed by a hydrolysis and a second oxidation to give * To whom correspondence should be addressed. Phone, (870) 5437205; fax, (870) 543-7136; e-mail, [email protected]. † National Center for Toxicological Research. ‡ Jo ´ zsef Fodor National Center for Public Health. # Be ´ la Johan National Center for Epidemiology. § National Institutes of Health. | Centers for Disease Control and Prevention.

the ultimate carcinogen trans-7,8-dihydroxy-anti-9,10epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (reviewed in ref 3). BPDE reacts with DNA to give a number of DNA adducts, with the major DNA adduct being 10(deoxyguanosin-N2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDE) (reviewed in ref 4). Given 1 Abbreviations: BP, benzo[a]pyrene; BPDE, trans-7,8-dihydroxy-anti9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPDE-d8, [1,2,3,4,5,6,11,12-2H8]trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPDE-DNA, DNA reacted with BPDE; CIA, chemiluminescence immunoassay; dG-BPDE, 10-(deoxyguanosin-N2-yl)-7,8,9trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; DELFIA, dissociationenhanced lanthanide fluoroimmunoassay; ES, electrospray; IAC, immunoaffinity chromatography; LOD, limit of detection; LOQ, limit of quantitation; MRM, multiple reaction monitoring; MS/MS, tandem mass spectrometry; NCTR, National Center for Toxicological Research; PAH, polycyclic aromatic hydrocarbon; RSD, relative standard deviation; SFS, synchronous fluorescence spectroscopy; S/N, signal-to-noise ratio; THF, tetrahydrofuran; PS, phosphorescence spectroscopy.

10.1021/tx050068y CCC: $30.25 © 2005 American Chemical Society Published on Web 06/25/2005

HPLC-ES-MS/MS Quantitation of Benzo[a]pyrene-DNA Adducts

the importance of BP in carcinogenesis research, many approaches have been developed to detect dG-BPDE in human tissues. Of these, 32P-postlabeling and immunoassays are probably the most widely used (5-7); but despite being highly sensitive, these methods do not provide structural characterization of the adducts detected. To circumvent this problem, preparative steps, including immunoaffinity chromatography (IAC) using DNA adduct-specific antisera and HPLC fractionation to obtain adduct-containing eluates, have been employed before final analysis by fluorescence or mass spectrometry (5-8). These methods require large quantities (115 mg) of DNA, and whereas the dG-BPDE adduct was identified in samples of human placental DNA from smokers (9-12), the approach is not feasible for use in routine human biomonitoring. In the last 10-15 years, mass spectrometry has become highly sophisticated, and the techniques currently employed permit the identification of specific DNA adducts in smaller quantities (10-100 µg) of human DNA. We have previously used HPLC with electrospray mass spectrometry (HPLC-ES-MS) to detect and quantify DNA adducts from the arylamine carcinogen 4-aminobiphenyl (13, 14). More recently, we have used HPLC-ES tandem mass spectrometry (HPLC-ES-MS/MS) to quantify etheno-DNA adducts (15), 8-oxodeoxyguanosine (16), malondialdehyde-derived DNA adducts (16), tamoxifenDNA adducts (17), and acrylamide-derived DNA adducts (18). We have now expanded the approach by developing and validating an HPLC-ES-MS/MS method to detect and quantify dG-BPDE. The technique has been applied to DNA modified in vitro with dG-BPDE, to liver DNA from mice treated with BP, to lung DNA from mice fed coal tar, and to human lung DNA samples.

Materials and Methods Caution: BPDE is carcinogenic in laboratory animals. It should be handled with extreme care, using proper personal protective equipment and a well-ventilated hood. Chemicals. [2H10]Pyrene was purchased from Cambridge Isotope Laboratory, Woburn, MA. Calf thymus DNA and the enzymes used for hydrolysis of the DNA samples were purchased from Sigma Chemical Co., St. Louis, MO. Except where noted, all other reagents were purchased from Aldrich Chemical Co., Milwaukee, WI. General Instrumentation. Mass spectra of synthetic products were recorded with an HP5988A dual-source mass spectrometer (Hewlett-Packard Co., Palo Alto, CA). UV-visible absorption spectra were obtained with a Beckman DU-40 UV/ visible spectrometer (Beckman Coulter, Fullerton, CA). 1H NMR spectra were recorded with a Bruker AM500 spectrometer (Bruker Instruments, Inc., Billerica, MA) operating at 500 MHz. Chemical shifts are reported in ppm downfield from tetramethylsilane; coupling constants are reported in Hz. Resonances were assigned through comparison to literature values for unlabeled derivatives. Melting points were determined with an Electrothermal melting point apparatus (Electrothermal, Ltd., London, U.K.) and are uncorrected. HPLC analyses of the synthetic BPDE-DNA adducts were conducted with a µBondapak C18 column (0.39 cm × 30 cm; Waters Associates, Milford, MA) using a Waters Associates system consisting of two model 510 pumps and a model 660 automated gradient controller, equipped with a Rheodyne model 7125 injector and a Hewlett-Packard 1050 diode array spectrophotometric detector. The peaks were monitored at 254 and 280 nm. Syntheses (Figure 1). 1. [1,2,3,4,5,6,11,12-2H8]9,10-Dihydrobenzo[a]pyren-7(8H)-one (1). Succinoylation of [2H10]-

