Chemicals and Enzymes
All chemicals were of the highest purity grade commercially available. α-AcetoxyNPYR (2
) and 4-iodobutyraldehyde were prepared as described (17
]dGuo was obtained from Cambridge Isotope Laboratories (Andover, MA). Ethanol was procured from AAPER Alcohol and Chemical Co. (Shelbyville, KY). 2-Propanol was purchased from Acros Organics (Morris Plains, NJ). Puregene DNA purification solutions were obtained from QIAGEN (Valencia, CA). Alkaline phosphatase (from calf intestine) was purchased from Roche Diagnostics Corp. (Indianapolis, IN). 2′,3′,5′-Triacetylguanosine, herring testes DNA, DNase I, phosphodiesterase I, dGuo, dAdo, and dThd were obtained from Sigma-Aldrich Co. (St. Louis, MO). All other chemicals were obtained from Sigma-Aldrich or Fisher Scientific (Fairlawn, NJ). Water was purified in a Milli-Q system (Millipore, Bedford, MA).
), and the respective isotopic standards [15
), and [15
) were prepared as described (27
). These were used for quantitation of adducts 13
, after NaBH3
CN reduction. [An interfering peak prevented quantitation of the NaBH3
CN reduction product of adduct 16
in this study, and levels of adduct 17
in vivo were too low for quantitation previously (28
)]. The 7,8-butanoguanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f
) was prepared as described and its spectral properties agreed with those reported (18
H NMR, 600 MHz, (DMSO-d6
): δ 1.85(m, H7,8
partially obscured by solvent), 4.03(m, 1H, H6a
), 4.22 (m, 1H, H6b
), 4.64 (m, 1H, H9
), 6.10 (s, 2H, NH2
); UV, λmax
285 nm; positive ESI-MS: m/z
222 [M + H]+
204 [M – H2
O + H]+
)one (adduct 9
) and [15
were prepared as described (34
α-AcetoxyNPYR (3) (48 mg, 0.3 mmol) was allowed to react with [15N5]Guo (12 mg, 0.04 mmol) in 6 mL of 0.1 M phosphate buffer (pH 7) at 37 °C for 17 h. Then, more α-acetoxyNPYR (30 mg) was added and the reaction mixture was incubated an additional 6 h, then heated at 100 °C for 1 h. The product, [15N5]6, was collected and purified by HPLC systems 2 and 1, respectively, and then neutralized, concentrated to dryness, and extracted 3 times with 10 mL of 1/1:CH3OH/C2H5OH. The extracts were concentrated to dryness and redissolved in H2O. The resulting [15N5]6 adduct was purified, identified and quantified by comparison to a standard in HPLC Systems 1 and 2.
HPLC analyses were carried out with Waters Associates (Waters Division, Millipore, Milford, MA) instruments with a model RF10 AXL fluorescence detector (Shimadzu Scientific Instruments, Columbia, MD) for HPLC systems 1 and 2, or a model 996 photodiode array UV detector (Waters) for HPLC system 3, or a model SPD-10 A UV-vis detector (Shimadzu) for HPLC system 4. Elution systems were as follows.
System 1 consisted of two 25 cm × 4.6 mm Partisil-10 SCX strong cation exchange columns (Whatman, Clifton, NJ) eluted isocratically with 5% CH3OH in 40 mM ammonium phosphate buffer, pH 2.0, at a flow rate of 1 mL/min, with detection by fluorescence (excitation 290 nm, emission 380 nm) for identification and quantitation of 6 and [15N5]6.
System 2 was a 9.4 mm × 50 cm Partisil 10 SCX column eluted isocratically with 40 mM ammonium phosphate buffer, pH 2.0, at a flow rate of 3 mL/min, and detection as in system 1, for collection and purification of [15N5]6.
System 3 was two 4.6 mm × 25 cm Supelcosil LC 28-BD columns (Supelco, Bellefonte, PA) with isocratic elution by 5% CH3OH in 40 mM ammonium acetate buffer, pH 6.6, for 5 min and then a gradient from 5 to 35% CH3OH in 40 min at a flow rate of 1 mL/min with UV detection (285 nm) for determination of the UV spectrum of adduct 6.
System 4 used a 250 mm × 4.6 mm i.d., 5 μm, Luna C18(2) column (Phenomenex, Torrance, CA) eluted with a gradient of H2
O and CH3
OH at a flow rate of 0.7 mL/min: from 0 to 15 min, 5 to 22% CH3
OH; from 15 to 17 min, 22 to 80% CH3
OH; from 17 to 20 min, 80% CH3
OH; from 20 to 22 min, 80 to 5% CH3
OH; from 22 to 35 min, 5% CH3
OH. The UV detector was set at 254 nm for dGuo, dThd, dAdo, and Gua quantitation in DNA samples, essentially as described previously (35
LC-MS Analysis of Synthetic Adduct 6
LC-MS analyses were carried out on a Thermo Finnigan LCQ Deca instrument (Thermoquest LC/MS Division, San Jose CA) interfaced with a Waters Alliance 2690 HPLC multi-solvent delivery system and equipped with an SPD 10 A UV-vis detector (Shimadzu). The ESI source was set as follows: voltage, 2.0 kV; current, 10 μA; and capillary temperature, 250 °C. HPLC system 3 was used, except that the flow rate was 0.7 mL/min.