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pyrene (1.99 g, 9.4 mmol) with succinic anhydride (940 mg, 9.4 mmol) was conducted in 150 mL of nitrobenzene, using anhydrous AlCl3 catalysis, at 0 °C for 3 h (19). The resulting ketoacid (2.14 g; 74% yield) was reduced to [2,3,4,5,6,7,8,9,10-2H9]4-(1-pyrenyl)butanoic acid by a Clemmensen reduction, and the latter product was cyclodehydrated to 1 with SnCl4. Mp 179180 °C [literature for unlabeled 1, 170-171 °C (20)]. 1H NMR (CDCl3): δ 1.95-2.25 (m, 2H, H9), 2.69 (t, 2H, H8), 3.25 (t, 2H, H10). MS: m/z 278 (M+). 2. [1,2,3,4,5,6,11,12-2H8]9,10-Dihydrobenzo[a]pyrene (3). Ketone 1 (200 mg; 720 µmol) in 50 mL of tetrahydrofuran (THF) was reduced with excess NaBH4 (120 mg) in 60 mL of MeOH at ambient temperature for 30 min. The reaction mixture was poured into ice water and neutralized with HCl, and the precipitate was collected to afford alcohol 2 in 99% yield. Mp 144-145 °C [literature for unlabeled 2, 144 °C (20)]. 1H NMR (CDCl3): δ 1.82-2.16 (m, 4H, H8, H9), 3.10-3.45 (m, 2H, H10), 4.98-5.16 (m, 1H, H7). MS: m/z 280 (M+). Dehydration of 2 (210 mg, 750 µmol) was conducted with 140 mg of p-toluenesulfonic acid in 150 mL of refluxing benzene for 100 min. Upon cooling, the benzene solution was washed with 10% NaHCO3 and water, concentrated, and chromatographed on a silica column with hexane to yield the alkene 3 as a light yellow solid (187 mg; 95% yield). Mp 152-153 °C [literature for unlabeled 3, 148-149 °C (20)]. 1H NMR (CDCl3): δ 1.95-2.38 (m, 2H, H9), 3.30 (t, 2H, H10), 6.25 (dd, 1H, H8, J8,9 ) 2.2 Hz), 6.64 (dd, 1H, H7, J7,8 ) 9.6 Hz, J7,9 ) 1.8 Hz). MS: m/z 262 (M+). UV (MeOH): 402 and 276 nm. 3. [1,2,3,4,5,6,11,12-2H8]trans-7,8-bis(Benzoyloxy)-7,8,9,10-tetrahydrobenzo[a]pyrene (4). A solution of alkene 3 (140 mg, 530 µmol) in 30 mL of benzene was added to a solution of silver benzoate (270 mg, 1.18 mmol) and iodine (150 mg, 590 µmol) in 20 mL benzene that had been refluxed previously for 30 min. The resulting solution was refluxed for 24 h under nitrogen. The reaction mixture was filtered through Celite, the Celite was washed with an additional 30 mL of benzene, and the filtrate was partitioned between ethyl acetate and dilute NaOH. The organic phase was washed with cold water, dried over MgSO4, and evaporated under reduced pressure. The crude product was chromatographed on a silica gel column with hexane/ethyl acetate (6:1) to give the dibenzoate 4 as a white solid (220 mg, 82% yield). Mp 219-220 °C [literature for unlabeled 4, 215-216 °C (21)]. 1H NMR (CDCl3): δ 2.50-2.51 (m, 2H, H9), 3.82-3.86 (m, 2H, H10), 5.72-5.77 (m, 1H, H8), 6.93 (d, 1H, H7, J7,8 ) 6.3 Hz), 7.25 (t, 4H, benzoyl, J ) 7.0 Hz), 7.62 (t, 2H, benzoyl, J ) 7.0 Hz), 8.01 (d, 4H, benzoyl, J ) 7.0 Hz). MS: m/z 504 (M+). UV (MeOH): 402, 382, 283, and 240 nm. 4. [1,2,3,4,5,6,11,12-2H8]trans-7,8-bis(Benzoyloxy)-7,8-dihydrobenzo[a]pyrene (5). A solution of the dibenzoate 4 (190 mg, 380 µmol) and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (100 mg, 440 µmol) in 30 mL of benzene was refluxed under an argon atmosphere for 3 h. The resulting deeply colored mixture was filtered through Celite and chromatographed on a silica column with hexane/ethyl acetate (4:1) to give the alkene 5 as a white solid (120 mg, 63% yield). Mp 213-214 °C [literature for unlabeled 5, 196-198 °C (21)]. 1H NMR (CDCl3): δ 4.95 (ddd, 1H, H8, J7,8 ) 7.5 Hz, J8,9 ) 4.1 Hz, J8,10 ) 1.8 Hz), 5.02 (d, 1H, H7), 6.24 (dd, 1H, H9, J9,10 ) 9.8 Hz), 7.51 (dd, 1H, H10), 7.30 (t, 4H, benzoyl, J ) 7.0 Hz), 7.65 (t, 2H, benzoyl, J ) 7.0 Hz), 8.10 (d, 4H, benzoyl, J ) 7.0 Hz). MS: m/z 502 (M+). UV (MeOH): 409, 393, 347, 297, 256, and 249 nm. 5. [1,2,3,4,5,6,11,12-2H8]trans-7,8-Dihydroxy-7,8-dihydrobenzo[a]pyrene (6). A solution of alkene 5 (60 mg, 120 µmol) in 5 mL of THF and sodium methoxide (14 mg, 250 µmol) in 5 mL MeOH was stirred at 60 °C for 1 h. The reaction mixture was partitioned between ethyl acetate and water, and the organic layer was separated, dried over MgSO4, and evaporated to give the dihydrodiol 6 as a colorless solid (34 mg, 97% yield). Mp 219-221 °C [literature for unlabeled 6, 216-217 °C (21)]. 1H NMR (methanol-d ): δ 4.58 (m, 1H, H8), 5.05 (d, 1H, H7, 4 J7,8 ) 11.2 Hz), 6.38 (dd, 1H, H9, J9,10 ) 10.3 Hz, J8,9 ) 2.2 Hz),

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7.51 (dd, 1H, H10). MS: m/z 294 (M+). UV (MeOH): 414, 345, 301, and 257 nm. 6. [1,2,3,4,5,6,11,12-2H8]trans-7,8-Dihydroxy-anti-9,10epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-d8). A solution of 6 (20 mg, 68 µmol) in 15 mL of anhydrous THF and a 20-fold excess of phosphate buffer-purified (22) m-chloroperoxybenzoic acid (235 mg) was stirred at ambient temperature under a nitrogen atmosphere for 1.5 h. The reaction mixture was added to 100 mL cold diethyl ether and rapidly extracted three times with cold 10% NaOH (3 × 40 mL) and twice with cold water (50 mL). The organic layer was dried over anhydrous K2CO3 and the solvent removed under reduced pressure to give BPDE-d8 (16 mg, 80% yield). Mp 214-216 °C (dec) [literature for BPDE, 214 °C (23)]. 1H NMR (acetone-d6): δ 3.78 (dd, 1H, H9, J8,9 ) 9.4 Hz, J9,10 ) 5.0 Hz), 3.82 (d, 1H, H8, J7,8 ) 5.0 Hz), 4.65 (d, 1H, H7), 5.10 (d, 1H, H10). MS: m/z 310 (M+). 7. [1,2,3,4,5,6,11,12-2H8]10-(Deoxyguanosin-N2-yl)-7,8,9trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-BPDEd8). BPDE-d8 (4.5 mg) was dissolved in 50 µL of THF and added to 18 mg of calf thymus DNA dissolved in 18 mL of 1 mM TrisHCl buffer (pH 7.5). The mixture was incubated at 37 °C for 4 h and then extracted sequentially with Tris-HCl-saturated n-butanol (three times), water-saturated isoamyl alcohol (three times), and water-saturated ethyl acetate. The modified DNA was precipitated by the addition of 1.8 mL 5 M NaCl and 20 mL ice-cold ethanol and redissolved in 5 mM Bis-Tris and 0.1 mM EDTA buffer (pH 7.1) at a concentration of 0.6 mg/mL. The extent of modification, as determined by measuring the UV absorbance of the DNA (24), was 0.95%. A similar reaction was conducted with BPDE (Midwest Research Institute, Kansas City, MO) to give DNA modified at a level of 0.33%. A portion (2.1 mg) of the DNA that had been reacted with BPDE-d8 was hydrolyzed to nucleosides with DNase I, snake venom phosphodiesterase, and alkaline phosphatase (25). The hydrolysate was extracted with water-saturated n-butanol (three times), the n-butanol was washed with n-butanolsaturated water (three times) and evaporated, and the residue was dissolved in methanol for analysis by HPLC. The dG-BPDEd8 was isolated by reverse-phase HPLC using a 30-min linear gradient of 20-100% methanol at 2 mL/min. DNA (1.3 mg) that had been reacted with BPDE was hydrolyzed in a similar manner, and the dG-BPDE was isolated by HPLC. HPLC-ES-MS/MS Analyses. DNA samples (typically ∼100 µg) were enzymatically hydrolyzed to nucleosides with DNase I, snake venom phosphodiesterase, and alkaline phosphatase (25). After adding 10 pg of dG-BPDE-d8 and 50 µL of methanol, to give a final concentration of ∼30% methanol, the samples were injected on the HPLC for ES-MS/MS analyses. The liquid handling system consisted of an Alliance 2795 Separations module (Waters Associates), a Dionex GP40 quaternary gradient pump (Dionex, Sunnyvale, CA), and an automated switching valve (TPMV, Rheodyne, Cotati, CA). The switching valve allowed for on-line sample enrichment. The valve was used to divert the trap column effluent to either waste or to the analytical column. The Alliance 2795 system was used for sample injection, sample concentration, and regeneration of the trap column. The Dionex pump, containing 72% of 0.1% formic acid and 28% of acetonitrile, was used to back-flush the trap column to the analytical column during analysis and to keep a constant flow of mobile phase through the analytical column into the mass spectrometer during the sample loading and preparation periods. Each sample was loaded onto a reverse-phase trap column [Luna C18(2), 2 mm × 30 mm, 3 µm, Phenomenex, Torrance, CA] with 0.1% formic acid at a flow rate of 0.5 mL/min for 1.5 min, and polar materials were washed to waste for 3 min with 90% of 0.1% formic acid and 10% of acetonitrile at a flow rate of 0.5 mL/min. After switching the divert valve, the concentrated sample was back-flushed from the trap column onto the analytical column [Luna C18(2), 2 mm × 150 mm, 3 µm, Phenomenex] with 72% of 0.1% formic acid and 28% of acetonitrile at 0.2 mL/ min, and the sample components were eluted into the mass