Capillary HPLC-ESI-MS/MS-SRM Analyses of Adducts 6 and 18–20
Adduct quantitation in DNA samples was carried out with a Finnigan Quantum Discovery Max (ThermoElectron Division, San Jose, CA) triple quadrupole mass spectrometer interfaced with an Agilent 1100 Series capillary flow HPLC. A 250 mm × 0.5 mm i.d., 5 μm, Zorbax SB C18 (Agilent) column was used with different solvent elution programs for quantitation of 7,8-butanoguanine 6 or 18 – 20. The elution systems were as follows.
System 5 was used for quantitation of 7,8-butanoguanine 6. The column was eluted with a gradient of 25 mM ammonium acetate (solvent A) and 25% CH3CN in CH3OH (solvent B) at a flow rate of 10 μL/min and a column temperature of 40 °C, as follows: from 0 to 10 min, 2 to 10% of solvent B; from 10 to 15 min, 10 to 30% of solvent B; from 15 to 22 min, 30% of solvent B; from 22 to 25 min, 30 to 70% of solvent B; from 25 to 30 min, 70% of solvent B; from 30 to 35 min, 70 to 2% of solvent B; from 35 to 45 min, 2% of solvent B. The first 12 min of eluant was directed to waste and the 12 – 45 min fraction was diverted to the ESI source.
System 6 was used for quantitation of adducts 18–20, from NaBH3CN reduction of adducts 13 – 15. The column was eluted with a gradient of 15 mM ammonium acetate, pH 6.6, and CH3CN at a flow rate of 10 μL/min and 25 °C, as follows: from 0 to 10 min, 0% of CH3CN; from 10 to 39 min, 0 to 20% CH3CN; from 39 to 44 min, 20 to 75% CH3CN; from 44 to 49 min, 75% CH3CN; from 49 to 54 min, 75 to 0% CH3CN; from 54 to 64 min, 0% of CH3CN. The first 31 min of eluant was directed to waste and the 31 – 49 min fraction was diverted to the ESI source.
The ESI-MS parameters for quantitation of 7,8-butanoguanine 6 were set in the positive ion mode as follows: spray voltage, 3 kV; sheath gas pressure, 20 psi; capillary temperature, 325 °C; collision energy, 25 V; scan width, 0.1 amu; scan time, 0.5 s; Q1 peak width, 0.7 amu; Q3 peak width, 0.7 amu; Q2 collision cell gas pressure, 1.0 mTorr; source CID, 8 V; and tube lens offset, 85 V.
The ESI-MS parameters for quantitation of 18 – 20 were set in the positive ion mode as follows: spray voltage, 4 kV; sheath gas pressure, 30 psi; capillary temperature, 250 °C; collision energy, 13V; scan width, 0.1 amu; scan time, 0.2 s; Q1 peak width, 0.7 amu; Q3 peak width, 0.7 amu; Q2 collision cell gas pressure, 1.0 mTorr; source CID, 10 V; and tube lens offset, 85V.
Adduct analyses were carried out with selected reaction monitoring (SRM) as follows: 6 m/z 222 → m/z 204; [15N5]6 m/z 227 → m/z 209; 18 m/z 340 → m/z 224; [15N5]18 m/z 345 → m/z 229; 19 m/z 324 → m/z 208; [15N5]19 m/z 329 → m/z 213; 20 m/z 315 m/z 199; [15N2]20 m/z 317 → m/z 201. Data were acquired and processed with Xcalibur software version 1.4 (ThermoElectron).
HPLC-ESI-MS/MS-SRM Analysis of Adduct 9 Diastereomers
This was carried out essentially as described previously (34
Six week old male F344 rats were purchased from Charles River (Wilmington, MA) and housed in the Research Animal Resources facility of the University of Minnesota. They were maintained on NIH-07 diet (Harlan, Madison, WI) and tap water.
Three groups of five rats, weighing approximately 250 g each, were treated by gavage with a single dose of NPYR (46, 92, or 184 mg/kg body weight) in 0.5 mL of saline. Another group of five rats received only 0.5 mL of saline by gavage. They were sacrificed after 16 h and their organs were collected and frozen at −80 °C.
Hepatic DNA was isolated as described in the Puregene® protocol for 1 g animal tissue (QIAGEN) with several modifications. Briefly, rat liver tissue samples (0.5 g) were homogenized with 15 mL of Puregene cell lysis solution on ice. Proteinase K (75 μL of a 20 mg/mL solution) was added and the homogenate was allowed to stand at room temperature overnight. RNase A (75 μL of a 4 mg/mL solution) was added to the cell lysate and, after 2 h at room temperature, protein was precipitated by addition of 5 mL of Puregene protein precipitation solution and centrifugation at 2000 × g for 10 min. The supernatant was poured into a tube containing cold 2-propanol and the precipitated DNA was collected, dissolved in 4 mL of 10 mM Tris/1 mM EDTA pH 7.0 buffer, and the solution was extracted 3 times with 4 mL of CHCl3 containing 4% isoamyl alcohol. The DNA was precipitated from the aqueous phase by addition of 0.4 mL of 5 M NaCl and 8 mL of ice-cold ethanol, washed three times with 3 mL of 70% ethanol and three times with 3 mL of 100% ethanol, and dried with a stream of N2. DNA concentration was determined by measuring UV absorption at 260 nm, and DNA purity was assessed by the UV absorbance ratios at 260/230 nm and 260/280 nm.