Beland et al. spectrometer. After 3 min, the valve was switched back to the load position and the trap column was washed (to waste) with 60% of acetonitrile and 40% of 0.1% formic acid for 5 min at a flow rate of 0.5 mL/min. The trap column was then equilibrated with the starting mobile phase of 0.1% formic acid. The total run time for sample preparation and analysis was 15 min. A Quattro Ultima quadrupole mass spectrometer (Waters Associates), equipped with an ES interface, was used with a source block of 120 °C and a desolvation temperature of 400 °C. Nitrogen was the desolvation (750 L/h), cone (100 L/h), and nebulizing gas. Argon was the collision gas, at a collision cell pressure of 1.5 × 10-3 mbar. Positive ions were acquired in the multiple reaction monitoring (MRM) mode (dwell time of 0.25 s and interchannel delay of 0.03 s) for transitions of the protonated dG-BPDE molecule [(M + H)+] (m/z 570) to m/z 257 and for the corresponding (M + H)+ (m/z 577) to m/z 264 transitions of dG-BDPE-d7. The cone voltage was 40 V, and the collision energy was 32 eV for the transitions. Samples were quantified by comparing the area of the dG-BPDE chromatogram peak to that of the dG-BPDE-d7 chromatogram peak. In Vitro and in Vivo DNA Samples. The following in vitro and in vivo DNA samples were obtained from the sources indicated: calf thymus DNA modified with [1,3-3H]BPDE (26), liver DNA from male B6C3F1 mice administered 0.5 or 1.0 mg [7,8-3H]BP (26), lung DNA from female B6C3F1 mice fed a diet containing 0.0 or 0.3% coal tar for 4 weeks (27), human lung DNA from tumor and normal peripheral tissue (28), and human lung DNA from normal tissue obtained at autopsy (29-31). The calf thymus DNA, modified with [1,3-3H]BPDE, was synthesized at the National Center for Toxicological Research (NCTR) in 1995 and was stored in 1-mL aliquots at -80 °C. Male B6C3F1 mice were treated with [7,8-3H]BP at NCTR in 1996, and the hepatic DNA was stored at -80 °C. Female B6C3F1 mice were fed coal tar at NCTR in 1992, and the lung DNA was stored at -80 °C. Normal peripheral lung tissue and tumor tissues (samples BRH-871 to BRH-948) were collected in Budapest in 2000-2001, and the DNA samples were stored at -100 °C. The DNA samples were evaporated before being shipped to NCTR. Upon arrival at NCTR, the samples were reconstituted with water, UV spectra were obtained to verify the DNA content, and the samples were stored at -80 °C. A control experiment was conducted in which calf thymus DNA, modified with [1,3-3H]BPDE, was evaporated and shipped from NCTR to the National Institute of Environmental Health, Budapest, and back. No decomposition of the adduct occurred during shipment. Normal lung tissues (samples 1069-1653) were collected in Bethesda in 1989, and the DNA samples were stored at 4 °C in 10 mM Tris-HCl and 1 mM EDTA buffer (pH 7.5). The samples were shipped on dry ice to NCTR and stored at -80 °C upon arrival. UV spectra were obtained before analysis to verify the DNA content. HPLC-ES-MS/MS analyses were conducted at NCTR from 2002 to 2004. The samples analyzed by HPLC-ES-MS/MS were identical to those assayed by the other DNA adduct detection methods.

Results Synthesis of the Deuterated Adduct Standard dG-BPDE-d8. BPDE-d8 was synthesized from the alkene 3, which was prepared in five steps starting with the succinoylation of [2H10]pyrene (Figure 1). Reduction of the ketone 1 with NaBH4 generated the alcohol 2 in nearly quantitative yield. Upon acid-catalyzed dehydration, 2 was converted to the alkene 3. It should be noted that stoichiometric amounts of p-toluenesulfonic acid were used in this reaction because a side product resulting from an intermolecular dehydration was obtained with catalytic quantities of p-toluenesulfonic acid. A Pre´vost reaction of 3 with silver benzoate and iodine resulted in the formation of the trans-dibenzoate 4. It was necessary to use freshly recrystallized silver benzoate for this

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Figure 1. Synthesis of BPDE-d8 and dG-BPDE-d8. The abbreviations used are p-TsOH, p-toluenesulfonic acid; Bz, benzoyl; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; m-CPBA, m-chloroperoxybenzoic acid.

Figure 2. HPLC-UV analysis of a hydrolyzed DNA sample from calf thymus DNA reacted with BPDE-d8. The inset shows the UV spectrum of the dG-BDPE-d8 that eluted at 18 min. The elution conditions are outlined in Materials and Methods.

Figure 3. Limited-range full-scan mass spectrum of dG-BPDEd8. In addition to the protonated molecule [(M + H)+] for dGBPDE-d8, protonated molecules were observed for dG-BPDEd7, dG-BPDE-d6, and dG-BPDE-d5.

reaction, otherwise the yield of 4 decreased drastically. Dehydrogenation of 4 with 2,3-dichloro-5,6-dicyano-1,4benzoquinone in refluxing benzene afforded 5, which upon methanolysis with sodium methoxide in methanol and THF gave the trans-dihydrodiol 6 in 97% yield. BPDE-d8 was then prepared by reacting 6 with a 20-fold excess of m-chloroperoxybenzoic acid in the usual manner (23). BPDE-d8 was incubated with a 4-fold excess (by weight) of calf thymus DNA at 37 °C. After 4 h, the reaction mixture was extracted sequentially with organic solvents, the modified DNA was precipitated and redissolved in buffer, and a portion was enzymatically hydrolyzed to nucleosides and extracted with n-butanol. HPLC analysis of the n-butanol extract indicated a major peak eluting at 18 min (Figure 2) that had a UV spectrum (Figure 2, inset) consistent with dG-BPDE-d8 (24). This material was collected and quantified by its molar extinction coefficient (24). dG-BPDE, which was prepared