Sample Preparation for MS/MS Analyses
DNA samples were subjected to neutral thermal hydrolysis for quantitation of 7,8-butanoguanine 6
and to enzymatic hydrolysis for analysis of the other adducts. For neutral thermal hydrolysis, DNA (0.1 – 1 mg weighed) was dissolved in 1 mL of 10 mM sodium cacodylate buffer, pH 7.0, containing 5000 fmol of [15
. The solution was incubated at 100 °C for 1 h, an aliquot of 50 μL was separated for Gua quantitation (see below), and the remaining 950 μL hydrolysate was partially purified by centrifugation using a Centrifree MPS device (MW cutoff of 30,000 Da, Amicon, Beverly, MA) to remove high molecular weight substances, and by solid-phase extraction (Strata-X cartridges, 33 μm, 30 mg/1 mL, Phenomenex) to desalt and partially isolate the adduct from the normal base pool. The Strata-X cartridge was washed with 1 mL of 5% CH3
OH in H2
O and 1 mL of 40% CH3
OH in H2
O. The 40% CH3
OH fraction was collected and evaporated to dryness. The residue was dissolved in 95 μL of H2
O, and 8 μL aliquots were analyzed by HPLC-ESI-MS/MS-SRM as described above. Prior to each set of analyses, a calibration curve containing varying amounts of 7,8-butanoguanine 6
(10, 50, 100, 200, 400, 800, 1600, 3200, and 6400 fmol) and a fixed amount of [15
(400 fmol) was constructed. Quantitation of Gua was carried out by a method similar to that described previously (35
). A 50 μL aliquot reserved from the hydrolysate was added to 150 μL of 0.1 N HCl and incubated at 80 °C for 1 h. After dilution with 300 μL of H2
O, 10 μL was injected in HPLC system 4.
For quantitation of 18
, the procedure was essentially as described (28
) with some modifications. Briefly, DNA samples (~ 1 mg weighed) were dissolved in 1 mL of 10 mM Tris/5 mM MgCl2
buffer, pH 7.0, containing DNase I (650 units). NaBH3
CN (10 mg) was added to the mixture three times. After the first two additions, the sample was allowed to stand at room temperature for 30 min. After the final addition, it was incubated at 37 °C for 30 min. The pH was adjusted to 7 with 40 μL of HCl, and the internal standards [15
(250 fmol), [15
(1250 fmol), and [15
(2500 fmol) were added. The resulting solution was incubated with an additional amount of DNase I (650 units) at 37 °C for 10 min. Phosphodiesterase I (0.02 units) and alkaline phosphatase (150 units) were then added and the incubation was continued for another 60 min (final volume = 1435 μL). A 20 μL aliquot was separated for dGuo, dThd, and dAdo quantitation using HPLC System 4 after dilution with 80 μL of H2
O. The remaining solution (1415 μL) was applied to a solid-phase extraction cartridge (Strata-X, 33 μm, 30 mg/1 mL, Phenomenex) that was washed with 1 mL of H2
O, 2 mL of 20% CH3
OH in H2
O, and 2 mL of 100% CH3
OH. The methanol containing eluants were combined, evaporated to dryness, redissolved in 200 μL of 50% CH3
OH in H2
O, transferred into an autosampler vial with an infused 300 μL insert (Chrom Tech), and dried again. The residue was dissolved in 49.3 μL of H2
O, and 8 μL aliquots were analyzed by HPLC-ESI-MS/MS-SRM as described above.
Blank samples with and without purchased herring testes DNA, containing all the solutions, enzymes, and internal standards used in the procedure were processed concomitantly to all DNA samples following the procedures described above to check for any contamination.
Validation of Method for Quantitation of 7,8-Butanoguanine 6
Method accuracy was determined by adding varying amounts of 7,8-butanoguanine 6 and a fixed amount of [15N5]6 to 1 mg of herring testes DNA and carrying out the analysis. The intraday precision was determined by adding 6 and [15N5]6 to 1 mg herring testes DNA (N = 4) and carrying out the analysis. Recovery was calculated by adding the internal standard [15N5]6 (5000 fmol) to 1 mg herring testes DNA and processing the samples as described above (N = 8). The internal standard areas were compared to those of the same quantities (1 mg) of herring testes DNA processed without addition of the internal standard (N = 4), which was added just prior to the HPLC-ESI-MS/MS-SRM analysis. The limit of detection (LOD) was estimated from the lowest amount of 6 added to the herring testes DNA sample.