in a similar manner, was also collected by HPLC and quantified by its molar extinction coefficient (24). A limited-range full-scan mass spectrum of dG-BPDEd8 indicated a protonated molecule [(M + H)+] at m/z 578 (Figure 3). Product ion analysis of the protonated molecule for dG-BPDE-d8, at a collision energy of 25 eV, gave major fragments consistent with a sequential loss of deoxyguanosine (m/z 311), H2O (m/z 293), and CO (m/z 265), as well as fragments indicative of guanine (m/z 152) and deoxyribose (m/z 117) (Figure 4). At a collision energy of 15 eV, a major ion, representing the protonated purine adduct, was also present at m/z 462 (not shown). In addition to the protonated molecule for dG-BPDE-d8, protonated molecules were observed for dG-BPDE-d7 (m/z 577), dG-BPDE-d6 (m/z 576), and dG-BPDE-d5 (m/z 575) (Figure 3). Product ion analyses of these protonated molecules gave fragments analogous to those observed for dG-BPDE-d8 (not shown).

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Figure 4. MS/MS characterization of dG-BPDE-d8, using ES ionization and a collision energy of 25 eV. The major fragments derived from the protonated nucleoside adduct molecule [(M + H)+] are indicated in the structures.

Figure 5. HPLC-ES-MS/MS analyses of calf thymus DNA modified in vitro with BPDE. The traces correspond to the MRM (relative signal intensity vs time) of the transitions for the protonated nucleoside adduct molecule [(M + H)+] to 7,9-dihydro-8H-cyclopenta[2,1-b]pyren-8-one for the internal standard dG-BPDE-d7 (m/z 577 f 264) (A) and the sample (m/z 570 f 257) (B). The retention time and area are indicated for the major peak in each chromatogram.

Inasmuch as the protonated molecule for dG-BPDEd7 gave a peak area greater than that observed for dGBPDE-d8 (Figure 3), the signal produced by dG-BPDEd7 was used for quantitation purposes. HPLC-ES-MS/MS Analyses of DNA Modified in Vitro with BPDE. The HPLC-ES-MS/MS method was validated with respect to intra-assay and inter-assay precision and accuracy by analyzing, on different days, 82 µg of hydrolyzed salmon testis DNA to which had been added 10 pg dG-BPDE. This corresponded to an adduct level of 122 fg dG-BPDE/µg DNA or 6.9 dG-BPDE adducts/108 nucleotides. To minimize the loss of the dGBPDE due to precipitation, the samples were adjusted to contain ∼30% methanol before analysis. On day 1, the dG-BPDE level measured in the sample was 122 ( 2 fg/µg DNA [1.6% relative standard deviation (RSD), n ) 3]. On day 2, the dG-BPDE level measured in the sample was 120 ( 2 fg/µg DNA (1.7% RSD, n ) 3). Single aliquots of 82 µg of hydrolyzed DNA were also spiked on each day with 2 and 20 pg of dG-BPDE. These

levels correspond to 1.4 and 14 adducts/108 nucleotides. Recovery levels for these single samples ranged from 95 to 104%. dG-BPDE was not detected in the hydrolyzed DNA in the absence of added adduct standard. The limit of detection (LOD), based upon a signal-to-noise ratio (S/ N) of 3, was approximately 500 fg on-column (equivalent to 0.3 adducts/108 nucleotides when analyzing 100 µg of DNA), and the limit of quantitation (LOQ; S/N ) 10) was approximately 1 adduct/108 nucleotides. A calibration curve was prepared using 10 pg of the internal standard dG-BPDE-d8 and 0.5-100 pg of dG-BPDE. A plot of the response ratio to the concentration ratio (not shown) was linear, with a correlation coefficient of >0.99 and a slope of 1.85. Calf thymus DNA modified in vitro with [1,3-3H]BPDE (26) was enzymatically hydrolyzed to nucleosides, 10 pg of dG-BPDE-d8 was added to serve as a quantitation standard, and the samples were analyzed by HPLC-ESMS/MS. A typical chromatogram is shown in Figure 5, where the traces correspond to MRM of the transitions

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Table 1. Analyses of DNA Modified in Vitro with [1,3-3H]BPDE and Liver DNA from B6C3F1 Mice Treated with [7,8-3H]BPa analytical method

dG-BPDE/108 nucleotides ( SD reference

DNA Modified in Vitro with [1,3-3H]BPDE radiolabeling 111 ( 2 HPLC-ES-MS/MS 115 ( 21b 32P-postlabeling 103 ( 67 BPDE-DNA DELFIA 135 ( 79 BPDE-DNA CIA 121 ( 25

26 this work 32 26 26

Liver DNA from B6C3F1 Mice Treated with 0.5 mg of [7,8-3H]BP radiolabeling 86 ( 2 26 HPLC-ES-MS/MS 7 ( 1c this work 32P-postlabeling 11 ( 4 32 BPDE-DNA DELFIA 4(2 26 BPDE-DNA CIA 11 ( 1 26 Liver DNA from B6C3F1 Mice Treated with 1.0 mg of [7,8-3H]BP radiolabeling 138 ( 3 26 HPLC-ES-MS/MS 16 ( 1c this work 32P-postlabeling 22 ( 9 32 BPDE-DNA DELFIA 8(5 26 BPDE-DNA CIA 17 ( 4 26 a HPLC-ES-MS/MS analyses were conducted as indicated in Materials and Methods. Results for measuring the adduct levels by direct determination of radioactivity, 32P-postlabeling, BPDEDNA DELFIA, and BPDE-DNA CIA are from the references indicated. b HPLC-ES-MS/MS analyses were conducted with 100 µg of DNA samples, of which 10 µg was injected on the column, and are presented as the mean ( SD of 12 independent determinations. c HPLC-ES-MS/MS analyses were conducted with 160 µg of DNA samples, of which 45 or 160 µg was injected on the column, and are presented as the mean ( SD of two independent determinations.

for the protonated nucleoside adduct molecule [(M + H)+] to 7,9-dihydro-8H-cyclopenta[2,1-b]pyren-8-one for the internal standard dG-BPDE-d7 (m/z 577 f 264; Figure 5A) and the sample (m/z 570 f 257; Figure 5B). In each trace, one peak is clearly present at 11.4-11.6 min. Based upon comparison to the peak area of the deuterated internal standard, the level of dG-BPDE-modified calf thymus DNA was determined to be 115 ( 21 dG-BPDE adducts/108 nucleotides (Table 1). This compares very well with 111 ( 2 dG-BPDE adducts/108 nucleotides as determined by measuring the radioactivity of the sample (26). The levels of dG-BPDE as determined by 32Ppostlabeling (32), dissociation-enhanced lanthanide fluoroimmunoassay (BPDE-DNA DELFIA) (26), and chemiluminescence immunoassay (BPDE-DNA CIA) (26) are also similar, as shown in Table 1. HPLC-ES-MS/MS Analyses of Liver DNA from B6C3F1 Mice Treated with [7,8-3H]BP. Male B6C3F1 mice were administered a single intraperitoneal injection of 0.5 or 1.0 mg [7,8-3H]BP, and after 24 h, hepatic DNA was isolated (26). HPLC-ES-MS/MS analyses of the liver DNA were conducted in a manner identical to those performed on the calf thymus DNA modified in vitro with [1,3-3H]BPDE. Representative chromatograms from mice receiving 0.5 and 1.0 mg [7,8-3H]BP are shown in Figure 6. In both instances, a peak is clearly evident in the samples (Figure 6B for 0.5 mg [7,8-3H]BP and Figure 6D for 1.0 mg [7,8-3H]BP) with the expected retention time based on the internal standard (Figure 6, panels A and C, respectively). The amount of dG-BPDE in these samples was only 10-13% of the total binding based upon the amount of radioactivity associated with the DNA (Table 1); nonetheless, these values were quite similar to the values determined by other DNA adduct detection methodologies (Table 1).

Figure 6. HPLC-ES-MS/MS analyses of liver DNA from male B6C3F1 mice treated with 0.5 or 1.0 mg of [7,8-3H]BP. The traces correspond to the MRM for the internal standard dG-BPDE-d7 (A and C), liver DNA from a mouse treated with 0.5 mg of [7,83H]BP (B), and liver DNA from a mouse treated with 1.0 mg of [7,8-3H]BP (D). The retention time and area are indicated for the major peak in each chromatogram.

Figure 7. HPLC-ES-MS/MS analyses of lung DNA from female B6C3F1 mice fed a 0 or 0.3% coal tar diet for 4 weeks. The traces correspond to the MRM for the internal standard dG-BPDE-d7 (A and C), lung DNA from a mouse fed control diet (B), and lung DNA from a mouse fed a 0.3% coal tar diet (D). The retention time and area are indicated for the major peak in each chromatogram.

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Table 2. HPLC-ES-MS/MS Analyses of Lung DNA from B6C3F1 Mice Fed a 0.3% Coal Tar Diet total DNA adducts/108 nucleotides ( SD as determined by 32P-postlabeling analysesa

estimated dG-BPDE adducts/108 nucleotides ( SD based upon BP content of coal tar dietb

dG-BPDE adducts/108 nucleotides ( SD based upon HPLC-ES-MS/MS analysesc

102 ( 14

0.8 ( 0.2

0.6 ( 0.04

a

Female B6C3F1 mice were fed a 0.3% coal tar diet for 4 weeks. DNA was isolated from lung tissue and assessed for total DNA adduct levels by 32P-postlabeling analyses (27). b The estimated dG-BPDE level was based upon the BP content of the coal tar and the dG-BPDE level determined by 32P-postlabeling analyses of lung DNA from mice fed BP (27). c HPLC-ES-MS/MS analyses were conducted with 100 µg of DNA samples from four mice and are presented as the mean ( SD.

HPLC-ES-MS/MS Analyses of Lung DNA from B6C3F1 Mice Fed Coal Tar. Female B6C3F1 mice were fed a 0.3% coal tar diet (containing approximately 2 mg BP/g coal tar) for 4 weeks, after which lung DNA was isolated (27). Figure 7 shows representative HPLC-ESMS/MS analyses of the lung DNA from a control mouse and from a mouse fed coal tar. In lung DNA from the mouse fed coal tar, a peak is clearly evident (Figure 7D) that has the retention time predicted based on the internal standard (Figure 7C). The peak was not present in lung DNA from the control mouse (Figure 7B). In chromatograms for both the control mouse (Figure 7B) and coal tar-fed mouse (Figure 7D), an additional peak was evident at 10.75 min. The identity of this peak is not known, but it is clearly not due to exposure to coal

tar because it was present in the control sample. Based upon comparison to the internal standard, the amount of dG-BPDE in lung DNA from mice fed 0.3% coal tar was 0.6 ( 0.04 dG-BPDE/108 nucleotides (Table 2). This is considerably lower than the total DNA adduct level determined by 32P-postlabeling analyses (Table 2) (27); nonetheless, it is similar to that expected based upon the BP content of the coal tar (27) and the dG-BPDE level in lung DNA, as determined by 32P-postlabeling analyses in B6C3F1 mice fed BP for the same length of time (Table 2) (27). HPLC-ES-MS/MS Analyses of Human Lung DNA. DNA was obtained from 10 normal lung tissue samples collected at autopsy and nine peripheral lung tissue samples from lung cancer patients. For seven of the lung cancer patients, DNA was also obtained from tumor tissue. When HPLC-ES-MS/MS analyses were conducted with these samples, dG-BPDE was detected in only one sample, 1582 (Table 3). Representative chromatograms are presented in Figure 8 for samples 1499 and 1582. For sample 1499 (Figure 8B), there is no indication of an adduct peak eluting in the region of the internal standard dG-BPDE-d8 (Figure 8A), whereas for sample 1582 (Figure 8D), a peak is clearly evident that corresponded to the internal standard (Figure 8C). As a control experiment, aliquots of the human lung DNA were combined with the calf thymus DNA that had been modified in vitro with [1,3-3H]BPDE, and the mixture was enzymatically hydrolyzed and analyzed by HPLCES-MS/MS. The peak obtained for dG-BPDE was identical to that obtained when the hydrolysis was in the

Table 3. Analyses of Human Lung DNA (Adducts/108 Nucleotides) method of analysis samplea

smoking status

BRH-871 BRH-879

former current

BRH-882

current

BRH-924 BRH-925

never former

BRH-929

current

BRH-941

never

BRH-947

former

BRH-948

never

1069 1119 1143 1242 1499 1516 1524 1582 1606 1653

current unknown former or never unknown former or never current former or never current former or never current

lung tissue

BPDE-DNA CIAb

normal tumor normal tumor normal normal tumor normal tumor normal tumor normal tumor normal tumor normal normal normal normal normal normal normal normal normal normal normal

2.1 1.1 8.8 5.3 9.1 11.3 3.1 3.3 3.9 8.4 0.5 4.2 2.8 9.1 0.4 3.8

32P-postlabelingc

19.5 10.7 19.3 5.4 15.8 1.9 7.8 8.6 2.2 2.7 1.5 0.8 0.4 1.6 1.2 0.8 12.8 17.9

23.7 36.5

IAC-SFSd

IAC-PSe

1.3-4.8 0.7 1.1-1.9 NDg

9.0 12.5

52.8 6.5

HPLC-ESMS/MSf